Technical
Abstract
Summaries
European Conferences on
Biomedical Optics
17–21 June 2007
ICM—International Conference Centre Munich
Germany
Program Chairs:
Wolfgang Drexler, Cardiff
Univ. (United Kingdom)
Mary-Ann Mycek, Univ. of
Michigan (United States)
Conf. 6626 Molecular Imaging . . . . . . . . 2-7
Conf. 6627 Optical Coherence
Tomography and
Coherence Techniques . . . 8-18
Conf. 6628 Diagnostic Optical
Spectroscopy . . . . . . . . . . 19-30
Sponsored by:
Conf. 6629 Diffuse Optical Imaging
of Tissue . . . . . . . . . . . . . . 31-43
Conf. 6630 Confocal, Multiphoton, and
Nonlinear Microscopic
Imaging . . . . . . . . . . . . . . . 44-52
Conf. 6631 Novel Optical Instrumentation
for Biomedical
Applications . . . . . . . . . . . 53-63
Conf. 6632 Therapeutic Laser
Applications and LaserTissue Interactions . . . . . . 64-78
Conf. 6633 Biophotonics 2007: Optics
in Life Science . . . . . . . . . 79-95
European Conferences on Biomedical Optics 2007 •
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Conference 6626: Molecular Imaging
Room 3 • Sunday-Monday 17-18 June 2007
Part of Proceedings of SPIE Vol. 6626 Molecular Imaging
6626-01, Session 1
Optical molecular imaging of stroke-induced
brain inflammation in the mouse
J. Klohs, J. M. Steinbrink, R. Bourayou, Charité-Univ. Medicine Berlin
(Germany); M. Grafe, Deutsches Herzzentrum Berlin (Germany); G.
Kronenberg, Charité-Univ. Medicine Berli (Germany); K. Greger, E. H. K.
Stelzer, European Molecular Biology Lab. (Germany); U. Lindauer, U.
Dirnagl, A. Wunder, Charité-Univ. Medicine Berlin (Germany)
Experimental and clinical evidence indicate that following stroke CD40
signalling is crucially involved in sustaining the inflammation and thereby
contributes to the expansion of the lesion. Non-invasive imaging of CD40
receptor expression could provide a powerful tool to diagnose stroke-induced
inflammation, to assess disease progression, to stratify patients for therapy,
and to monitor response to therapeutic intervention.
In the present study, we utilised non-invasive planar near-infrared fluorescence
(NIRF) imaging to detect stroke-induced brain inflammation in a mouse stroke
model. A monoclonal antibody against CD40 labelled with the NIRF dye Cy5.5
was injected intravenously 96 hours after transient middle cerebral artery
occlusion (MCAO) in mice. NIRF imaging was performed 16 hours after
injection of the compound. In MCAO-mice, high fluorescence intensities were
detected through the skull and skin over the affected hemisphere.
Corresponding ex-vivo NIRF images of the brain and brain sections confirmed
localisation of the fluorescence in the ischemic territory. MCAO-mice receiving
Cy5.5-labeled IgG as a control for non-specific accumulation did not show
fluorescence enhancement over the ischemic hemisphere in-vivo. Single Plane
Illumination Microscopy (SPIM) revealed the cellular localisation of the CD40
targeting contrast agent which was found to locate at activated microglia
and endothelium after subsequent immunhistochemistry. Co-injection
experiments with the green fluorescent cell tracker 6-carboxylfluorescein
diacetate into the spleen of MCAO-mice revealed the presence of bloodderived mononuclear cells that were labelled with the CD40 targeting contrast
agent.
In conclusion, the results show that non-invasive NIRF imaging can be used
to visualize stroke-induced brain inflammation in mice with high sensitivity
and specificity.
6626-02, Session 1
G. Zheng, Ontario Cancer Institute (Canada) and Univ. of Toronto
(Canada) and Univ. of Pennsylvania (USA); J. Chen, Ontario Cancer
Institute (Canada); K. Stefflova, Univ. of Pennsylvania (USA); B. C.
Wilson, Ontario Cancer Institute (Canada)
Precisely localizing neoplastic areas within the body would greatly reduce
the toxicity and improve the efficacy of cancer treatment. To achieve this aim
without further burdening the patient by administering yet another drug,
imaging and therapy can be combined using a single molecule capable of
both. The obstacle standing in the way of a practical synergy of many imaging
and treatment modalities lies in the drastically different doses needed for
these two purposes. Here we report the successful combination of
photodynamic therapy, a novel but already well established cancer treatment,
and near-infrared fluorescence imaging, a sensitive and noninvasive method
of in vivo cancer detection, into one probe - photodynamic molecular beacons.
The primary component of photodynamic molecular beacons is a fluorescent
photosensitizer responsible both for the imaging and therapy. By attaching
other components, e.g. various DNA- or peptide-based linkers, quenchers
with differing quenching abilities or cancer cell-specific delivery vehicles, we
can modulate the beacon’s primary function as well as target specificity and
pharmacological properties. This modular design makes these beacons very
flexible, anticipating future applications in which few simple building blocks
are assembled into one target-specific multifunctional probe. In this report
we outline the basic principles of photodynamic molecular beacons, the
current achievements, and future directions including possible cancer targets
and different therapeutic applications.
Functional relations between GFP-like
chromoproteins and red fluorescent proteins
S. Gundel, G. U. Nienhaus, J. Wiedenmann, Univ. Ulm (Germany)
We cloned a red fluorescent protein (asFP595) and a chromoprotein (asCP562)
from the sea anemone Anemonia sulcata var. rufescens. While asFP595 emits
bright fluorescence peaking at 595 nm, asCP562 is virtually non-fluorescent.
The amino acid sequences of both proteins are highly similar (~ 90 %). Key
residues responsible for the dramatic differences in fluorescence intensity
were identified. Adapted from these results, we suggest a strategy to create
fluorescent markers with novel properties based on non fluorescent proteins.
6626-05, Session 1
Optical properties of green fluorescent proteins
and their applications on virus infection
Recently, exogenous fluorescent agents have been widely used as biological
indicators in bioimaging techniques. Among the variety of exogenous
fluorescent agents, green fluorescent protein (GFP) possesses the advantages
of robustness and high quantum efficiency. Therefore, GFP has become
indispensable in molecular and cell biology as noninvasive luminescent labels
for monitoring gene expression, protein localization and protein interactions.
Although GFP and its mutant have been used in many applications, their
optical properties and reactions have not been completely understood,
especially when they are under various environmental conditions and different
cloning representation genes by virus infection. In this research, we developed
a spectrum-analyzing system to investigate the fluorescent properties of GFP
in the environments of different temperatures. We found that the fluorescent
spectrum of GFP consisted of two components that might come from the
transitions between different electronic energy levels where the quantum
efficiencies of the two components varied with different temperature. This
effect was expected to come from the thermal effect on the electron
distributions in the molecular energy levels of GFP. Furthermore, GFP was
used as fluorescent marker to monitor the infection process of cells by viruses
with a dynamic spectral imaging system. The recombinant baculoviruses
containing the red and green fluorescent protein gene that can simultaneously
produce dual fluorescence were used as vectors in Spodoptera frugiperda
21 cells under the control of a polyhedrin promoter. The system was used to
monitor the spatial distribution of fluorescent spectra of cells infected by
virus during the process of infection.
6626-06, Session 2
Three dimensional bioluminescence tomography
6626-03, Session 1
Sensitive detection of protoporphyrin-IX
accumulation in genetically modified colon
cancer cells: a new tool for molecular imaging
B. Ebert, S. Rüttinger, J. Voigt, R. Macdonald, Physikalisch-Technische
Bundesanstalt (Germany); W. Kemmner, K. Wan, U. Klamm, P. M.
Schlag, Charité-Univ. Medizin Berlin (Germany)
Optical modalities for fluorescence imaging and spectroscopy are of
increasing interest. Fluorescence based determinations of crucial metabolic
compounds are key applications. Specific accumulation of the fluorescent
heme precursor Protoporphyrin IX (PpIX) in carcinomas and lymph node
European Conferences on Biomedical Optics 2007 •
6626-04, Session 1
J. Lee, C. Kao, Y. Chen, T. Wu, I. Hsu, Chung Yuan Christian Univ.
(Taiwan)
Detection and treatment of cancers using
photodynamic molecular beacons
2
metastases has been shown as well as accumulation of PpIX achieved by
exogenous addition of 5-aminolevulinic acid (ALA). However, the mechanisms
which lead to the increased PpIX-accumulation in malignant tissue are not
fully understood so far. Exploiting the PpIX fluorescence decay time, which
is longer than that of the majority of unspecific tissue fluorophores, timedependent fluorescence spectroscopy has been shown to considerably
facilitate determination of PpIX concentration. Detection of PpIX in minute
amounts was achieved using an advanced pulsed solid-state laser system
combined with an intensified CCD camera for time-delayed observation of
fluorescence. In order to prove that PpIX-accumulation is caused by a
decrease of ferrochelatase (FECH), a genetic approach was used to suppress
FECH-expression. Due to reduced expression of ferrochelatase in pellets of
cancer cells an increase in fluorescence intensity of PpIX by a factor of 50
was demonstrated. This finding was supported by two-photon scanning
microscopy images which showed strong cytoplasmatic red fluorescence.
This demarcation is a prerequisite for detection of malignant tissue and PpIXmediated photodynamic therapy. Genetically controlled FECH decrease
leading to accumulation of PpIX within the treated cells may substantially
enlarge the application of fluorescence detection in medicine and support
the identification and localization of targets in optical molecular imaging.
H. Dehghani, The Univ. of Exeter (United Kingdom); B. W. Pogue, S. C.
Davis, Dartmouth College (USA); M. S. Patterson, Juravinski Cancer Ctr.
(Canada)
Recent interest in modeling and reconstruction algorithms for
Bioluminescence Tomography (BLT) has increased and led to the general
consensus that non-spectrally resolved intensity-based BLT results in a nonunique problem. However, the light emitted from, for example firefly Luciferase,
is widely distributed over the band of wavelengths from 500 nm to 650 nm
and above, with the dominant fraction emitted from tissue being above 550
nm. This work will demonstrate the development of a 3D model and algorithm
used for multi-wavelength 3D spectrally resolved Bioluminescence
Tomography (BLT) image reconstruction. It will demonstrated that through
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Conference 6626: Molecular Imaging
the use of fast single-step algorithms, accurate 3D reconstruction of internal
Bioluminescence sources from deep within tissue can be achieved using
only a single angle measurement of data. It is shown that in a small animal
model, using a single view data, bioluminescence sources of up to 15 mm
deep can be successfully recovered. It will also be demonstrated that the
underlying spectral tissue heterogeneity in optical properties which is a
function of the tissue function and physiology, has a substantial effect on the
imaging problem. Reconstructed images are created to show that it is possible
to recover the intrinsic optical properties of tissue from transmission
measurements of Near Infrared light sources, which will in turn enable accurate
3D BLT imaging.
6626-07, Session 2
Post mortem evaluation of a new approach for
quantitative bioluminescence imaging in small
animals
D. C. Comsa, Juravinski Cancer Ctr. (Canada) and McMaster Univ.
(Canada); T. J. Farrell, M. S. Patterson, Juravinski Cancer Ctr. (Canada)
We report the performance of a simple method for making quantitative
bioluminescence measurements of a point-like source embedded in small
animals. In this method, video reflectometry is first used to obtain an estimate
of the bulk optical properties of the tissue containing the bioluminescent
source [1]. A 2-dimensional image of the bioluminescence signal emitted
from the surface of the animal is then acquired with a CCD. Using the
measured optical properties, and a simple diffusion theory model, an inversion
algorithm is then applied to retrieve the source depth and power from a region
of interest of the bioluminescence images [1]. Two major factors determine
the accuracy of the reconstruction: tissue heterogeneity and curvature of the
imaged surface. The use of measured optical properties to characterize bulk
tissue surmounts, to a degree, the heterogeneity problem: post mortem data
from mice and rats show that the relative power can be retrieved within a
factor of 2 and frequently within 20 %, and the depth within 1.0 mm for
implanted depths of 4-10 mm. For depths shallower than 4 mm, the errors in
the retrieved depth are consistently larger, possibly because the bulk optical
properties are less representative of this superficial region, as confirmed by
Monte Carlo simulations. The effects of surface curvature depend on animal
size and are greater in mice than rats. We show that careful selection of the
region of interest in the bioluminescence images minimizes the effects of
tissue curvature.
[1] D C Comsa, T J Farrell, M S Patterson, Quantification of bioluminescence
images of point source objects using diffusion theory models 2006 Phys.
Med. Biol. 51 3733-3746.
6626-08, Session 2
Spectral unmixing of multi-color tissue specific
in vivo fluorescence in mice
G. Zacharakis, R. Favicchio, A. Garofalakis, S. Psycharakis, C.
Mamalaki, J. Ripoll, Foundation for Research and Technology-Hellas
(Greece)
Fluorescence Molecular Tomography (FMT) has emerged as a powerful tool
for monitoring biological functions in vivo in small animals. It provides the
means to determine volumetric images of fluorescent protein concentration
by applying the principles of diffuse optical tomography. Using different probes
tagged to different proteins or cells, different biological functions and pathways
can be simultaneously imaged in the same subject. In this work we present a
spectral unmixing algorithm capable of separating signal from different probes
when combined with the tomographic imaging modality. We show results of
two-color imaging when the algorithm is applied to separate fluorescence
activity originating from phantoms containing two different fluorophores,
namely CFSE and SNARF, with well separated emission spectra, as well as
Dsred- and GFP-fused cells in F5-b10 transgenic mice in vivo.
The same algorithm can furthermore be applied to tissue-specific
spectroscopy data. Spectral analysis of a variety of organs from control,
DsRed and GFP F5/B10 transgenic mice showed that fluorophore detection
by optical systems is highly tissue-dependent. Spectral data collected from
different organs can provide useful insight into experimental parameter
optimisation (choice of filters, fluorophores, excitation wavelengths) and
spectral unmixing can be applied to measure the tissue-dependency, thereby
taking into account localized fluorophore efficiency. Summed up, tissue
spectral unmixing can be used as criteria in choosing the most appropriate
tissue targets as well as fluorescent markers for specific applications.
6626-09, Session 3
Molecular imaging of experimental arthritis
using an EDB targeting antibody NIR-dye
conjugate
A. Vater, K. Licha, S. Vollmer, Bayer Schering Pharma AG (Germany); I.
Gemeinhardt, O. Gemeinhardt, J. Schnorr, Charité-Univ. Medizin Berlin
(Germany); J. Voigt, J. Berger, B. Ebert, Physikalisch-Technische
European Conferences on Biomedical Optics 2007 •
Bundesanstalt (Germany); M. Taupitz, Charité-Univ. Medizin Berlin
(Germany); M. Schirner, Bayer Schering Pharma AG (Germany)
Rheumatoid arthritis is a chronic inflammatory disease of the joints,
characterized by synovitis and synovial hyperplasia, which are accompanied
by angiogenesis. Chronic synovitis leads to irreversible bone and cartilage
destruction, and in consequence to joint deformities and disabilities. Early
detection of synovitis may thus help to delay or prevent disease progression.
We have studied a molecular imaging approach to detect collagen-induced
arthritis (CIA) in rats by targeting the Extra Domain B (EDB) of fibronectin.
EDB is produced by alternative splicing during embryonic development and
conditions of vascular remodeling. We have confirmed the expression of EDB
in the hyperplastic synovium of CIA rats by immunohistochemistry. For in
vivo diagnostics, the EDB-binding single-chain antibody fragment AP39 was
used as a targeting probe. It was covalently linked to the near-infrared (NIR)
dye Tetrasulfo¬cyanine(TSC) and visualized by fluorescence reflectance
imaging with a 740 nm NIR laser source and an intensified CCD-camera. A
conjugate consisting of TSC and Ovalbumin, having a similar molecular weight
as the spontaneously forming AP39-TSC dimer, was used as a control for
targeting specificity.
AP39-TSC showed a markedly enhanced fluorescence contrast in arthritic
joints that allowed discrimination from control joints in the CIA-rat model up
to 24 h after i.v. application. With ovalbumin-TSC, only a minor accumulation
of the conjugate in affected joints was observed.
Since AP39 is a fully human scFv initially directed against human EDB, a
translation of this molecular imaging approach for early arthritis detection in
man seems feasible.
6626-10, Session 3
Ligand-conjugated lipoprotein nanocarriers for
molecular imaging and therapy of cancer
I. Corbin, J. Chen, Ontario Cancer Institute (Canada); G. Zheng, Ontario
Cancer Institute (Canada) and Univ. of Toronto (Canada) and Univ. of
Pennsylvania (USA)
Advances in genomic and proteomic technologies have identified numerous
cell and receptor targets for diagnostic and therapeutic applications in
oncology. Previous approaches using antibody / ligand conjugated drugs or
imaging agents have effectively targeted cancer cells. Nanotechnology
employs devices on the order of nanometers for novel applications in cancer
diagnostics and therapeutics. When combined with a targeting moiety, these
nano-scale delivery vehicles are able to selectively home in on tumor cells
and deliver their payload of diagnostic and/or cytotoxic agents. However,
many challenges (e.g., biocompatibility, reproducibility and delivery efficiency)
remain before the promise of nanomedicine can be realized in clinical practice.
Lipoproteins are endogenous, non-immunogenic nano-scale delivery vehicles
that ferry cholesterol and other molecules through the bloodstream. However,
the drawback of using them for drug delivery is the narrow purview of
lipoprotein receptor-positive cancer cells. We recently developed methods
that not only allow lipoproteins to be loaded with imaging agents or toxic
payloads but we can also redirected these loaded carriers from their normal
destination (lipoprotein receptor-positive cells) to a variety of cancer cell types,
through targeting specific tumor surface markers. Herein we first describe
the utility of lipoproteins as a versatile class of natural targeted nanoplatforms.
From there we go on to demonstrate both in vitro and in vivo that loaded
lipoproteins can be effectively rerouted to alternate receptors or surface
markers expressed on cancer cells (eg. Folate receptor). Targeted lipoprotein
nanoparticles open opportunities for new strategies of targeted diagnostic
and therapeutics in cancer medicine.
6626-11, Session 3
Synthesis, functionalization, and
characterization of rod-shaped gold
nanoparticles as potential optical contrast
agents
R. G. Rayavarapu, C. Ungureanu, W. Petersen, S. Manohar, T. G. van
Leeuwen, Univ. Twente (Netherlands)
Interest in the interaction of light with plasmonic nanoparticles is growing
tremendously due to potential applications especially in the biomedical
imaging and therapeutic fields. Gold nanoparticles exhibit intense and narrow
optical extinction bands due to plasmon resonance making them useful as
contrast agents. Localized heating results from the absorbed light energy,
which may be applied to photothermally induced hypothermia for therapeutic
applications. The bioconjugation of gold nanoparticles to appropriate
antibodies targeted to tumours in vivo, could make highly selective detection
and therapy of tumors possible. We have synthesised gold nanorods based
on seed mediated protocols. Gold nanorods produced using a single
surfactant silver assisted method have aspect ratios between 2.3 - 3.6 having
plasmon peaks between 670-850 nm within the “optical imaging and
therapeutic window”. The bisurfactant method has yielded nanorods with
peaks in the range of 850-1100 nm having aspect ratios between 5 - 11.
Typical, concentrations of these particles in aqueous dispersions are in the
range of 1x1010 - 1x1011 particles per ml. We have also coated the gold
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Conference 6626: Molecular Imaging
nanorods with polyethylene glycol (PEG) for biocompatibility in vivo and
bioconjugated gold rods with anti-HER2/neu mouse monoclonal antibodies.
Characterization and size estimation were performed using electron
microscopies, optical spectroscopy and confocal microscopy. We present
these results and implications for use of these nanoparticles for in vivo
biomedical applications.
6626-12, Session 3
Nanoparticle assisted optical molecular imaging
(NAOMI) using biodegradable nanoparticles
D. J. Faber, M. D. de Bruin, M. C. G. Aalders, F. D. Verbraak, T. G. van
Leeuwen, Univ. van Amsterdam (Netherlands)
We present a novel method for designing biodegradable nanoparticles which
are suitable as contrast agents in optical techniques such as Optical
Coherence Tomography. Our initial results presented in this contribution are
based on Rhodamine B. -doped PLGA nanoparticles, which exhibit
‘anomalous’ scattering behavior around the absorption peak of Rhodamine
B. at 542 nm as predicted by a Kramers-Kronig / Mie theory analysis.
6626-13, Session 4
We present a lifetime fluorescence imaging system for small animal imaging.
The system uses a linear fiber array with given separations between a single
source fiber and several detection fibers. The goal is to localize tumors and
monitor their progression, using specific fluorescent markers. We have chosen
a near infrared dye, Alexa Fluor 750, as a contrast agent. To validate the
system we performed measurements of fluorescence lifetime for targets
embedded in tissue-like phantoms and compared obtained fluorescence
decay curves with a random walk based theoretical forward model to
substantiate depth-related corrections to intrinsic fluorescence time-of-flight
distributions. Good correlation between measurements and developed model
has been demonstrated. Observed pH sensitivity of the chosen near infrared
dye, conjugated with to tumor specific antibodies (Herceptin), makes it a
promising contrast agent for pH mapping of the tumor area. Initial results on
in vivo (mouse model) pH mapping in the vicinity of the superficial tumor
show lower pH values inside the tumor, comparing to that of the surrounding
normal tissues.
6626-16, Session 4
Non-invasive scalping: increasing the sensitive
of non-invasive fluorescence brain imaging in
mice by using a two wavelength approach
P. Bahmani, J. Klohs, A. Wunder, U. Lindauer, R. Bourayou, U. Dinagl, J.
M. Steinbrink, Charité-Univ. Medizin Berlin (Germany)
MR-guided near-infrared fluorescence
spectroscopy of brain tumor
B. W. Pogue, Dartmouth College (USA)
A unique spectroscopy system has been constructed and tested for imaging
with near-infrared (NIR) inside a small animal body coil. The broad spectrum
capability provides access to transmission, fluorescence or bioluminescence
signals as needed, while not inhibiting the ability for MR imaging of the animal.
The key factor in making a truly useful system is to have the optical sources
and detectors built into the MR coil in a way which provides data from both
at the same time, and does not compromise on quality of the data from
either. This system was constructed for a Philips 3T MR system by modifying
the rodent coil, and is being used to study production of the fluorescent
compound protoporphyrin IX in glioma tumors.
A 3T rodent coil was specially designed and fabricated by Philips Research
Hamburg, complete with holes for the fibers to be placed through the coil,
and onto the rodent inside. The system was tested with rat and mouse
tumors. The spectroscopy is completed with sixteen bifurcated fibers, where
one end of the bifurcation goes to a laser sequencer, and the other ends all
go to individual spectrometers. Reconstruction of the interior tissue values is
done with spatial mapping onto the MR image, using standard segmentation
methods of the tissue volumes. Each region is given a unique set of optical
spectra as estimated by literature values. Fluorescence imaging and
absorption imaging of molecular features of the tissue is possible, and MRguided imaging of the brain tumors show clear ability to quantify the tumor
volume through endogenous PpIX fluorescence.
Fluorescence imaging has been promoted for its high sensitivity (detection
limit < 10-9 to 10-12 mol/l [Massoud and Gambhir, Genes Dev, 2003] for
microscopic set-ups). However, for subsurface sounding during in-vivo animal
studies, the detection limit is strongly hampered due to the i) attenuation of
the excitation and fluorescence light by the superficial layers ii) the autofluorescence in the outer layers and the iii) fluorescence of non-specific
fluorochromes. As a result the lowest detection limit reported for the mouse
brain is about 10-5 mol/l in a volume 0.1 microliter [Klohs et. al. Mol. Img.
2006].
Here we propose a dual-wavelength excitation approach to improve the invivo fluorescence detection sensitivity in deeper tissue and show an
application for the mouse brain. The excitation wavelength range for the
chosen Cy5.5 spans from around 600 to about 690nm. In this wavelength
range the penetration depth of light varies strongly due to the increased
absorption by deoxy-hemoglobin in the low wavelength range. Thus applying
600 to 630nm excites the fluorochrome pre-dominantly in the scalp, whereas
light at 670 to 690 nm excites the fluorchrome also in the brain. Thus deducting
the image obtained at an excitation wavelength of 630nm from the image
obtained at 680m allows to measure a ‘brain weighted’ fluorescence image
and thus improves the detection limit mentioned above.
We show the successful application of this imaging approach in a simple
brain pathology model consisting of a fluorescence capsule implanted in the
mouse brain. The method is further validated in tissue simulating phantoms.
6626-17, Session 4
6626-14, Session 4
Autofluorescence removal from fluorescence
tomography data using multispectral imaging
Time-resolved scanning system for double
re?ectance and transmittance ?uorescence
imaging of small animals
S. Psycharakis, G. Zacharakis, A. Garofalakis, J. Ripoll, Foundation for
Research and Technology-Hellas (Greece)
M. Brambilla, L. Spinelli, A. Pifferi, A. Torricelli, R. Cubeddu, Politecnico
di Milano (Italy)
Autofluorescence has been a significant disadvantage when dealing with
tomographic imaging of biological samples or tissue phantoms. Consequently,
the accurate removal of autofluorescence signal has been a major concern
in fluorescence tomography. Here we present a study on three-dimensional
mapping and removal of autofluorescence signal from fluorescence molecular
tomography (FMT) data, both for phantoms and small animal in vivo. The
technique is based on the recording of tomographic data in multiple spectral
regions with different excitation light and on the application of a linear unmixing
algorithm for targeting multiple fluorescent probes. Two types of
measurements are taken, one with the excitation being in the region of the
maximum absorption of the targeted fluorophore and one in a region away
from the maximum. The relative strengths of the different spectra are employed
to calculate the signal to be removed from the tomographic reconstruction.
Autofluorescence spectra are recorded using identical reflection geometry
as during the FMT acquisitions allowing for the correct mapping of the
autofluorescence signal. Results from phantoms exhibiting different
background autofluorescence strengths are presented and discussed. In this
work we have also studied in vivo fluorescent activity in mice, involving both
subcutaneously implanted fluorescent phantoms and b10 transgenic mice.
We developed a time-resolved scanning system for fluorescence molecular
imaging in diffusive media, such as biological tissues. In the present work
the system is described and characterized in terms of linearity against optical
parameters of the sample and against homogeneously diffuse fluorescent
dye. Finally, preliminary measurements performed on phantom are presented,
pointing out the ability of our system to produce projective images of the
fluorophore distribution into the sample with a 200 fmol sensitivity and to
decouple fluorescence amplitude from depth by means of fluorescence
transmittance imaging.
6626-15, Session 4
Fluorescence Lifetime Imaging of Targets, a
Step to a Functional Imaging of Tissue
Abnormalities, Deeply Embedded in Turbid
Medium
V. V. Chernomordik, M. Hassan, J. D. Riley, A. H. Gandjbakhche,
National Institutes of Health (USA)
4
European Conferences on Biomedical Optics 2007 •
6626-18, Session 4
Recent advances in time-resolved confocal
fluorescence microscopy
U. Ortmann, F. Koberling, P. Kapusta, PicoQuant GmbH (Germany)
Today, time resolved measurements allow with one universal technique to
follow fluorescence dynamics starting in the sub-nanosecond range up to
fluctuations in the second range and beyond. Moreover, the underlying
technique (Time-Tagged Time-Resolved (TTTR) single photon recording) offers
not only to acquire timing information but at the same time to store also
spectral and spatial information for every detected photon from the sample.
Microscopy based on this unrestricted photon data acquisition approach
enables one to easily study dependencies between various fluorescence
parameters. Furthermore, the significance and accuracy in common FCS
and FRET analysis schemes can be improved applying sorting and weighting
of the detected photons on the basis of the photon arrival time.
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Conference 6626: Molecular Imaging
We will demonstrate the power of this approach for different techniques: The
nanosecond lifetime information allows easily to remove scattered light and
common detector artefacts in standard FCS experiments. Moreover,
Fluorescence Lifetime Correlation Spectroscopy (FLCS) offers the possibility
to separate FCS curves for species which differ only in their fluorescence
lifetime but, for example, cannot be distinguished spectrally [1]. We will discuss
the accuracy of deduced diffusion constants and show also first results from
dual focus FCS experiments which circumvent the necessity to have prior
information about the size and shape of the confocal volume for the FCS
analysis. The determining parameter in this application is the easily accessible
distance between the two foci which can be individually adressed by using
pulsed interleaved excitation (PIE) [2].
We are using the confocal microscope MicroTime 200 which can be equipped
either with compact picosecond pulsed diode lasers as excitation sources
or utilized in conjunction with a high power femtosecond solid state laser to
allow for two photon imaging. The timing performance is significantly
increased by using new SPAD detectors which offer together with the
PicoHarp TCSPC electronics an overall system IRF of less than 120 ps FWHM
while maintaining single molecule sensitivity. The count rate independent
IRF position in time of these detection modules allows for the first time
accurate fluorescence lifetime imaging (FLIM) with count rates up to the MHz
range.
6626-19, Session 4
Applying time-dependent data for fluorescence
tomography
R. B. Schulz, J. Peter, W. Semmler, Deutsches Krebsforschungszentrum
(Germany); C. D’Andrea, G. Valentini, R. Cubeddu, Politecnico di Milano
(Italy); M. Schweiger, S. R. Arridge, Univ. College London (United
Kingdom)
Can time-resolved, high-resolution data as acquired by an intensified gated
CCD camera (ICCD) aid in the tomographic reconstruction of fluorescence
concentration? Usually it is argued that fluorescence is a linear process and
thus does not require non-linear, time-dependent re°(c)constructions
algorithms, unless absorption and scattering coefficients need to be
determined as well. Furthermore, the acquisition of a number of time frames
is usually prohibitive for fluo°(c)res°(c)cence measurements, at least in small
animals, due to the increased total measurement time.
On the other hand, it is obvious that diffusion is less pronounced in images
at early gates, due to se°(c)lective imaging of photons of lower scatter order.
This will be the case also for photons emitted by fluorescent sources. Earlygated imaging leads to higher contrast and possibly improved fluorescence
localization.
Herein, we present early gated fluorescence images obtained from phantoms
and compare them to con°(c)tinuously acquired data. Increased contrast
between background and signal maximum can be ob°(c)served in time-gated
images as compared to continuous data. To make use of the properties
ex°(c)hibited by early gated frames, it is necessary to use a modified
reconstruction algorithm. We pro°(c)pose a variant of the well-known Born
approximation to the diffusion equation that allows to take into account single
time frames. The system matrix for the time-dependent Born approach is
more complex to calculate, however the complexity of the actual inverse
problem (and the acquisition times) of single-frame reconstructions remains
the same as compared to continuous mode.
6626-20, Session 4
Pump-lasers-induced multi-structures
photoprocesses or the near-lying singlet and
triplet excited electronic states in the
geteroaromatic molecules
A. E. Obukhov, Moscow Mining Institute (Russia)
This paper considers the relation between the mechanisms behind optical
and non-optical energy deactivation of electron-vibrational excitation
associated with inner and spin-orbital inter-combination convertion in series
N-, O-, S- multi-atomic molecules, which are capable of fluorescing and
generation of light within the range of wavelengths = 260 - 760 nm. To solve
such a problem of the spectroscopy, one has to reveal general relationships
between the structure of excited electronic states and transitions of different
spin and orbital nature and photo physical properties of organic molecules.
In the case of UV laser pumping of organic molecules and effective population
of the high-lying and states the processes of photo-ionization and the
subsequent photo-destruction are virtually inevitable.
During a high-power pump pulse of the width comparable with active molecule
lifetime in the excited state, the active molecule excited into the lower singlet,
, or the triplet, , states can reabsorb excitation energy as a result of electron
transitions into (induced reabsorption spectrum ) and (induced reabsorption
spectrum ) states prior to light emission. For molecules the growth in the
energy stored in excitation, which occurs within the time interval no less than
the characteristic time of initiation of multi-photon processes in excited
molecules with , is shown to give rise transitions populating those highly
European Conferences on Biomedical Optics 2007 •
excited states that are involved in spin-orbital interaction with triplet states
( ). Therefore, the drop in the fluorescence quantum yield in such a situation
is several order of magnitude stronger and limit time pump pulse of the laser
< 0,01 nc.
6626-21, Poster Session
Discovery of innovative fluorescent probes for
cells imaging using a fast parallel approach
M. Gruit, Univ. de Rennes I (France)
This project consists in synthesizing new probes to visualize the membranes
of cells.
These probes present an hydrophilic head, a combined bridge (double or
triple bond) and an hydrophobic tail, allowing a good insertion in to the lipidic
membrane (GUV or cells).
The two very important parameters defining the efficiency of the probes is
their capacity of insertion in the membranes, determined by broad band
microscopy (camera CCD) and their intensity of fluorescence, determined by
confocal microscopy of fluorescence with one photon.
We developed a method including:
- a fast stage of synthesis, by carbon-carbon coupling, in order to create a
diversity of potential probes.
- a stage of parallel screening of a great number of probes, by a comparative
analysis of their efficiency with Di-4-ANEPPS (commercial dye). These
analyses are made on the models of GUV (Giant Unilamellar Vesicles), of
which the structure is completely similar with those lipidic membranes of
alive cells.
- a stage of detailed study of the best candidates on cells P19 (cellular lines
of mouse neurons).
- a stage of validation by the study of the toxicity of the probes within the
cells.
The method is fast and easily accessible to get a first idea of interesting
probes.
6626-22, Poster Session
Activatable quantum dots for mouse noninvasive
fluorescence imaging
I. F. Texier Nogues, J. Marchand, E. Heinrich, A. Da Silva, Commissariat
à l’Energie Atomique (France)
Fluorescence imaging is a very attractive tool for fastening the development
of new therapeutics. Two classes of labels exist for the near infrared domain:
organic dyes and quantum dots (QDs). QDs are inorganic luminescent semiconductor nano-crystals which display very attractive optical features. They
are now commercially available for in vivo mouse tests, and new compositions
with less toxic elements are currently being developed.
The concept of activatable probes, which fluorescence is activated specifically
upon the biological process to be visualized, has also been demonstrated to
improve the fluorescence image contrast.
The construction of activatable probes based on quantum dot labels was
therefore undertaken. Commercial PEGylated quantum dots bearing around
80-100 amino pending groups were used. Long PEG chains are demonstrated
to be essential in order to increase the blood circulation time of the particles
and avoid their massive storage into the liver. The amino groups coating the
QD surface can be used for their further functionalization by either a tumortargeting ligand, a cleavable spacer bearing a fluorescence inhibitor I, or both.
Functionalization of 80% of the amino groups by the inhibitors I leads to
more than 99% fluorescence quenching. Cleavable spacers X-S-S-I in which
S-S is a disulfide bridge cleavable by cell internalization, and X a chemical
group for QD grafting have been synthesized. The functionalization of the
QD by 12 cleavable spacers leads to more than 80% fluorescence inhibition,
which can be recovered upon cleavage of the disulfide bridges.
6626-23, Poster Session
Molecular targeting as a contrast agent
mechanism for fluorescence endoscopy
A. J. Healey, R. Bendiksen, A. Tornes, E. W. Johannesen, GE Healthcare
Bio-Sciences (Norway)
Several mechanisms of action can be employed for a molecular imaging
contrast agent for use with endoscopy. Targeting of cell surface molecules
that are up regulated at an early disease stage, with a fluorescent reporter is
one attractive approach. However, it suffers from the inherent limitation that
the concentration of agent available is fundamentally limited by the
concentration of receptor molecules available. Simple models indicate that
for successful imaging with a targeting approach, the imaging system should
be able to adequately image concentrations in the nanomolar region. Such
low reporter molecule concentrations have implications for the choice of
contrast agent. Target tissue size and location, the tissue native fluorescence
contribution, the brightness of the reporter molecule, and photobleaching
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Conference 6626: Molecular Imaging
thresholds are all factors which contribute to the choice of reporter. For
endoscopic imaging of millimetre sized target tissue volumes close to the
surface we demonstrate that Cy5 (650-700nm) wavelengths are preferable
to Cy3 (550-600nm) and Cy7 (750-800nm).
We have constructed a system optimised for sensitivity by tailoring light
delivery, collection, filtering and detection, in order to address the fundamental
technical performance limits for endoscopic applications. It is demonstrated
through imaging system calibration, phantom based measurement and animal
imaging data that low nanomolar concentrations of Cy5 based fluorescent
contrast agent in millimetre sized superficial lesions are adequately imaged
with a clinically relevant endoscope system in real time. It is concluded that
targeting is a technically viable approach for endoscopic applications.
6626-24, Poster Session
Ethidium bromide as a probe of mtDNA
replication in living cells
A. M. Villa, P. Fusi, C. Pozzi, M. Valtorta, Univ. degli Studi di Milano
Bicocca (Italy); G. Amicarelli, D. Adlerstein, DiaSorin S.p.A. (Italy); S. M.
Doglia, Univ. degli Studi di Milano Bicocca (Italy)
Recent studies suggest that mitochondrial DNA (mtDNA) plays an important
role in maintaining the malignant phenotype of tumor cells, since cell
differentiation affects mtDNA cell content, mitochondrial gene expression
and D-loop frequency. By laser scanning confocal fluorescence microscopy
(LSCFM) we observed in single living carcinoma cells that the fluorescence
of ethidium bromide (EB) localized in nucleoids showed different intensity,
suggesting that a different interaction with EB might occur. Since it is known
that EB interacts by intercalation with the mtDNA double helix, a higher EB
fluorescence intensity may reflect a higher DNA accessibility, possibly due
to the presence of replicating D-loops. To clarify this point, we investigated
by LSCFM the EB fluorescence distribution in mitochondria nucleoids of living
human neuroblastoma cells (SHSY-5Y),before and after cell differentiation
induced by retinoic acid. A drastic EB fluorescence decrease was observed
after differentiation. To probe the mtDNA replication state, we evaluated the
D-loop content by ligation-mediated real time PCR. The threshold cycle
obtained on mtDNA from differentiated cells was found to be consistently
lower than that obtained from cancer cells, indicating the presence of a halved
amount of replicating D-loops in differentiating cells. These results suggest
that the low EB fluorescence of nucleoids in differentiated cells may be related
to a low content of replicating mtDNA, indicating that EB may be used as a
marker of mtDNA replication in intact living cells.
6626-25, Poster Session
Correlation between direct microscopy and
FDG-PET in the study of cerebral brain flow in
rats
O. Blagosklonov, Univ. de Franche-Comte (France) and Jean Minjoz
Univ. Hospital (France); G. I. Podoprigora, S. V. Pushkin, Y. R.
Nartsissov, Institute of Cytochemistry and Molecular Pharmacology
(Russia); L. Comas, J. Cardot, H. Boulahdour, Univ. de Franche-Comte
(France)
Isotope studies provide valuable data about an organ’s function in vivo. Thanks
to positron emission tomography (PET) using the radiolabeled natural
metabolites, such as [18F]-2-fluoro-deoxy-d-glucose (FDG), biological and
physiological meaning of nuclear medicine scans has been considerably
increased. Therefore it is of interest to elucidate the possibilities of the
technique in a study of some natural metabolites like glycine influencing the
blood microcirculation.
Glycine, as a medicine, was recently shown to have a positive therapeutical
effect in the treatment of patients with ischemic stroke and some other
neurological disorders based on vascular disturbances. By previous direct
biomicroscopic investigations of pial microvessels in laboratory rats an
expressed vasodilatory effect of topically applied glycine was proved. The
arterioles diameters depending on initial size have been increased from 200%
to 250% for arterioles of 20-40 µm and from 150% to 200% for arterioles of
50-80 µm.
The PET images were acquired before and after sublingual application of
glycine (200 mg). The quantitative analysis of FDG volume concentration
(Bq/ml) in the rat brain demonstrated that, in studies after glycine
administration, maximal, minimal and mean FDG volume concentration in
the brain increased from 150% to 250% in comparison with the baseline
data.
Thus, our results revealing evident correlation between FDG-PET images
and direct biomicroscopic observations confirm the great potential of
molecular imaging techniques to explore in vivo process in the brain.
6
European Conferences on Biomedical Optics 2007 •
6626-26, Poster Session
Multiresolution transform denoising and
segmentation of single molecule motility image
series
F. von Wegner, T. Ober, O. Friedrich, R. H. A. Fink, Ruprecht-Karls-Univ.
Heidelberg (Germany); M. Vogel, Harvard Univ. (USA) and RuprechtKarls-Univ. Heidelberg (Germany)
We present a multiresolution transform-based method for the extraction of
moving filament trajectories from single molecule motility data. Noisecorrupted fluorescence image series are denoised using the multiscale median
transform and trajectories are detected on the denoised data set. The
presented method reduces noise more efficiently than 2D-anisotropic diffusion
and several wavelet based techniques. Fibre trajectories are extracted by
segmentation of the denoised image stacks and non-crossing trajectories
are unambiguously identified combining the information of 2D (XY) and 3D
(XYT) segmentation.
The algorithm is applied and evaluated using experimental data sets - image
sequences of fluorescently labeled F-actin and their 2D-trajectories on a
myosin coated surface. This so-called ‘motility assay’ is used to analyze
kinetics, biochemical regulation and pharmacological modulation of these
biologically relevant molecules. The presented method improves the signalto-background discriminiation and facilitates filament identification and may
contribute to significantly improve the performance of this assay.
6626-27, Poster Session
Fluorescence diffuse tomography for detection
of RFP-expressed tumors in small animals
I. V. Turchin, Institute of Applied Physics (Russia); A. P. Savitsky, A.N.
Bach Institute of Biochemistry (Russia); V. A. Kamensky, V. I. Plehanov,
A. G. Orlova, M. S. Kleshnin, M. V. Shirmanova, I. I. Fix, Institute of
Applied Physics (Russia); V. O. Popov, A.N. Bach Institute of Biochemistry (Russia)
Capabilities of tumor detection by different optical methods can be
significantly improved by labeling of tumors with fluorescent markers. Creation
of tumor cell lines transfected with fluorescent proteins provides the possibility
not only to detect tumor, but also to conduct the intravital monitoring studies.
Cell lines of human melanomas Mel-P, Mel-Kor and human embryonic kidney
HEK-293 Phoenix were transfected with DsRed-Express and Turbo-RFP
genes. Emission of RFP in the long-wave optical range permits detection of
the deeply located tumors, which is essential for whole-body imaging. Only
special tools for turbid media imaging, such as fluorescent diffusion
tomography (FDT), enable noninvasive investigation of the internal structure
of biological tissue. FDT setup for monitoring of tumor growth in small animals
has been created. An animal is scanned in the transilluminative configuration
by low-frequency modulated light (1 kHz) from Nd:YAG laser with second
harmonic generation at the 532 nm wavelength. In vivo experiments were
conducted immediately after the subcutaneously injection of fluorescing cells
into small animals. It was shown that FDT method allows to detect the
presence of fluorescent cells in small animals and can be used for monitoring
of tumor growth and anticancer drug responce.
6626-28, Poster Session
Tumor vascular permeability correlated with
acute response to antivascular therapy
assessed by time domain fluorescence imaging
U. Sunar, D. J. Hall, Univ. of California/San Diego (USA)
Combretastatin A4 phosphate (CA4P) is a vascular targeting agent, which
modifies tumor vasculature in the tumor. The drug targets tumor blood vessels
in the tumor and there is a shut-down of the blood flow in tumors by disrupting
the capillaries that feed the tumors. MRI studies showed that tumor endothelial
cells are more permeable to CA4P compared to surrounding normal tissue.
Preferential tumor targeting should improve the efficacy of the drug in clinical
trials. Measurement of tumor microvascular density (MVD) from tumor biopsies
is a common way to assess the efficacy of the antivascular drugs, however it
is not suitable for repeatable measurements. To facilitate clinical translation
of agents that target tumor vasculature, an ability to assess tumor vessel
permeability with repetitive measurements is desirable. Potential clinical
applications in mind we have tested CA4P on murine tumor models with fullfield time domain optical molecular imaging system incorporating a highpower laser for area illumination and a gated-intensified CCD camera for
area detection. Vascular permeability and blood flow changes were assessed
by pixel-by-pixel fitting of the pharmacokinetics of Indocyanine Green (ICG)
and the results showed that changes in these functional parameters were
highly correlated.
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Conference 6626: Molecular Imaging
6626-29, Poster Session
Three-dimensional optical metrology and
models for non-contact diffuse optical
tomography of small animals
Y. Bérubé-Lauzière, M. Comtois, Univ. de Sherbrooke (Canada)
Traditionally, obtaining diffuse optical tomography (DOT) data for deep 3D
optical imaging of thick (\>1cm) biological tissues has been carried with optical
fibers in direct contact with the tissue or by using a matching fluid interface.
Aside from simplifying the geometry and having to account for a single
propagation mode (diffusive mode), the use of fibers and fluids in small animal
imaging implies light signal attenuation, regular system maintenance, possible
drowning and undesirable squeezing of the animal. Non-contact DOT is thus
clearly more suitable for small animal imaging and brings the challenge of
measuring the animal’s outer surface shape. We present a 3D optical
metrology system for measuring this shape and its integration into the noncontact small animal DOT scanner we are developing. The key feature of our
approach is to use the same laser beam as that for the tomographic
measurements, and a stereo camera pair, thus considerably reducing system
complexity. Moreover, the 3D measurements can be carried while the DOT
data are being acquired. For fast acquisition and precise measurements
(<1mm), we rely on a novel axis (rotational and translational) optical calibration
technique allowing the acquisition of full 3D models. A benefit of our system
is to measure, rather than indirectly infer, the exact position where laser light
is injected into the animal. This is extremely useful information as regards the
tomographic reconstruction algorithm, which is not available in other systems.
Experimental 3D measurements showing the precision and effectiveness of
our system are presented.
European Conferences on Biomedical Optics 2007 •
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Conference 6627: Optical Coherence Tomography and Coherence Techniques
Room 5
Sunday-Tuesday 17-19 June 2007
Part of Proceedings of SPIE Vol. 6627 Optical Coherence Tomography and Coherence Techniques III
6627-01, Session 1
6627-03, Session 1
Advances in ultrahigh speed OCT with Fourier
domain mode locked (FDML) lasers
Ultrahigh resolution optical coherence
tomography at two infrared wavelength regions
using a single light source
R. A. Huber, Ludwig-Maximilians-Univ. München (Germany); D. C. Adler,
V. J. Srinivasan, I. M. Gorczynska, J. G. Fujimoto, Massachusetts
Institute of Technology (USA)
Fourier Domain Mode Locking (FDML) is a novel laser operating mode
enabling ultrahigh speed, narrowband wavelength-swept lasers with a wide
tuning range. In FDML lasers sweep rate limitations caused by lasing build
up time are eliminated by synchronizing the drive period of the intra-cavity
optical bandpass filter to the optical roundtrip time of the cavity. The
application of these lasers for ultrahigh speed swept source / Fourier domain
OCT imaging and phase-sensitive OCT measurements has been
demonstrated in the 1300 nm range at up to 370 kHz sweep rate. However,
for applications such as retinal imaging or optical coherence microscopy
(OCM), operation at shorter wavelengths is required. For retinal imaging,
operation near 800 nm or 1050 nm is preferred due to high water absorption
near 1300 nm. For OCM applications, shorter wavelengths enable improved
transverse resolution. In addition, for eye-safe profilometry applications or to
reduce scattering in the sample, longer wavelength ranges near the 1550 nm
telecom band are also of interest. In contrast to FDML operation at 1300 nm,
which is the zero dispersion point of standard single mode fiber, the nonnegligible dispersion at other wavelengths must be taken into account in the
design of FDML lasers. We discuss design, operation parameters, dispersion
compensation schemes, component choice and performance of an FDML
laser in the 1050 nm range. We apply the 1050 nm FDML laser for 3dimensional real time imaging of the human retina in vivo at an A-scan rate
of 236 kHz. Finally, the potential for ultra-high resolution OCT systems based
on FDML lasers will be analyzed.
F. Spöler, S. Kray, P. Grychtol, B. Hermes, J. Bornemann, M. Först, H.
Kurz, RWTH Aachen (Germany)
6627-02, Session 1
Optical coherence tomography (OCT) probes scattering and absorption
properties within turbid media. Since interaction cross sections of biological
tissues are strongly wavelength dependent, the use of more than one
wavelength band in OCT imaging offers the potential to increase information
about the sample morphology. Hence, additional contrast enhancement can
be achieved.
In this study ultrahigh resolution optical coherence tomography is
demonstrated at two infrared wavelength regions delivered by a single
commercial supercontinuum light source. This source comprises a passively
mode-locked fibre laser, a fibre amplifier and a highly nonlinear fibre.
Optimisation of the pump power of the fibre amplifier and appropriate filtering
results in two spectral bands centred at 835 nm and 1250 nm. The FWHM of
these two spectral regions is approximately 230 nm each with only minor
spectral modulation within the wavelength bands. The output power of each
wavelength band exceeds 50 mW.
An ultrahigh resolution OCT system, based on a free space Mach-Zehnder
interferometer setup which is optimised for the full bandwidth of the light
source, is presented. Both wavelength bands are collinearly coupled into the
interferometer, thus only the photodiode detectors have to be exchanged
between measurements at different wavelengths. Balanced detection is used
to reduce common-mode noise. A single reflection from a BK7 window was
analysed to characterise the system performance. The axial free space
resolution of the system is determined to 1.7 µm at 835 nm and 3.1 µm at
1250 nm. Imaging of biological tissue for both wavelength regions is
demonstrated.
Novel superluminescent diodes and SLD-based
light sources for optical coherence tomography
6627-04, Session 1
V. R. Shidlovski, S. D. Yakubovich, E. V. Andreeva, P. I. Lapin, V.
Prokhorov, M. V. Shramenko, Superlum Diodes Ltd. (Russia)
Performance characteristics of recently developed superluminescent diodes
(SLDs) based on double quantum-well (InGa)As heterostructure and InAs/
AlGaAs/GaAs quantum-dot heterostructure are presented. Emission spectra
of these SLDs cover spectral bands of 960-1080 nm and 1100-1230 nm
respectively. Depending on the design and operation conditions these SLDs
exhibit “bell-like” spectra with 30-50 nm linewidth or “double-hump” spectra
with linewidth of more than 100 nm. Owing to their usage the family of SLDbased combined light sources (BroadLighter series) [1] was significantly
broadened. Their emission spectra now cover the entire NIR-range 770-1230
nm. Among the new models the four-channel device Q-940 deserves special
attention. It possesses spectral power density ex SM fiber of more than 20dBm/nm in the band exceeding 300 nm centered at 940 nm. Its coherence
length of 2.9 um is a record value for semiconductor light sources.
New prototypes of swept-wavelength light sources in the range of 820-1080
nm based on quantum-well broadband semiconductor optical amplifiers
(SOAs) and tunable acousto-optic filters (AOTFs) are described. In particular
the output power of such light source with SOA-37 as active element and
high-selective AOTF was significantly increased (up to 5.0 mW ex PM fiber,
APC-mode) in comparison with the first prototype [2]. Spectral and dynamic
parameters are preserved at practically the same level: tuning range - 820 870 nm; instant linewidth - <0.05 nm; sweep speed - \>104 nm/sec. Using
another AOTF with worse spectral selectivity, but faster switching time for
the same tuning range the following parameters were obtained: output power
- \>5.0 mW; instant linewidth - <0.4 nm; sweep speed - \>106 nm/sec.
The authors believe that the developed light sources may find effective
applications in OCT systems.
REFERENCES
1. P.I.Lapin, D.S.Mamedov, S.D.Yakubovich, M.Wojtkovsky, J.G.Fujimoto
“Novel Near-IR Broad-Band Light Sources for Optical Coherence Tomography
Based on Superluminescent Diodes”, SPIE-OSA / Vol. 5861, 586108-1 (2005).
2. M.V.Shramenko, E.V.Andreeva, D.S.Mamedov, V.R.Shidlovski,
S.D.Yakubovich “NIR Semiconuctor Laser with Fast Broadband Tuning”,
Proc. of SPIE Vol. 6079, 60791M-1 (2006).
8
European Conferences on Biomedical Optics 2007 •
High speed wavelength-swept laser source with
a simple configuration for optical coherence
tomography
C. Chong, A. Morosawa, T. Sakai, Santec Corp. (Japan)
This paper reports on a high-speed, wavelength-swept laser operating at
1310nm for optical coherence tomography (OCT) applications. The laser
comprises a pigtailed semiconductor optical amplifier (SOA) and a
wavelength-scanning filter in a fiber ring cavity configuration. The tunable
filter consists of a diffraction grating and polygon mirror scanner in Littrow
configuration. A photodiode is used to generate a start trigger signal
synchronized to start of each frequency sweep. Intracavity prisms are utilized
to maintain constant and narrow laser linewidth and linear frequency sweep
during operation. The laser exhibits a peak power of over 20mW. The
measured tuning range of the laser is 120nm maximum, and 100nm FWHM
at a scanning frequency of 20kHz. The coherence length of the laser output
was measured to be 4mm. Implementation of a novel double pass
configuration in the scanning filter demonstrated an improvement in
coherence length to 7mm. An OCT system has been developed using the
scanning laser that exhibits 106dB sensitivity and image axial resolution of
12micrometer.
6627-05, Session 1
Wide tuning range wavelength-swept laser with
single semiconductor optical amplifier for OCT
A. Morosawa, C. Chong, T. Sakai, Santec Corp. (Japan)
This paper reports on a wide-range, high-speed, wavelength-swept laser
operating at 1310nm for optical coherence tomography (OCT) applications.
The laser comprises a pigtailed wideband, high-gain semiconductor optical
amplifier (SOA) and a wavelength-scanning filter in a fiber ring cavity
configuration. The tunable filter consists of a diffraction grating and polygon
mirror scanner in Littrow configuration. A photodiode is used to generate a
start trigger signal synchronized to start of each frequency sweep. Intracavity
prisms are aligned to provide constant and narrow laser linewidth and linear
frequency sweep. This arrangement also generates a wide tuning range for a
given beam deflection angle by the polygon scanner while maintaining narrow
laser linewidth. The laser exhibits a peak power of over 15mW. The measured
tuning range of the laser is 160nm maximum, with 110nm FWHM at a scanning
frequency of 20kHz using a single custom engineered SOA device. Laser
output is coupled via HI1060 fiber with a cut-off wavelength of 980nm ensuring
single-mode propagation over the 1230nm to 1390 nm tuning range. A tuning
range over 100nm FWHM corresponds to a theoretical axial resolution of
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Conference 6627: Optical Coherence Tomography and Coherence Techniques
7micrometer in air and 5.2micrometer in tissue, which promises high resolution
imaging in OCT applications.
6627-06, Session 2
Three-dimensional Fourier-domain optical
coherence tomography of alveolar mechanics in
stepwise inflated and deflated isolated and
perfused rabbit lungs
A. Krueger, L. Knels, S. Meissner, M. Wendel, A. R. Heller, T. Lambeck,
T. Koch, E. Koch, Technische Univ. Dresden (Germany)
Understanding lung mechanics on an alveolar scale is essential for the
development of more protective modes of mechanical ventilation. In a previous
study we showed that Fourier-domain optical coherence tomography (FDOCT) at 840 nm center wavelength and 50 nm spectral width is capable of
imaging lung parenchyma down to the alveolar scale and we also showed
that ventilation with a positive end expiratory pressure leads to persisting
opening of alveoli in isolated and perfused rabbit lungs, while zero end
expiratory pressure causes repetitive collapse and reopening of the alveoli.
However, cross-sectional imaging of the ventilated lung did not allow us a
detailed analysis of the geometrical changes individual alveoli undergo during
the ventilation cycle, because the position of the cross-sectional plane with
respect to the moving lung was changing. Therefore, in this study we
performed three dimensional FD-OCT imaging on perfused isolated rabbit
lungs during stepwise volume uptake and release using a fast threedimensional FD-OCT scanner head mounted on a motorized three axis
positioning arm with a ball joint. A built in camera helped to trace a marker
spot on the pleura and to reposition the scanner head. By analysing series of
three dimensional image stacks of the same alveolar structure at different
constant airway pressures, pressure-volume-curves of individual alveoli were
determined. In addition, perfusion fixation of the aerated lung with
glutaraldehyde was monitored with three dimensional FD-OCT and was
proven to preserve the realistic geometry without shrinking or expanding of
alveolar volume.
6627-07, Session 2
Diagnostic potential of optical coherence
tomography in non-melanoma skin cancer: a
clinical study
M. Mogensen, Univ. of Copenhagen (Denmark); L. Thrane, P. E.
Andersen, Technical Univ. of Denmark (Denmark); G. B. E. Jemec, Univ.
of Copenhagen (Denmark)
Introduction: Non-melanoma skin cancer (NMSC) is the most prevalent cancer
in the Western World. OCT has proved great potential in assisting clinical
diagnosis and perhaps reducing the need for biopsies in NMSC. As noninvasive treatment is increasingly used for NMSC patients with superficial
lesions, the development of non-invasive diagnostic technologies is highly
relevant.
Methods: The aim of this cross-sectional clinical study aimed at enrolling
100 NMSC patients, is to investigate the diagnostic accuracy and applicability
of OCT in NMSC diagnosis. OCT-images will be compared to clinical and
histopathological diagnosis and computer assisted image analysis has also
been applied. Our OCT-system has been developed at Risoe National
Laboratory, Denmark and offers polarisation sensitive-OCT that may have
additional advantaged as BCC differ in content of birefringent collagens from
normal skin.
Results: Basal cell carcinomas (BCC) can in some cases be distinguished
from normal skin in OCT-images, as normal skin exhibits a layered structure
this layering is not present in BCC and sometimes not in actinic keratosis
(AK). BCC lesions seem to be clearly less reflective than normal tissue. PSOCT seems to add some information in differentiating AK from normal skin.
The predictive value of OCT in NMSC will be presented from a clinical point
of view and from computer assisted analysis of OCT-images. The results to
be presented will be based on OCT-imaging of more than 75 patients with
125 skin cancer lesions.
Discussion: The earlier a skin cancer is diagnosed, the better the prognosis.
Estimation of diagnostic accuracy and abilities of OCT in clinical studies of
skin cancer patients is essential to establish the role and future set-ups for
diagnostic OCT-systems.
6627-08, Session 2
In vivo and 3D visualization of coronary artery
development by optical coherence tomography
L. Thrane, Technical Univ. of Denmark (Denmark); K. Norozi,
Medizinische Hochschule Hannover (Germany); J. Männer, GeorgAugust-Univ. Göttingen (Germany); F. Pedersen, Technical Univ. of
Denmark (Denmark); S. Mottl-Link, Deutsches Krebsforschungszentrum
(Germany); H. E. Larsen, P. E. Andersen, Technical Univ. of Denmark
(Denmark); A. Wessel, T. M. Yelbuz, Medizinische Hochschule Hannover
(Germany)
European Conferences on Biomedical Optics 2007 •
One of the most critical but poorly understood processes during
cardiovascular development is the establishment of a functioning coronary
artery (CA) system. Due to the lack of suitable imaging technologies, it is
currently impossible to visualize this complex dynamic process on living
human embryos. Furthermore, due to methodological limitations, this
intriguing process has not been unveiled in living animal embryos, too. We
present here, to the best of our knowledge, the first in vivo images of
developing CAs obtained from the hearts of chick embryos grown in shellless cultures. The in vivo images were generated by optical coherence
tomography (OCT). The OCT system used in this study is a mobile fiberbased time-domain real-time OCT system operating with a center wavelength
of 1330 nm, an A-scan rate of 4 kHz, and a typical frame rate of 8 frames/s.
The axial resolution is 17 µm (in tissue), and the lateral resolution is 30 µm.
The OCT system is optimized for in vivo chick heart visualization and enables
OCT movie recording with 8 frames/s, full-automatic 3D OCT scanning, and
blood flow visualization, i.e., Doppler OCT imaging. Using this OCT system,
we generated in vivo OCT recordings of chick embryo hearts to study the
process of connection of the future right coronary artery (RCA) to the aorta.
Recordings were made at three critical stages during development: day 8
(no clear connection yet), day 9 (established connection of RCA with the
aorta with clear blood flow) and day 10 (further remodeling of the established
RCA).
6627-09, Session 2
Ultrahigh-speed optical coherence tomography
imaging and visualization of the embryonic avian
heart using a buffered Fourier domain mode
locked laser
M. W. Jenkins, Case Western Reserve Univ. (USA); D. C. Adler,
Massachusetts Institute of Technology (USA); M. Gargesha, Case
Western Reserve Univ. (USA); R. Huber, Massachusetts Institute of
Technology (USA); F. G. Rothenberg, Univ. of Cincinnati (USA); M.
Watanabe, D. L. Wilson, Case Western Reserve Univ. (USA); J. G.
Fujimoto, Massachusetts Institute of Technology (USA); A. M. Rollins,
Case Western Reserve Univ. (USA)
The embryonic avian heart is a commonly used model for studying cardiac
developmental biology. The mechanisms that govern the development of a
four-chambered heart from a peristaltic heart tube are largely unknown due
to a lack of adequate imaging technology. Due to the small size and rapid
transient events associated with the in vivo embryonic avian heart, an imaging
system with high spatial and temporal resolution is required to study these
models. Here, an optical coherence tomography (OCT) system with a buffered
Fourier Domain Mode Locked (FDML) laser is used for ultrahigh-speed noninvasive imaging of embryonic quail hearts at 100,000 axial scans per second.
The high scan rate enables the acquisition of high temporal resolution 2D
datasets (195 frames per second) and 3D datasets (10 volumes per second).
Spatio-temporal details of cardiac motion not resolvable using previous OCT
technology are analyzed. Visualization and measurement techniques are
developed to non-invasively observe and quantify cardiac motion throughout
the brief period of systole (less than 50 msec) and diastole. This marks the
first time that the preseptated embryonic avian heart has been imaged in 4D
without the aid of gating and the first time it has been viewed in cross section
during looping with extremely high temporal resolution, enabling the
observation of morphological dynamics of the beating heart during systole.
6627-10, Session 3
Dynamic imaging of penetration and
decontamination after chemical eye burn using
optical coherence tomography
F. Spöler, M. Först, H. Kurz, RWTH Aachen (Germany); M. Frentz, N. F.
Schrage, Aachen Ctr. of Technology Transfer in Ophthalmology
(Germany)
Chemical eye burns cause approximately one forth of all traumatic ocular
injuries. To improve the efficiency in the emergency treatment of such injuries
the penetration and the effects of decontamination within tissue have to be
qualified and quantified. Conventional methods like intraocular pH
measurements only give limited insight into the mechanism of chemical
trauma. With its ability to non-destructively generate cross sectional images
of tissue morphology at high speed with micrometer scale resolution we
demonstrate that optical coherence tomography (OCT) offers large potentials
to close this analytical gap.
In this study OCT is used to evaluate the penetration characteristics and the
decontamination of corrosives within the cornea of a well defined and reliable
ex vivo animal model. Here, changes of the microstructure induced by the
chemical are accompanied with a substantial increase in the scattering
properties of the tissue. This process is efficiently monitored by OCT with
high spatial and temporal resolution. Hydrofluoric acid burn was studied in
detail. In contrast to findings for other acids, where coagulated proteins within
the epithelium act as a barrier for further penetration, full corneal penetration
was observed even for low concentrations, e.g. within 240 seconds for 2.5
% HF. Using different rinsing solutions, such as tap water, calcium gluconate
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Conference 6627: Optical Coherence Tomography and Coherence Techniques
solution, and a commercial antidot for HF, the deep corneal stroma remained
clear until rinsing was stopped after 15 minutes. This status was preserved
over one hour after rinsing for the HF antidot only, while further penetration
was observed for other rinsing solutions.
6627-11, Session 3
Operating microscope with time domain optical
coherence tomography (OCT) for neurosurgery
E. Lankenau, D. Klinger, H. Müller, A. Malik, Univ. zu Lübeck (Germany);
C. Winter, Thorlabs GmbH (Germany); A. Giese, Georg-August-Univ.
Göttingen (Germany); S. Oelckers, Möller-Wedel Optical GmbH
(Germany); G. Hüttmann, Univ. zu Lübeck (Germany)
Optical coherence tomography (OCT) allows non-contact / non-invasive
analysis of tissues of the central nervous system with a penetration depth of
2-3 mm reaching a spatial resolution of approximately 4-15 µm. This resolution
is compatible with the resolution of modern operation microscopes. With the
aim to provide a three-dimensional intraoperative visualization of tissue
structures, a spectral domain OCT (a modified version of the Sectral Radar
from Thorlabs) was adapted to a motorized operation microscope (HR 1000
Möller-Wedel). Via a specially designed scanner, the 840 nm OCT was adapted
directly to a camera port the operation microscope. Group velocity dispersion
of the microscope optics was compensated successfully to provide bandwidth
limited depth resolution. Together with a stepper-motor controlled reference
arm, an automatic control of the working distance (232 mm to 290 mm), the
scan field (4 mm to 24 mm) and the position of the OCT focus within the
sample is possible. The system was tested successfully in a preclinical setting
with different brain tissues and will be used in a first clinical trail for demarcation
of borders between tumor and brain in neurosurgery.
6627-12, Session 3
Investigation of murine vasodynamics by Fourier
domain optical coherence tomography
S. Meissner, J. Walther, G. Müller, A. Krüger, H. Morawietz, E. Koch,
Technische Univ. Dresden (Germany)
Fourier domain optical coherence tomography was used to image the
vasodynamics of the murine arteria saphena for the study of the early
pathogenesis of atherosclerosis. In contrast to established isometric force
measurements OCT imaging allows the investigation of vasoconstriction and
vasodilatation without inducing preparation traumata and changing the
physiological situation of the vessel. The murine arteria saphena is suitable
for the in vivo examination particularly due to the small diameter, the significant
response of the blood vessel to vasomotor stimuli and the advantageous
anatomical position for transluminal imaging. The reaction of the blood vessel
was induced by dermal application of the vasodilator Sodium-Nitroprussid
(SNP) and the vasoconstrictor Potassium. OCT images were acquired with a
two axis galvanometer scanner head which operated either in 2D or 3D mode.
Three dimensional image stacks were used to image the morphology of the
arteria, vena and nervus saphena. Time series (3 frames per second, 300x512
pixel per frame) of cross sectional images were analysed with image
processing software which measured the time course of the vessel lumen.
The diameter of the arteria saphena of male ten-week old C57BL/6 mice
changed from 91?15 µm before to 33?9 µm (36 %) after application of
Potassium within 30 seconds. After application of SNP a vasodilatation of
226?13 µm (248 %) of the initial inner diameter could be quantified. The
protocol can be used to study differences in the vasodynamics of wild type,
knock-out and transgenic mouse models under different types of diets.
6627-13, Session 3
Robust intravascular optical coherence
elastography
G. van Soest, Erasmus Univ. Medical Ctr. (Netherlands); R. R.
Bouchard, Erasmus Univ. Medical Ctr. (Netherlands) and Duke Univ.
(USA); F. Mastik, Erasmus Univ. Medical Ctr. (Netherlands); N. de Jong,
Erasmus Univ. Medical Ctr. (Netherlands) and Univ. of Twente (Netherlands) and Interuniv. Cardiology Institute of The Netherlands (Netherlands); A. F. W. van der Steen, Erasmus Univ. Medical Ctr. (Netherlands)
and Interuniv. Cardiology Institute of The Netherlands (Netherlands)
High strain spots in the vessel wall indicate the presence of vulnerable plaques.
The majority of acute cardiovascular events are preceded by rupture of such
a plaque in a coronary artery. Intracoronary optical coherence tomography
(OCT) can be extended, in principle, to an elastography technique, mapping
the strain in the vascular wall. However, the susceptibility of OCT to frameto-frame decorrelation, caused by tissue and catheter motion, inhibits reliable
tissue displacement tracking and has to date obstructed the development of
OCT-based intravascular elastography.
We introduce a new technique for intravascular optical coherence
elastography, which is robust against motion artifacts. Using acoustic radiation
force, we apply a pressure to deform the tissue synchronously with the line
scan rate of the OCT instrument. Radial tissue displacement can be tracked
10
European Conferences on Biomedical Optics 2007 •
based on the correlation between adjacent lines, instead of subsequent frames
in conventional elastography. The viability of the method is demonstrated
with a simulation study. The root mean square (rms) error of the displacement
estimate is 0.55 µm, and the rms error of the strain is 0.6%. It is shown that
high-strain spots in the vessel wall, such as observed at the sites of vulnerable
atherosclerotic lesions, can be detected with the technique.
Experiments to realize this new elastographic method are presented.
Simultaneous optical and ultrasonic pulse echo tracking demonstrate that
the material can be put in a high frequency oscillatory motion with an amplitude
of several micrometers, more than sufficient for accurate tracking with OCT.
The resulting data are used to optimize the acoustic pushing sequence and
geometry.
6627-14, Session 4
In vivo optophysiology of the human retina
B. M. Hermann, A. Binns, B. PovaÏay, A. Unterhuber, B. Hofer, T. H.
Margrain, W. Drexler, Cardiff Univ. (United Kingdom)
A functional extension of ultrahigh resolution OCT (UHR OCT) has been
developed, that has the potential to establish this technique as an optical
analogue to electrophysiology, by detecting depth resolved variations in
optical backscattering caused by physiological tissue changes. After
successful demonstration in in vitro studies on excised, but physiologically
intact, rabbit retinas this technique has been transferred to in vivo experiments
in the human retina. UHR OCT has been synchronized with the white light
stimulus to properly detected spatially resolved alterations in optical
backscattering over time caused by light-induced intraretinal, physiological
changes. Preliminary results demonstrate the potential of this novel extension
of UHR OCT for in vivo detection of time-dependent optical backscattering
changes after application of a white light stimulus in specific retinal layers,
especially in the photoreceptor layer. Possible explanation of the detected
optophysiological signals include hyperpolarizaton of photoreceptors, altered
metabolic rates that cause changes in the mitochondrial refractive index, as
well as Actin-dependent retinomotor activity, which is a general phenomenon
of the non-mammalian vertebrate photoreceptors and pigment epithelial
processes. Challenges of this technique applied in vivo include proper time
resolution (time vs. frequency domain OCT technique), eye motions as well
as proper post processing of the detected optophysiological signals.
6627-15, Session 4
OFDI for retinal imaging
J. F. DeBoer, Massachusetts General Hospital (USA)
We will present Optical Frequency Domain Imaging for retinal imaging at a
wavelength of 850 and 1050 nm. Laser sources and system designs will be
presented to maximize system sensitivity. Acquisition rate for both retinal
systems is at least 30,000 depth profiles per second. Differences in scattering
properties and contrast for retinal structures at these wavelengths will be
presented.
6627-16, Session 4
Phase retardation measurement of retinal nerve
fiber layer using polarization-sensitive spectral
domain optical coherence tomography and
scanning laser polarimetry
M. Yamanari, Univ. of Tsukuba (Japan); M. Miura, Tokyo Medical Univ.
Kasumigaura Hospital (Japan); S. Makita, T. Yatagai, Y. Yasuno, Univ. of
Tsukuba (Japan)
Three-dimensional phase retardation map around the optic nerve head is
measured by polarization-sensitive spectral domain optical coherence
tomography using the B-scan-oriented polarization modulation method.
Birefringence of the optical fiber and the cornea is compensated by Jones
matrix based analysis. En-face phase retardation map of the retinal nerve
fiber layer is shown and compared with the result of scanning laser polarimetry
(SLP). Both systems showed similar pattern of the phase retardation for
healthy and glaucomatous eyes. Unlike SLP, our system can measure the
phase retardation quantitatively without using bow-tie pattern of the
birefringence in the macular region, which enables diagnosis of glaucoma
even if the patients have macular disease.
6627-17, Session 4
Intensity based quantification of fast retinal
blood flow in 3D via high resolution resonant
Doppler spectral OCT
R. Michaely, A. H. Bachmann, M. L. Villiger, C. Blatter, T. Lasser, R. A.
Leitgeb, École Polytechnique Fédérale de Lausanne (Switzerland)
Resonant Doppler Fourier Domain Optical Coherence Tomography is a
functional imaging modality for quantifying fast tissue flow. The method profits
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Conference 6627: Optical Coherence Tomography and Coherence Techniques
from the effect of interference fringe blurring in spectrometer-based FDOCT
in the presence of sample motion. If the reference path length is changed in
resonance with the Doppler frequency of the sample flow the signals of resting
structures will be suppressed whereas the signals of blood flow are enhanced.
This allows for an easy extraction of vascularization structure. 3D images of
blood vessels at the human optic nerve head are obtained with high axial
resolution of 8µm in air and an imaging speed of 17.400 depth profiles per
second. An electro-optic modulator allows controlled reference phase shifting
during camera integration. A differential approach is presented for the
quantification of fast flows that are un-accessible via standard phase sensitive
Doppler analysis. Flow velocity analysis extracts only the axial component
which is dependent on the orientation of the vessel with respect to the optical
axis. 3D information of the segmented vessel structure is readily used to
obtain the flow velocity vectors along the individual vessels and to calculate
the true angle-corrected flow speed.
6627-18, Session 4
6627-21, Session 5
En-face visualization methods for analyzing
three-dimensional UHR OCT retinal imaging
data
I. M. Gorczynska, J. J. Liu, V. J. Srinivasan, Massachusetts Institute of
Technology (USA); R. W. Chen, Tufts Univ. School of Medicine (USA); M.
Wojtkowski, Nicolaus Copernicus Univ. (Poland); E. Reichel, J. S. Duker,
Tufts Univ. School of Medicine (USA); J. G. Fujimoto, Massachusetts
Institute of Technology (USA)
Ultrahigh resolution (UHR), three dimensional (3-D) OCT imaging provides
more comprehensive information on retinal morphology than any other
currently available diagnostic technique. However, since it may consist of
over a hundred cross sectional images, visualization methods must be
developed to help interpret the data. In this study we investigate applications
of OCT fundus images, en-face sections, and weighted sums of en-face
sections for visualization of features characteristic of age related macular
degeneration (AMD). Maps of elevations, contours and thicknesses will be
shown.
OCT imaging was performed using a spectral / Fourier domain instrument
operating at 25,000 axial scans per second with 3.5 µm axial resolution. Sets
of 180, horizontal OCT cross-section images, consisting of 500 axial scans
with 1024 axial points each, were acquired in a 6mm by 6mm macular area
of the eye. Patients with non-exudative AMD were imaged in the
ophthalmology clinic.
OCT fundus imaging with en-face sectioning of the data is a promising
visualization method. It enables quick analysis of the condition of the retina
and localization of pathological changes that may be markers for the
development of the disease. In order to provide quantitative information, enface sections should be complemented with appropriate retinal maps. These
visualization methods may aid in tracking the disease development,
recognizing early markers of progression to more advanced stages or
assessing effectiveness of treatment. The eventual goal of this study is to
establish efficient methods for visualization of three-dimensional UHR OCT
data for the wide range of retinal pathologies.
6627-19, Session 4
Towards isotropic resolution in ophthalmic
ultrahigh-resolution optical coherence
tomography by using pancorrection
E. J. Fernández, C. Torti, B. PovaÏay, B. M. Hermann, A. Unterhuber, B.
Hofer, W. Drexler, Cardiff Univ. (United Kingdom)
A novel adaptive optics modality, pancorrection, consisting of the
simultaneous correction of both monochromatic and chromatic ocular
aberrations, was applied to ophthalmic ultrahigh resolution optical coherence
tomography (UHR OCT). The system combined a novel deformable mirror
based on magnetic forces, and an achromatizing lens for compensating the
chromatic aberration of the eye. Pancorrection significantly improved both
contrast and transverse resolution of retinal images. Volumetric, in vivo, UHR
OCT images of the retina with pancorrection, obtained with up 25000 A scans/
s and high resolution (~ 2 x 2 x 2 µm; transverse (x) x transverse (y) x axial (z))
were recorded at different eccentricities. The three-dimensional structure of
the photoreceptors mosaic was obtained at 2.25 deg of eccentricity.
6627-20, Session 5
Scatterer size-based analysis of optical
coherence tomography signals
A. Kartakoulis, C. Pitris, Univ. of Cyprus (Cyprus)
The early stages of malignancy, in most tissues, are characterized by unique
cellular changes. More specifically, the number of cells and nuclei increases,
the nuclei themselves become enlarged and hyperchromatic, nuclear sizes
change from the normal 5-10 µm to 1.5x-2x the size, i.e. 10-20 µm. Currently,
these early changes are detectable only by confocal or multi-photon
European Conferences on Biomedical Optics 2007 •
microscopy. Unfortunately, neither of the two imaging techniques can
penetrate deep enough into the tissue to investigate the borders of thick
lesions. A technique which would allow extraction of information regarding
scatterer size from Optical Coherence Tomography (OCT) signals could prove
a very powerful diagnostic tool and produce significant diagnostic insight. In
this work we describe a method which would allow extraction of information
regarding scatterer size from Optical Coherence Tomography (OCT) signals.
The technique is based on AR spectral estimation and analysis of the resulting
spectra. Statistical analysis proves that there exist significant differences in
the spectra of signals resulting from solutions of microspheres of diameters
smaller than the resolution of the OCT system. In addition, linear equations
can be used to estimate the diameter size. The results are very encouraging
and they show that the spectral content of OCT signals can be used to extract
scatterer size information. This technique can result in an extremely valuable
tool for the investigation of disease tissue features which now remain below
the resolution of OCT.
Stereoscopic optical coherence tomography in
the frequency domain for refractive index
sensitive imaging
P. H. Tomlins, M. Tedaldi, R. A. Ferguson, National Physical Lab. (United
Kingdom); R. K. Wang, Oregon Health and Science Univ. (USA) and
Cranfield Univ. (United Kingdom)
In this article we present a novel stereoscopic approach to optical coherence
tomography from which the refractive index, rather than group index, of bulk
layers within a material are measured irrespective of the interface roughness.
Depth (axial) scans are made using a frequency domain OCT system that
acquires two spectra at different angles. In each axial scan the optical
thickness of each layer is used in conjunction with the knowledge of the
angles of incidence of the light upon the sample to solve the equation of
Snell’s law for each layer refractive index. To demonstrate this technique we
have measured the refractive indices within a well characterised tri-layer
optical phantom. Initial results show accurate refractive index determination
in the second decimal place, however this is not a fundamental limit of the
technique.
This method is potentially useful for providing extra clinical diagnostic
information in vivo. It also provides useful information for tissue optics and
understanding the fundamental optical properties of biological tissue.
6627-22, Session 5
Speckle reduction in optical coherence
tomography images of human skin by a spatial
diversity method
T. M. Jørgensen, L. Thrane, A. Zam, P. E. Andersen, Technical Univ. of
Denmark (Denmark)
Many techniques have been suggested for dealing with the signal degrading
speckle noise in optical coherence tomography (OCT). Some of these
approaches require substantial modifications of the OCT system itself. Here,
we consider a method that in principle can be fitted to most OCT systems.
Specifically, we address a spatial diversity technique for suppressing speckle
noise in OCT images of human skin. The method is a variant of changing the
position of the sample relative to the measuring probe. Instead of physically
moving the sample, which is often not feasible for in vivo imaging, the focal
plane of the probe beam inside the sample is shifted. We have tested the
scheme with a mobile fiber-based time-domain real-time OCT system. We
compare the resulting image enhancement to the results obtained by using
digital image algorithms for speckle noise reduction. The results show promise
for obtaining better image contrast when imaging highly scattering tissue in
vivo.
6627-23, Session 5
Contribution of various scattering orders to OCT
images of skin
M. Y. Kirillin, Univ. of Oulu (Finland) and M.V. Lomonosov Moscow State
Univ. (Russia); A. V. Priezzhev, M.V. Lomonosov Moscow State Univ.
(Russia); R. A. Myllylä, Univ. of Oulu (Finland)
Simulated OCT images of skin were obtained implementing Monte Carlo
simulations. The multilayer skin model used in simulations was based of the
experimental OCT images obtained at the wavelength of 910 nm. The
following skin layers were considered in the model: stratum corneum,
epidermis prickle layer, epidermis basal layer,and dermis. The images were
obtained both with and without speckle accounting. The former case is
obtained from the envelopes of calculated interference signals while the latter
accounts for the interference fringe patterns. The contributions of least and
multiple scattering, diffusive and non-diffusive components of the
backscattered light to the resulting OCT image were separated and analyzed.
It was shown that least scattering contribution represents the imaging of the
upper skin layers, while multiple scattering contribution can be characterized
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Conference 6627: Optical Coherence Tomography and Coherence Techniques
as blurred image with reduced contrast preserving, however, essential details.
The least scattering component contributes to the image for optical depth
up to 1.1 mm. From the analysis of the contribution of non-diffusive and
diffusive components it follows that the diffusive component contributes to
imaging the object starting from the epidermis basal layer and is more blurred
compared to the multiple scattering contribution. The non-diffusive
component contributes to the image for optical depth up to 1.3 mm. The
effect of coherence length on the contributions of least and multiple scattering
was also studied. It was shown, that contribution of multiple scattering
increases with a decrease of the coherence length.
6627-24, Session 5
Speckle size in optical coherence tomography
G. Lamouche, National Research Council (Canada); C. Bisaillon, Conseil
National De Recherches Canada (Canada); R. Maciejko, École
Polytechnique de Montréal (Canada); M. L. Dufour, National Research
Council (Canada); J. Monchalin, Conseil National De Recherches
Canada (Canada)
A fiber-optic, time-domain optical coherence tomography (OCT) system
coupled with a pneumatically-actuated micro-lens is demonstrated. The OCT
system uses a superluminescent diode (SLD) emitting at a center wavelength
of 1300 nm. Microsystem fabrication technologies employing
polydimethylsiloxane (PDMS) are used to fabricate the micro-lens with an
aperture of 2mm. A B-scan is carried out while dynamically shifting the focal
length of the micro-lens along the axial scan. The OCT scan results show a
higher lateral resolution and higher contrast of the backscattered interference
signals when using the tunable lens; hence, deeper axial scans are possible.
The ability to miniaturize the dimensions of the micro-lens will allow the system
to be applicable to en-face optical coherence tomography and endoscopic
applications.
6627-57, Poster Session
Slit-lamp adapted OCT for the visualization of
retinal structures
G. Hüttmann, Univ. zu Lübeck (Germany); C. Winter, P. Koch, Thorlabs
GmbH (Germany); H. Müller, E. Lankenau, Univ. zu Lübeck (Germany)
Speckle is inherent to any Optical Coherence Tomography (OCT)
measurements. It is often seen as degrading the signal and many techniques
were proposed to circumvent it. Recently, a few papers attempted to use
speckle to differentiate tissues. One approach relies on first-order speckle
statistics, using speckle contrast[1]. This approach is of interest mainly when
there are few scatterers within the probed volume. A broader range of first
order and second order statistical parameters were also studied[2]. Some
results are encouraging but the renormalization and analysis do not lead to a
simple physical interpretation of the observed variations. In our study, we
pursue similar interests by concentrating on the variation of speckle size
with the tissue microstructure. From a theoretical point of view, the speckle
size is studied using a simple model treating the tissue as an ensemble of
discrete scatterers. On the experimental side, a new approach to produce
solid and deformable phantoms with a controlled density of scatterers is
developed and speckle size is measured using a time-domain OCT system.
The speckle size is seen to vary with the density of scatterers. Upper and
lower bounds for its variation are estimated from theory and validated
experimentally. As a tool to differentiate tissues, the speckle size provides
similar results to those obtained with the contrast inspection. Nevertheless,
the study of speckle size variation with microstructure is of larger interest,
providing, for example, useful information for planning elastography
measurements.
[1] T. R. Hillman et al., Opt. Lett. 31, 190 (2006)
[2] K.W. Gossage et al. , Phys Med Biol. 51, 1563 (2006).
Optical coherence tomography (OCT) is a non-invasive imaging technique
with a resolution below 10 micrometer. Retinal imaging by OCT was extremely
successful and has entered already clinical practice. Several instruments a
now on the market. Up to now all commercially available systems are based
on a dedicated instrument working like a fundus camera or confocal retina
scanner. However the main optical instrument for the ophthalmologist is still
the slit-lamp. In the past adapting OCT to a slit-lamp was hampered by the
slow imaging speed which did not allow real-time imaging. With the advent
of spectral-domain OCT A-scan rates of 30,000 A-scans per second became
possible. In order to test the clinical usefulness of a slit-lamp adapted OCT
we designed an OCT scanner which can be used together with different
types of slit-lamps. The system is based on the Spectral Radar of Thorlabs
Inc., which was modified for this special use. Retinal imaging is possible
with contact glasses as well as with a Volk lens. The length of the reference
arm is automatically adjusted. With a two-axis scanner fields of 8° are at a
resolution of 8 µm are possible. The performance of the slit-lamp OCT is
compared with commercially available OCT-system.
6627-53, Poster Session
Optical coherence tomography (OCT) system that can resolve sub-wavelength
structures in full field is highly desirable as it allows following the functionality
of human tissue activity in real time. Full field OCT (FF-OCT) using the Linnik
microscope can reveal images of high resolution using high NA objectives,
however then the focal depth is also short. This later fact, although has the
advantage of allowing high axial resolution even with a source of narrow
spectral range such as a laser, it prevents real time depth imaging using the
Fourier domain OCT. We are working on some novel concepts to improve
the performance of FF-OCT both to increase the signal to noise ratio using
common path configurations and to increase the depth of field using masks
in the back aperture plane of the microscope objective. Theoretical and
experimental results will be presented using the common path Mirau
interference microscope and using the Linnik microscope with annular masks
to increase the depth of field.
Depth-resolved simplified characterization of
collagen depletion in dermis with polarization
sensitive optical coherence tomography
applicable to non-laboratory conditions
V. Tougbaev, T. Eom, W. Shin, B. Yu, Y. Lee, C. Kee, D. Ko, J. Lee,
Gwangju Institute of Science and Technology (South Korea)
A further insight into the system for polarization sensitive optical coherence
tomography applicable to non-laboratory conditions is brought forward and
basic design parameters are evaluated numerically. In contrast to the earlier
disclosed approach based on conventional polarization-maintaining fibers
integrated in the tandem interferometer of special type, the newly presented
design of the flexible compact probe involves fiber optic analogs of quarterwave achromatic retarders emerged recently in the art. Fabrication of such
kind of fibers is shown to be feasible by making use of the photonics
technology possessed by the authors. Potentialities of endoscope
embodiment are considered as well. A phenomenological model is adopted
from the theory of light depolarization in crystalline polymers and yields an
algorithm for depth-resolved computing of light depolarization that
distinguishes between subsurface skin layers with depleted birefringence
and upper dermis of normal human skin characterized by collagen birefringent
fiber bundles highly randomized in planes parallel to the skin surface. Both
the design concept and the algorithm imply the well proofed prognostic factors
which corroborate the tumor thicknesses of less than half a millimeter as an
important criterion for complementary functional diagnostics of malignant
melanoma at its early stage. Choice of the model is inspired by close similarity
of structural and optical properties between liquid-crystal collagen fibers in
dermis and birefringent crystalline lamellae in polymer materials. Unlike the
contradictory interpretations known in the art the numerical computation
based on the model and real peculiarity of dermis gives unambiguous
explanation for asymptotically delayed increase in depolarization with depth.
6627-58, Poster Session
Full field common path optical coherence
tomography with annular aperture
I. S. Abdulhalim II, R. Friedman, L. Liraz, Ben-Gurion Univ. of the Negev
(Israel)
6627-59, Poster Session
Maximum likelihood estimation of depth
reflectances in time-domain optical coherence
tomography
C. Flueraru, S. S. Sherif, S. Chang, Y. Mao, National Research Council
Canada (Canada)
We use a non-stationary random process model for the photocurrent in timedomain optical coherence tomography (TD-OCT) to obtain a maximum
likelihood estimate of the reflectance at different depths of an object. This
statistical image restoration approach is more effective than the previously
reported deterministic methods, as the dominant photoelectric noise is signal
dependent. We also present an expression for the Fisher information transfer
in TD-OCT, which is important for the optimization of practical TD-OCT setups.
We present both theoretical and experimental results.
6627-60, Poster Session
6627-55, Poster Session
The effects of Gaussian beams on optical
coherence tomography
Optical coherence tomography using a
dynamically-focusing tunable micro-lens
C. Liu, National Chiao Tung Univ. (Taiwan); C. Cheng, C. Chiu, I. Hsu,
Chung Yuan Christian Univ. (Taiwan)
K. Aljasem, A. Werber, S. Reichelt, H. Zappe, Albert-Ludwigs-Univ.
Freiburg (Germany)
12
European Conferences on Biomedical Optics 2007 •
Optical coherence tomography (OCT) is a noninvasive imaging technique to
extract cross-sectional information from biological tissues. It has been rapidly
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Conference 6627: Optical Coherence Tomography and Coherence Techniques
developed and is useful in biomedical applications. A typical OCT system is
based on the configuration of a Michelson interferometer. The theory of OCT
was conventionally considered as the light beams propagating in the system
to be in the forms of planar waves. Under such an assumption, the theory of
OCT is quite simple and is consistent with most of the experimental results.
However, the actual behaviors of the light beams in an OCT system are more
likely to be Gaussian beams. While considering the effects of Gaussian beams,
the interference signals in an OCT system will be different to that resulting
from planar waves. Furthermore, the effects will be more crucial when an
ultra-broadband light source was used to achieve a high-resolution imaging,
which is one of the most important issues in the current development of
OCT. In our research, we investigated the effects of Gaussian beams on an
OCT system. With the consideration of the light beam passing through the
focal lens in the sample arm to be a Gaussian beam, we deduced the theory
of OCT in an analytic form. We also simulated and analyzed the interference
signals with different positions of the photodetector and the interface in the
sample as well as their transverse patterns spectrally. The results were
demonstrated by experiments with both the time-domain and Fourier-domain
OCT systems.
6627-61, Poster Session
Absorption effects in optical coherence
tomography modeling
T. Chow, Nanyang Technological Univ. (Singapore); J. C. Y. Kah,
National Univ. of Singapore (Singapore); B. Ng, Nanyang Technological
Univ. (Singapore); C. J. R. Sheppard, National Univ. of Singapore
(Singapore)
Estimation of the tissue optical characteristics using optical coherence
tomography (OCT) requires good modeling. Present modeling of the system
includes effects such as scattering of light in tissues. However, absorption
effects were often neglected in the model. They may be significant depending
on the tissue type and the wavelength of the light source. We present a study
where the effects of absorption in light propagation in biological tissue were
examined in the theoretical modeling of OCT signal based on the extended
Huygens-Fresnel principle. OCT M-scans were performed on liquid tissue
phantoms of 1% Intralipid. In order to mimic the effects of absorption, India
ink was added to the solution. Different concentrations of India ink were
used to vary the absorption coefficient in the tissue phantoms. Estimation of
the absorption, scattering coefficients and the anisotropy factor from the
OCT signal were obtained based on our proposed theoretical model with
absorption effects. In order to verify the accuracy of the model, these
coefficients were measured using integrating spheres. Substantial reduction
in the slope of the logarithmic depth dependent OCT signal was observed
when India ink was introduced to the scattering phantoms. Estimations of
the absorption coefficients agreed with values measured using integrating
spheres. The results suggest that the effects of the absorption clearly affect
estimation of the tissue optical characteristics. In order to improve the
accuracy of estimation of these tissue optical properties, absorption effects
should be taken into account.
6627-62, Poster Session
Diffractive optical coherent microtomography
S. G. Vertu, E. Maeda, M. Ochiai, I. Yamada, J. Delaunay, The Univ. of
Tokyo (Japan); O. Haeberlé, Univ. de Haute Alsace (France); Y.
Okamoto, Chiba Univ. (Japan)
In this study, we report first results in the development of a diffractive optical
coherent microtomography instrument for use in histology. We aim at the
development of a new approach to histology for thick section of tissues
without staining. Conventional histology involves sectioning and staining very
thin tissue slices less than 10 µm thick and imaging 10-30 µm diameter tissue
cells by using standard transmission optical microscopy. The thickness of
the sectioned tissue being smaller than the average cell diameter, observation
of complete and intact cells is very unlikely and observation of the threedimensional arrangement of cells impossible. Diffractive optical tomography
makes the observation of samples with thickness of the order of a few cell
diameters feasible and thus allows for the observation of complete cells as
well as cell arrangement in three dimensions. It is this morphological
information on individual cells and cell cluster that is thought to be crucial in
improving cancer diagnosis.
We developed a coherent optical diffraction tomographic microscope of the
Mach-Zehnder type that was used to record on a CCD detector interferograms
resulting from the interference between the reference wave and the wave
transmitted through the sample. The sample holder consisted of a microcapillary in which the sample for observation can be inserted and of a
microfabricated guide to control the position of the microcapillary with a
micrometer precision. The microcapillary was filled with an index matching
fluid, inserted in the microfabricated guide and fixed to the rotation axis. The
sample was illuminated by a plane wave and was rotated together with the
microcapillary so as to collect interferograms under different illumination
angles. A computer controlled the rotation of the sample holder by using a
stepping motor, the displacement of the piezostage for the phase-shifting
interferometry and the CCD acquisition of the interferograms. Soda-lime glass
European Conferences on Biomedical Optics 2007 •
beads of a micro-size were used as a phase object in this study and were
introduced into the micro-capillary sample holder. We report 3D images of
the glass beads computed using the Filtered Back Projection algorithm and
discuss potential applications of the developed instrument to histology.
6627-64, Poster Session
Effects of path-length gating to scattered light: a
Monte Carlo analysis of a focused beam in OCT
system
C. Tjokro, Singapore-Massachusetts Institute of Technology Alliance
(Singapore); T. Chow, Nanyang Technological Univ. (Singapore); J. C. Y.
Kah, National Univ. of Singapore (Singapore); C. J. R. Sheppard,
National Univ. of Singapore (Singapore) and Singapore-Massachusetts
Institute of Technology Alliance (Singapore)
A study is carried out to better understand the interplay of path-length
properties of scattered light with its scattering properties. This study is done
in the context of an optical coherence tomography system which employs a
focusing objective lens in its sample arm. Furthermore, a new mechanism to
model a perfect correction lens is introduced to avoid the contribution of any
spherical aberration due to the difference in refractive index of the medium
and air. A virtual semi-spherical lens is also introduced to replace the
conventional model of a level/flat lens plane which introduces path-length
deviations.
A Monte Carlo model is developed to simulate the light propagation inside
the medium. Each photon carries its positional, directional, weight, pathlength, and scattering-processes information for later analysis. The detection
criterion due to the path-length gating is imposed directly to the photon itself
and not to any target layer as suggested by other study.
In this paper, the optical-sectioning property of the path-length gating is
investigated. The observation is then further explored by comparing the phase
space of the scattered light under different influences, such as: the pathlength criterion set by the system reference arm; and the light scattering
number. Some discussion on the undetected photons is also presented to
enrich the understanding of the effect of path-length gating to the scattered
light.
6627-65, Poster Session
Optical coherence tomography (OCT) imaging
and computer aided diagnosis of human cervical
tissue specimens
F. Bazant-Hegemark, Gloucestershire Royal Hospital (United Kingdom)
and Cranfield Univ. (United Kingdom); N. Stone, M. D. Read,
Gloucestershire Royal Hospital (United Kingdom); K. McCarthy,
Gloucestershire Hospitals NHS Foundation Trust (United Kingdom); L. J.
Ritchie, Cranfield Univ. (United Kingdom); R. K. Wang, Oregon Health &
Science Univ. (USA)
Background: The keyword for management of cervical cancer is prevention.
The present programme within the UK, the NHS cervical screening programme
(NHSCSP), is based on cytology. Despite having reduced the incidence of
cervical cancer, this requires follow ups and relies on diagnostic biopsying.
There is potential for reducing costs and workload within the NHS, and
relieving anxiety of patients. Optical Coherence Tomography (OCT) is
investigated for its capability to improve this situation.
Methods: Our time domain bench top system uses a superluminescent diode
(Superlum), centre wave length ~1.3 µm, resolution (air) ~15 µm. Tissue
samples are obtained according to the ethics approval by Gloucestershire
LREC, Nr. 05/Q2005/123. Images of 186 participants have been compared
with histopathology results and categorised accordingly.
Results: Our OCT images do not reach the clarity and resolution of
histopathology. However, we believe to be able to identify structural features
of diagnostic significance, such as glands, cysts, blood vessels, and
appearance of tissue layers in near real time. Moreover, the use of computer
algorithms allows taking decision making from the subjective appraisal of a
physician to an objective assessment.
Discussion: The success of OCT will depend on its ability to differentiate
between low and high grade dysplasia, a matter of interest for several types
of epithelial malignancies. Completely replacing histopathology in CC
management is a fair, but ambitious aim. Three applications seem realistic in
short term: 1) Reducing workload for histopathology where low grade
dysplasia is clearly diagnosed; 2) Confirming lesion-free margins straight after
tissue removal; 3) Application of OCT for other epithelial disorders which are
not covered by a highly sophisticated disease management.
6627-66, Poster Session
Logarithmic transformation technique for exact
signal recovery in frequency domain optical
coherence tomography
C. S. Sekhar, R. A. Leitgeb, A. H. Bachmann, M. A. Unser, École
Polytechnique Fédérale de Lausanne (Switzerland)
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Conference 6627: Optical Coherence Tomography and Coherence Techniques
We address the problem of exact signal recovery in frequency domain optical
coherence tomography (FDOCT) systems. The standard technique for
tomogram reconstruction from the FDOCT data is the inverse Fourier
transform. However, the inverse Fourier transform is known to yield
autocorrelation artifacts which interfere with the signal structure. We propose
a new logarithmic transformation technique for extracting the signal structure
from the intensity measurements. Our technique relies on the fact that, in a
spectral interferometry setup, the intensity of the total signal reflected from
the object is smaller than that of the reference arm. Our technique is noniterative, non-linear and it leads to an exact solution in the absence of noise.
The reconstructed signal is free from autocorrelation artifacts that are
encountered in the widely-used inverse Fourier transform technique. We
present results on synthesized data as well as experimental FDOCT
measurements of the retina of the eye.
(A patent has been filed by the EPFL based on the research results reported
in this paper.)
6627-67, Poster Session
Spectroscopic Fourier domain optical
coherence tomography
M. R. Hofmann, C. Kasseck, K. Lehmann, N. C. Gerhardt, Ruhr-Univ.
Bochum (Germany)
Conventional optical coherence tomography (OCT) is either performed in
the time domain (TD-OCT) or in the Fourier domain (FDOCT). This technique
is conventionally used to achieve depth resolved structural information about
strongly scattering biological tissue. In TDOCT full recording of amplitude
and phase of the interferograms allows to achieve spectroscopic data in
addition to the pure structural information. However, the reconstruction of
this spectroscopic information is associated with large computational effort
and therefore not established, yet. We suggest an alternative concept for
achieving spectroscopic sample information from FDOCT by analyzing the
shapes of FDOCT depth profile peaks.
We implement this spectroscopic FDOCT concept and show first experimental
results of test samples with well defined absorbers. These results are
compared to theoretically calculated depth profiles for our test structure.
The results are analyzed and discussed with respect to their applicability to
biological tissue with complex content.
6627-68, Poster Session
Using a piezoelectric fiber stretcher to remove
the depth ambiguity in optical Fourier domain
imaging
S. Vergnole, G. Lamouche, M. L. Dufour, B. Gauthier, National Research
Council (Canada)
This paper reports the study of an Optical Fourier Domain Imaging (OFDI)
setup for optical coherence tomography. One of the main drawbacks of the
OFDI is the ambiguity between positive and negative depths. Some setups
have already been proposed to remove this depth ambiguity by shifting the
frequency by means of electro-optic or acousto-optics modulators.
Here, we implement a piezoelectric fiber stretcher in our OFDI setup which
enables to generate a phase shift in the reference arm and, thus, allows us to
cancel the depth ambiguity. We evaluate the performances of our system
(sensitivity, SNR, Full Width at Half Maximum) and a study of the evolution of
the differential chromatic dispersion is also performed.
At last, we present the advantages and the drawbacks of such a technique
compared to the technique using acousto-optics modulators.
6627-69, Poster Session
H. Huang, National Yang-Ming Univ. (Taiwan); W. Kuo, National Taiwan
Normal Univ. (Taiwan); S. Chang, Yuan Ze Univ. (Taiwan); C. Ho,
National United Univ. (Taiwan); C. Chou, National Yang-Ming Univ.
(Taiwan)
A novel optical low coherence differential-phase reflectometer is developed
in which a balanced detector detection and a phase modulation to amplitude
modulation conversion are integrated together to result a near shot-noiselimited detection on phase measurement. In the meantime, a common phase
noise rejection mode is setup in the reflectometer too, which is able to immune
the environmental disturbance. Experimentally, the phase detection sensitivity
at 0.1° is demonstrated. Meanwhile, a localized surface profile measurement
of reflectometer resolution is obtained via scanning an optical grating surface
in this experiment. Moreover, the requirement on equal amplitude of the
reference and signal beams in this reflectometer is derived and discussed
European Conferences on Biomedical Optics 2007 •
Signal-to-noise analysis of Fizeau-based Fourier
domain optical coherence tomography
P. A. Shilyagin, V. M. Gelikonov, G. V. Gelikonov, Institute of Applied
Physics (Russia)
Signal-to-noise analysis of a Fizeau-based Fourier domain optical coherence
tomography system with a linear photodiode array (Sensors unlimited Inc.
SU512LD-1.7T1-0500) is presented. Comparison of theoretical and
experimental results is made. Conclusions on dominant noise and ways of
system optimization are made.
6627-26, Session 6
Simultaneous optical coherence and
multiphoton microscopy of skin-equivalent
tissue models
J. K. Barton, The Univ. of Arizona (USA); S. Tang, R. Lim, Univ. of
California/Irvine (USA); B. J. Tromberg, Beckman Laser Institute and
Medical Clinic (USA)
Organotypic skin-equivalent tissue models offer an alternative to in vivo
systems for studying the effects of chemical compounds. Optical coherence
(OC), two-photon excited fluorescence (TPEF), and second harmonic
generation (SHG) microscopy can provide complementary information about
these models without the need for destructive processing. Twelve skinequivalent models (rafts) were created containing a fibroblast/collagen layer
and an air-exposed keratinocyte layer. Eight of the rafts were exposed to
Dimethyl Sulfoxide (DMSO) either topically or by immersion. Some rafts were
incubated with rhodamine 123 or Hoechst 33342 stains. A single instrument
with a 12-fs broadband source simultaneously acquired OC, TPEF, and SHG
microscopy images.
TPEF images revealed elongated fibroblasts and collagen bundles in the
bottom layer, and progressively enlarging keratinocyte nuclei and keratin in
the top layer. Keratinocyte nuclei appeared dark in OC images. The fibroblast/
collagen layer had a characteristic texture, but neither cell nuclei nor collagen
bundles were resolved. OC signal intensity was diminished at the layer
interface. Strong SHG was apparent in the collagen layer of native rafts. In all
modalities, endogenous signal intensity was diminished in the DMSO-exposed
rafts. TPEF images with exogenous stains revealed altered fibroblast
morphology in the exposed rafts. OC, TPEF, and SHG microscopy can supply
complementary, non-destructive information on native and DMSO-exposed
raft structure.
6627-27, Session 6
High-Speed, auto-focusing optical coherence
microscopy system for cellular resolution
imaging of human tissues
A. D. Aguirre, Massachusetts Institute of Technology (USA) and Harvard
Medical School (USA); J. G. Fujimoto, Massachusetts Institute of
Technology (USA)
An optical coherence microscopy (OCM) system for high-speed en face
cellular resolution imaging in human tissues was developed and demonstrated
for both ex vivo and in vivo imaging. The system incorporates an autofocusing feature that enables precise, near real-time alignment of the confocal
and coherence gates in tissue, thereby ensuring user-friendly optimization of
image quality during the imaging procedure. The system design is compact,
portable, and suitable for future clinical imaging applications.
6627-28, Session 6
Differential-phase optical low coherence
reflectometer for surface profile measurement
14
6627-70, Poster Session
Measurement of axial position of spherical
objects in a multiple delay element C-scan OCT
L. Plesea, A. G. Podoleanu, M. Gomez, Univ. of Kent at Canterbury
(United Kingdom)
In a previous report we have proposed a novel method using en-face OCT
for the evaluation of the curvature of an object, with immediate application
to measurement of corneal curvature. This method uses single shot C-scans
obtained by using a multiple delay element in the reference path of an OCT
system. In the present report we show how the same methodology can be
used for rapid measurement of the axial position of a spherical object. The
theoretical basis and the accuracy of assessing the axial position using this
method for a spherical object of known radius are explored. The potential
application of this method in the measurement and tracking of the in-vivo
axial position of the eye is also discussed.
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Conference 6627: Optical Coherence Tomography and Coherence Techniques
6627-30, Session 6
Filter bank approach to enhance signal
processing for FD OCT
B. Hofer, B. PovaÏay, B. M. Hermann, A. Unterhuber, Cardiff Univ.
(United Kingdom); G. Matz, F. Hlawatsch, Vienna Univ. of Technology
(Austria); W. Drexler, Cardiff Univ. (United Kingdom)
A central step in signal processing for frequency domain (FD) optical
coherence tomography (OCT) involves fast Fourier transformation based
remapping of spectrally encoded reflectivity depth profiles back into the spatial
domain. Especially for spectrometer based setups the transformation is
currently preceded by resampling procedures since the interference spectra
are acquired non-uniformly in k-space. The output signals show a depth
dependent signal roll-off and peak broadening which is also known as fringe
wash-out and has been previously identified to be dependent on various
finite precision effects of the involved optical system, such as limited
bandwidth, refraction limitations, pixilation, non-linear sampling and channel
crosstalk.
We use a filter bank (FB) framework to model the signal acquisition in FD
OCT which allows us to analyze the signal degrading system properties.
Thereby the individual samples of the A-line interference spectrum are
considered as being the output channels (subbands) of a critically sampled
analysis FB. This analysis FB basically represents a system of parallel narrowbandwidth band-pass filters. The individual transfer functions of these bandpass filters can be determined via system characterization either using single
frequency swept light sources or known spatial setups for calibration (i.e., a
reference mirror that can be precisely stepped). After characterization of the
analysis FB in force, the FB framework allows to design the optimal fitting
synthesis FB, which replaces the suboptimal Fourier transform based
algorithms. The synthesis FB implicitly handles the resampling procedures,
since it directly takes care of non-uniform sampled spectra. Furthermore
such synthesis FB can be realized in real-time and allows to incorporate
dispersion compensation.
We practically evaluate the FB approach on our 800 nm ophthalmic
spectrometer based FD system, showing reduced signal roll-off and enhanced
dynamic range. This is demonstrated both on data from mirror measurements
as well as on in vivo tomograms. We conclude that the FB framework allows
to numerically improve the imaging properties of a system by relying on the
real system description which was not possible by Fourier transform based
algorithms and enables optimal extraction of diagnostically relevant
information.
6627-31, Session 6
We present two approaches to SD PS-OCT systems with different detection
units to record the spectral interferograms from the two orthogonal polarization
channels of the PS interferometer. Whereas one employs two complete
spectrometers, i.e. one for each polarization channel, only a single
spectrometer is used for the other one. In the latter case the two polarization
channels’ beams share the same diffraction grating, imaging lens and line
scan camera. We point out the constructional differences of these two setups,
discuss advantages and limitations of the different methods and show results
of calibration and performance measurements for both setups. Furthermore,
PS-OCT images of human tissue are presented to demonstrate the
performance of both system designs.
6627-33, Session 7
MEMS based non-rotatory circumferential
scanning optical probe for endoscopic optical
coherence tomography
Y. Xu, National Univ. of Singapore (Singapore) and Institute of Microelectronics (Singapore); J. Singh, Institute of Microelectronics
(Singapore); H. S. Jason, K. Ramakrishna, N. Chen, C. T. Kuan, National
Univ. of Singapore (Singapore)
In this paper, we present a non-rotatory circumferential scanning optical probe
integrated with a MEMS scanner for in-vivo endoscopic optical coherence
tomography (OCT). OCT is an emerging optical imaging technique that allows
high resolution cross-sectional imaging of tissue microstructure. To extend
its usage to endoscopic applications, a miniaturized optical probe based on
Micro-Electro-Mechanical Systems (MEMS) fabrication techniques is currently
desired. A 3D electrothermally actuated micromirror realized using
micromachining single crystal silicon (SCS) process highlights its very large
angular deflection, more than 45 degree, with low driving voltage for safety
consideration. The micromirror is integrated with a GRIN lens into a waterproof
package which is compatible with requirements for minimally invasive
endoscopic procedures. To implement circumferential scanning substantially
for diagnosis on certain pathological conditions, such as Barret’s esophagus,
the micromirror is mounted on 90 degree to optical axis of GRIN lens. 3
Bimorph actuators that are connected to the mirror on one end via supporting
beams and springs are selected in this micromirror design. When three
actuators of the micromirror are driven by three channels of sinusoidal
waveforms with 120 degree phase differences, beam focused by a GRIN is
redirected out of the endoscope by 45 degree tilting mirror plate and achieve
circumferential scanning pattern. This novel driving method making full use
of very large angular deflection capability of our micromirror is totally different
from previously developed or developing micromotor-like rotatory MEMS
device for circumferential scanning.
Pushing the usable bandwidth of ophthalmic
ultra-high resolution Optical Coherence
Tomography
6627-34, Session 7
A. Unterhuber, B. PovaÏay, B. Hofer, B. M. Hermann, Cardiff Univ.
(United Kingdom); E. J. Fernández, Univ. de Murcia (Spain) and Cardiff
Univ. (United Kingdom); J. E. Morgan, Cardiff Univ. (United Kingdom); C.
Glittenberg, S. Binder, Ludwig Boltzmann Institut (Austria); W. Drexler,
Cardiff Univ. (United Kingdom)
M. Wojtkowski, A. Szkulmowska, M. Szkulmowski, T. Bajraszewski, W.
T. Fojt, A. Kowalczyk, Mikolaja Kopernika Univ. (Poland)
Optical coherence tomography (OCT) at 800 nm in the human eye is widely
utilized with superluminescent diodes, centred around 850 nm with
bandwidths commonly below 70 nm or broadband Ti:Sapphire lasers,
operating with bandwidths of ~130nm at full width at half maximum, achieving
~3µm axial resolution. Further increase seemed not to result in higher axial
resolution, but rather reduced the sensitivity. This limit is known to be set by
the chromatic aberrations in the human eye, which have been found to
increase at shorter wavelengths. With adaptive optics OCT, being more
sensitive to focal shifts, it could be demonstrated that a chromatic
compensation lens can reduce these adverse effects for a bandwidth of
160nm resulting effective axial and transversal resolution increase.
Implementation of this lens in an ophthalmic ultra-high resolution (UHR)OCT system in conjunction with an extremely wide emitting Ti:Sapphire laser
(up to 300nm), we investigate the effects of higher bandwidth on the OCT
tomogram in respect to resolution, but also different contrast due to the
spectrally modulated absorption profiles of endogenous chromophores.
Doppler spectral optical coherence tomography
with optical frequency comb
Estimation of velocities in in-vivo medical and biomedical samples is one of
the key aims of functional measurements in OCT. Different methods using
information about the initial phase of OCT interferometric signal were so far
proposed to solve the problem. Unfortunately strong phase noise, inherent
to OCT method, limits the applicability of such methods. In this contribution
we propose application of a discrete spectrum to Spectral Optical Coherence
Tomography (SOCT) to analyze Doppler signals without direct involvement
of the initial phase of the fringe signal. The discrete spectrum with equidistantly
distributed optical frequency components is known in the literature as optical
frequency comb (OFC). High stability and narrow line-width of the single
optical frequency component in OFC enable measuring the beat frequency
coming from the Doppler shift of light scattered from a moving object. In this
contribution we will demonstrate novel instrumentation and processing
techniques, which enable Doppler flow analysis by Spectral OCT device.
6627-35, Session 7
6627-32, Session 6
Optical coherence tomography controlled
femtosecond laser microsurgery system
Single- vs. two-camera based spectral-domain
polarization-sensitive OCT systems
O. Massow, Laser Zentrum Hannover e.V. (Germany); F. G. Will, Rowiak
GmbH (Germany); H. Lubatschowski, Laser Zentrum Hannover e.V.
(Germany)
B. Baumann, E. Götzinger, M. Pircher, C. K. Hitzenberger, Medizinische
Univ. Wien (Austria)
Recently, first spectral domain (SD) polarization sensitive (PS) optical
coherence tomography (OCT) systems have been presented, thus combining
the ability to gather birefringence information and the advantages of SDOCT which allows for high acquisition speed and increased sensitivity
compared to conventional time domain (TD) OCT. These instruments
employed different detection units to record the spectral information of the
polarized interferometric signal.
European Conferences on Biomedical Optics 2007 •
Due to nonlinear interaction with optical transparent probes the femtosecond
technology is a very useful tool for high precision micro surgery on biological
tissues. At the same time femtosecond lasers are ideal light sources for
imaging methods such as optical coherence tomography (OCT) due to the
broad spectrum of the laser, which is necessary for creating ultra short pulses.
Using OCT structures within biological tissues can be imaged non invasive
with a resolution within the low µm-range.
The combined use of an ultra short pulse laser for cutting of biological tissues
as well as imaging via OCT is a very interesting tool. It opens up a wide range
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Conference 6627: Optical Coherence Tomography and Coherence Techniques
of new surgery techniques and improves many existing methods due to high
precision and high flexibility of the cutting process.
Therefore we combined a femtosecond cutting system and a fourier domain
OCT. In a first attempt the OCT is driven with an SLD and is used alternately
to the cutting system. The OCT is integrated into the optical path which
enables in situ imaging of the surgery area.
6627-36, Session 7
Coherent amplified Fourier domain optical
coherence tomography
J. Zhang, B. Rao, Z. Chen, Univ. of California/Irvine (USA)
A technique to improve the signal-to-noise ratio of a high speed 1300 nm
swept source optical coherence tomography (SSOCT) system was
demonstrated. A semiconductor optical amplifier (SOA) was employed in
the sample arm to coherently amplify the weak light back-scattered from
sample tissue without increasing illumination laser power on sample. The
optical coherence tomography (OCT) signal was amplified by more than 25
dB with 2.4 dB signal to noise ration (SNR) improvement due to the limited
performance of swept source. The image quality improvement was visualized
by imaging the anterior segment of a rabbit eye at imaging speed of 20,000
A-lines per second.
6627-38, Session 8
Increase of the imaging depth in linear OCT
systems
G. Hüttmann, V. Hellemanns, Univ. zu Lübeck (Germany); P. Koch,
Thorlabs-HL (Germany)
6627-40, Session 8
Design criteria in choosing optimized OCT
scanning regimes
C. C. Rosa, Univ. do Porto (Portugal) and Instituto de Engenhariade
Sistemas e Computadores do Porto (Portugal); J. A. Rogers, P. Justin,
Ophthalmic Technologies Inc. (Canada); R. B. Rosen, The New York Eye
and Ear Infirmary (USA); A. G. Podoleanu, Univ. of Kent at Canterbury
(United Kingdom)
A comparative analysis of different scanning regimes depending on image
size to fit different areas to be imaged is presented. Safety thresholds due to
the different continuous irradiation time per transverse pixel in different
scanning regimes are also considered. We present the maximum exposure
level for a variety of scanning procedures, employing either A scanning (depth
priority) or T scanning (transverse priority) when generating cross section
images, en-face images or collecting 3D volumes.
We present a comparison between such B-scan images, and different criteria
to allow the user to choose the right mode of operation. Mainly, two criteria
are detailed, a scanning criterion and a safety criterion. The scanning criterion
depends on the number of pixels along the lateral and axial directions. The
analysis shows that en-face scanning allows wider images while the
longitudinal scanning is more suitable to deep cross sections. The safety
criterion refers to safety levels to be observed in each scanning mode. We
show that the flying spot OCT imaging has different safety limits for T- and Abased imaging modes. The analysis leads to maximum permissible optical
power levels that favors T-scan imaging of wide objects. We then apply the
analysis considering as object the eye.
6627-42, Session 8
OCT sensors traditionally use scanning optical delay lines with moving parts
and a single detector. OCT systems with a linear detector array (linear OCT
or L-OCT) are simple and robust, but a high number detector pixels (10,000
or more) are needed for an useful imaging depth, because the fringe pattern,
which results from the superposition of probe and reference beam has to be
sampled with at least two elements per period. In principal only 4 pixel per
coherence peak are necessary for a correct sampling of the image information.
We will discuss different approaches to increase the measurement range for
a given pixel number and demonstrate two different optical setups for LOCT with increased measurement range. With an additional grating in front
of the detector a reduction of the spatial frequencies of the fringe pattern on
the detector without loss of SNR is possible. An implementation of this system
will be shown and images are be compared to OCT-images of a commercially
available spectral domain system. Advantage of the linear OCT system is a
simple set-up with no demands on the quality of the optics. Disadvantage
with regard to the spectral radar technology is the lower SNR which is prone
to time domain OCT. Long measurement range application with low or
moderate resolution are well suited for the L-OCT. Possible applications of
L-OCT will be discussed.
6627-39, Session 8
In vivo imaging of mouse cornea by dualchannel detection based full-filed OCT
M. Akiba, K. Chan, Yamagata Promotional Organization for Industrial
Technology (Japan)
We report in vivo sub-cellular level imaging results of mouse cornea using
full-field optical coherence tomography (FF-OCT). A FF-OCT system capable
of real-time observation of horizontal cross-sectional image at a sub-cellular
level has been developed. The system is based on a white-light interference
microscope combined with a dual-channel detection technique using a pair
of CCD cameras. A dual-channel detection technique, which employs an
achromatic phase shifter consisting of a linear polarizer and a quarter
waveplate in the reference arm, enables capturing a pair of 90-degrees phase
difference images simultaneously. By exploiting a low-coherence nature of
the thermal light source, FF-OCT image is optically sectioned out with a
thickness of 1.2 um. The FF-OCT image consists of 500 x 500 pixels covering
an area of 420 um x 420 um with a transverse resolution of 1.7 um x 1.7 um.
Time sequence of FF-OCT images are recorded at 30 frames/s. In the present
study, four to six week-old albino mice were anesthetized by intramuscular
injection. A drop of hydroxyethylcellulose was put between cornea and
objective for index matching. Our FF-OCT results show that epithelium cells
and endothelium cells are clearly observed, where highly scattering epithelium
cell nuclei and the hexagonal structure of endothelial cells can be recognized,
demonstrating that ultrahigh resolution FF-OCT is feasible for visualizing the
corneal structure of mouse at the sub-cellular level in vivo.
Doppler calibration method for spectral-domain
OCT spectrometers
D. J. Faber, D. M. de Bruin, H. de Vries, T. G. van Leeuwn, Univ. van
Amsterdam (Netherlands)
We present a method for per-pixel calibration of spectrometers used in
spectral-domain OCT imaging. By linearly displacing the reference mirror as
in time-domain OCT, the time-dependent signal from each pixel of the
spectrometer is modulated by the Doppler frequency of the moving mirror.
With known velocity, this method allows determination of the exact wavelength
and bandwidth incident on each pixel.
6627-43, Session 8
Static depth dependant dispersion
compensation in a real-time static delay line
grating based correlation OCT system
L. Froehly, P. Sandoz, L. Furfaro, M. Ouadour, Univ. de Franche-Comté
(France)
In OCT the depth resolution is related to the source spectral bandwidth.
High depth resolution supposes wide spectral bandwidth and then increasing
of the dispersion. This broadens the OCT axial point spread function.
Numerical dispersion compensation has proven to be quite efficient for
imaging of retina up to the third order dispersion coefficients. This kind of
methods requires anyway significant data post processing. In TDOCT some
hardware depth dependant dispersion compensation systems where
proposed. They are based on a frequency domain optical delay line and the
dispersion property already known in pulse compression systems. A
dispersion law that is ‘linearly’ dependant on the scanner position (e.g. the
scanning depth position) is then achieved. This system requires anyway a
scanning to get access to the whole A-scan.
We demonstrate theoretically and experimentally in this conference a
possibility to take advantage from the dispersion grating property in a system
where a static delay line is used. This device is based on grating correlation,
already demonstrated in OCT and more recently in spectroscopic OCT. The
dispersion correction introduced is linearly dependant on the inspected depth.
The acquired A scan is compensated proportionally to the actual thickness
of the dispersive medium. Discussions about limitations of this system will
be proposed as well as the possibility to couple it to classical balancing of
dispersion by hardware or software post processing to access higher order
terms of the dispersion law.
6627-44, Session 8
Extended focus Fourier domain optical
coherence microscopy assists developmental
biology
M. L. Villiger, École Polytechnique Fédérale de Lausanne (Switzerland);
M. Beleut, C. Brisken, Swiss Institute for Experimental Cancer Research
(Switzerland); T. Lasser, R. A. Leitgeb, École Polytechnique Fédérale de
Lausanne (Switzerland)
16
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Conference 6627: Optical Coherence Tomography and Coherence Techniques
We present a novel detection scheme for Fourier domain optical coherence
microscopy (FDOCM). A Bessel-like interference pattern with a strong central
lobe was created with an axicon lens. This pattern was then imaged by a
telescopic system into the sample space to obtain a laterally highly confined
illumination needle, extending over a long axial range. For increased efficiency,
the detection occurs decoupled from the illumination, avoiding a double pass
through the axicon. Nearly constant transverse resolution of ~1.5µm along a
focal range of 200µm with a maximum sensitivity of 105dB was obtained. A
broad bandwidth Ti:Sapphire laser allowed for an axial resolution of 3µm in
air, providing the nearly isotropic resolution necessary to access the
microstructure of biological tissues. Together with the speed- and sensitivityadvantage of FDOCT, this system can perform in vivo measurements in a
minimally invasive way. Tomograms of the mouse mammary gland and the
mouse follicle, recorded in vitro, revealed biologically relevant structural
details. Images acquired with classical microscopy techniques, involving
stained and fluorescent samples, validate these structures and emphasize
the high contrast of the tomograms. It is comparable to the contrast achieved
with classical techniques, but employing neither staining, labeling nor slicing
of the samples, stressing the high potential of FDOCM for minimally invasive
in vivo small animal imaging.
6627-63, Session 8
Endoscopes for spectral radar OCT
E. Lankenau, Univ. zu Lübeck (Germany); K. Eder, Fraunhofer-Institut für
Produktionstechnologie (Germany); D. Boller, P. Koch, Thorlabs GmbH
(Germany); G. Hüttmann, Univ. zu Lübeck (Germany)
Optical coherence tomography (OCT) is limited to imaging of tissue surfaces
due to the limited penetration depth of visible and IR radiation. In order to
extend OCT to inner surfaces of the body endoscopic techniques were
developed. Up to now most OCT probes were constructed using rotating or
moving mono-mode fibers or micro scanners at the tip of the probe. We
describe two, a rigid and a flexible, endoscopic OCT system which both
uses an extracorporal scanner to create OCT images with a resolution below
10 µm. Both system work with a Spectral Radar OCT (Thorlabs Inc.) at 900
nm. The rigid OCT endoscope was constructed using a 270 mm gradient
lens with a diameter of 3 mm. Dispersion of the endoscope was compensated
in the OCT interferometer which was located in the hand piece. The flexible
OCT probe uses an imaging fiber bundles manufactured from specially made
mono-mode fibers. Principle considerations and first results will be presented.
6627-45, Session 9
Correcting ocular aberations with a high stroke
deformable mirror
S. G. Tuohy, A. Bradu, Univ. of Kent (United Kingdom); A. G. Podoleanu,
Univ. of Kent at Canterbury (United Kingdom); N. Chateau, Imagine
Eyes (France)
The capabilities of a novel deformable mirror to correct ocular aberrations
are analyzed. The deformable mirror, (MIRAO52 Imagine Eyes) is incorporated
within a retinal imaging system able to produce simultaneous en-face optical
coherence tomography (OCT) and scanning laser ophthalmoscopy (SLO)
images of the retina. The performances of the deformable mirror have been
examined by evaluating the amount of aberrations which the deformable
mirror is able to correct for, the improvement in the signal-to-noise ratio and
transversal resolution in both OCT and SLO images obtained from an artificial
eye.
6627-46, Session 9
Quantification of the photoreceptor layer
thickness: normative data and macular hole
patients
B. A. Sander, Copenhagen Univ. Hospital Glostrup (Denmark); T. M.
Jørgensen, Technical Univ. of Denmark (Denmark)
Purpose: To present normative data of photoreceptor layer thickness obtained
by a newly developed image analysis algorithm based on software enhanced
optical coherence tomography (OCT) images.
Methods: Methodological case series. Using the Stratus OCT3 instrument
we obtained twenty vertically directed OCT scans from identical retinal
locations from twenty-five normal eyes and 3 patients with unilateral idiopathic
macular hole. Eight good quality scans from each person were selected for
software enhancing. Enhanced OCT images were analysed by a new software
algorithm using the vertical intensity profile of the image to automatically
demarcate retinal layers allowing thickness measurements of the retinal
pigment epithelium- and photoreceptor outer segment layer (RPEOScomplex). Retinal thickness, pixel light intensity in the central part of the
retina and the width of central photoreceptor defects after anatomically
successful macular hole surgery were also obtained.
Results: In healthy subjects, the mean RPE-OScomplex thickness in the foveal
centre was 77.2 µm (SD = 3.95), and the mean RPE-OScomplex thickness
profile along the 6 mm long vertical scan showed a rise in the central 1000
µm of the scan, corresponding to the long cone photoreceptor outer segments
European Conferences on Biomedical Optics 2007 •
in this region. Mean normal retinal thickness in the foveal centre was 161.5
µm (SD = 15.2). In addition to thickness measurements we were able to
quantify central photoreceptor defects and the relative reflectivity of the outer
nuclear layer in patients after macular hole surgery on the postoperative
enhanced OCT images.
Conclusions: Software image enhancing of Stratus OCT images enables
quantitative analysis of outer retinal layers and small central photoreceptor
defects as for macular holes. In addition measurements of relative reflectivity
of the outer nuclear layer permits further insight in macular pathology.
6627-47, Session 9
Scattering optical coherence angiography with
1-um swept source optical coherence
tomography
Y. Yasuno, Univ. of Tsukuba (Japan); Y. Hong, Univ. of Tsukuba (Japan)
and Korea Advanced Institute of Science and Technology (Japan); S.
Makita, M. Yamanari, Univ. of Tsukuba (Japan); M. Akiba, Yamagata
Promotional Organization for Industrial Technology (Japan); M. Miura,
Tokyo Medical Univ. Kasumigaura Hospital (Japan) and Univ. of Tsukuba
(Japan); T. Yatagai, Univ. of Tsukuba (Japan)
Retinal, choroidal and scleral imaging by using swept-source optical
coherence tomography (SS-OCT) with a 1-um band probe light, and highcontrast and three-dimensional (3D) imaging of choroidal vasculature are
presented. This SS-OCT has a measurement speed of 28,000 A-lines/s, a
depth resolution of 10.4 um in tissue, and a sensitivity of 99.3 dB. Automatic
dispersion compensation, a dynamic spectral reshaping, a morphological
despeckle filter and a fast fundus preview method of 3D OCT are also
presented. Owing to the high penetration of the 1-um probe light and the
high sensitivity of the system, the in vivo sclera of healthy volunteer can be
observed. A software-based algorithm for scattering optical coherence
angiography (S-OCA) is developed for the high-contrast and 3D imaging of
the choroidal vessels. The S-OCA visualizes the 3D choroidal vasculature
ofin vivo human macula and the optic nerve head.
6627-48, Session 9
Optical coherence angiography for the retina
and choroid
S. Makita, Univ. of Tsukuba (Japan); Y. Hong, Univ. of Tsukuba (Japan)
and KAIST (South Korea); M. Miura, Tokyo Medical Univ. Kasumigaura
Hospital (Japan) and Univ. of Tsukuba (Japan); M. Yamanari, T. Yatagai,
Y. Yasuno, Univ. of Tsukuba (Japan)
Noninvasive ophthalmic angiographies are demonstrated for the in vivo
human. Three-dimensional structural and flow imaging have been performed
with a high-speed spectral-domain optical coherence tomography. The two
methods are presented, i.e. Doppler optical coherence angiography (DOCA)
and scattering optical coherence angiography (SOCA). For DOCA, bidirectional flow and power of Doppler shift imaging are used to contrast the
blood vessels. Bulk motion artifacts in flow images are eliminated by
histogram-based algorithm. Three-dimensional retinal and choroidal
vasculature images are simultaneously obtained by separating the volume
set into retinal part and choroidal part. Two-dimensional images of blood
vessels are obtained by integrating the volume sets of flow images along
axial direction. Since power of Doppler flow images exhibit higher sensitivity
than bi-directional flow images, three-dimensional volume sets of power of
Doppler shift images are used. Angiographic images of retinal vessels and
choroidal vessels are produced by selective integration for retinal part and
choroidal part, respectively. These images are in good agreement with
fluorescein angiography and indocyanine green angiography. SOCA utilized
the lower OCT signal of choroidal blood comparing to that of the choroid.
The choroidal vessels are segmented in en-face OCT slices which the relative
depth to the retinal pigment epithelium is constant. A vascular projection
image is obtained by integrating the segmented choroidal vasculature. The
segmented vascular image is compared with the images obtained using
existing invasive methods such as indocyanine green angiography (ICGA),
to check the feasibility of the alternative method.
6627-49, Session 9
In-vivo 3-D imaging of age-related macular
degeneration using optical frequency domain
imaging at 1050 nm
D. M. de Bruin, Massachusetts General Hospital (USA); J. F. DeBoer,
Harvard Medical School (USA) and Wellman Ctr. for Photomedicine
(USA); D. L. Burnes, J. Loewenstein, C. Kerbage, G. N. Maguluri, B. H.
Park, Massachusetts General Hospital (USA); A. Yun, Harvard Medical
School (USA) and Wellman Ctr. for Photomedicine (USA)
INTRODUCTION
Macular degeneration comprises a group of diseases, which are characterized
by progressive aggravation of the macular region. The most common clinical
appearance of macular degeneration is age related macular degeneration
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Conference 6627: Optical Coherence Tomography and Coherence Techniques
(AMD), which is one of the world’s leading causes of vision loss in people
over 65 in western countries. The golden standard methods for the diagnosis
of AMD include color fundus photography (CFP) and fluorescence angiograms
(FA) technique. [1] Although these methods provide detailed information about
the en-face location and global dimension of the macular abnormalities, they
do not offer valuable depth information throughout the macular region of the
retina and choroid. We present ophthalmic OFDI at 1050 nm, with an A-line
rate up to 55 kHz and demonstrate the improved ranging depth (defined as
the 6 -dB roll off in sensitivity) of 2-2.4 mm to study wavelength dependent
scattering properties of various retinal structures and penetration depth in
the choroids and to determine the sensitivity and specificity of OFDI at 1050
nm for the detection of AMD.[2]
6627-50, Session 9
Three-dimensional high speed OCT at 1050 nm
vs. 800 nm: Reduced scattering for enhanced
penetration and through cataracts and into
choroidal tissue
B. PovaÏay, B. M. Hermann, A. Unterhuber, Cardiff Univ. (United
Kingdom); H. Sattmann, Medical Univ. Vienna (Austria); F. Zeiler, Ludwig
Boltzmann Institut (Austria); J. E. Morgan, Cardiff Univ. (United
Kingdom); C. Falkner-Radler, C. Glittenberg, S. Binder, Ludwig
Boltzmann Institut (Austria); W. Drexler, Cardiff Univ. (United Kingdom)
Two high speed (~10 k depth scans/second) 3D-OCT systems were built
around broadband light sources operating at central wavelengths of 800 and
1050 nm. Both systems employed the spectrometer based frequency domain
(FD) design to support the full bandwidth of the sources (~160 nm and ~70
nm), resulting in effective axial resolutions of ~3 and ~7 µm respectively and
comparable sensitivity in normal subjects. Volumetric tomograms of normals
and patients with different stages of cataract were taken consecutively with
the same protocol by both FD-OCT systems. Image data was post processed
and rendered with the same procedures for both systems. The lower scattering
at 1050 nm significantly improved the imaging performance in cataract
patients; thereby widening the clinical applicability of ophthalmic OCT.
Additionally the lower susceptibility to scattering at 1050 nm allowed deeper
penetration into tissue compared with the conventionally used 800 nm
radiation. The use of longer imaging wavelengths should increase the usability
of OCT in the early diagnosis of diabetic retinopathy and age related macular
degeneration.
6627-51, Session 10
Polarization sensitive OCT in patients with
macula and nerve head disorders
C. K. Hitzenberger, E. Götzinger, M. Pircher, B. Baumann, S. Michels, W.
Geitzenauer, C. Vass, U. Schmidt-Erfurth, Medizinische Univ. Wien
(Austria)
Polarization properties of anterior segment disorders of the eyes were
evaluated using a fiber-based polarization-sensitive Fourier-domain optical
coherence tomography (PS-FD-OCT). The light source is a superluminescent
diode with a central wavelength of 840 nm, and bandwidth of 50 nm.
Synchronized two line-CCD cameras allow high-speed measurement of
birefringence of retina (line rate 27.7 kHz), and the sensitivity of the system is
100.7 dB. Birefringence of the optical fiber was compensated with the surface
reflection. Phase retardation and orientation of the birefringence were
measured with a Jones matrix based algorithm. The phase retardation map
of the anterior segment was visualized as a depth-resolved three-dimensional
image in addition to the conventional cross sectional OCT image. In the
polarization image of the normal eye, striking polarization change was
observed at the sclera. In the eyes with necrotizing scleritis, abnormal thinning
of the sclera could be confirmed. In the eyes after filtering glaucoma surgery,
polarization change in the conjunctiva due to the abnormal fibrosis after
surgery could be observed. PS-FD-OCT is an effective tool to understand
the polarization properties of different types of pathological changes in the
anterior segment of the eye.
6627-54, Session 10
Mueller coherency matrix method for contrast
image in tissue polarimetry
J. L. Arce-Diego, F. Fanjul-Vélez, D. Pereda-Cubián, Univ. de Cantabria
(Spain)
Optical characterization of biological tissue is improving greatly the information
that practitioners obtain from disease processes, and leads to a non-invasive
effective diagnosis tool. Polarimetry techniques are specially appropriate for
biological tissues, due to the fact that their properties show dependence
with polarization of light, and they usually exhibit a depolarising behaviour.
Methods of analysis that do not take into account tissue depolarisation, like
Jones matrix, produce limited results. The extension of these characterization
techniques to Mueller matrix measurement adds data to the image obtained,
but further information can be extracted.
In this work, we propose the use of the Mueller Coherency matrix of biological
tissues in order to increase the information from tissue images and so its
contrast. This method involves different Mueller Coherency matrix based
parameters, like the eigenvalues analysis, the entropy factor calculation,
polarization components crosstalks, linear and circular polarization degrees,
hermiticity or the Quaternions analysis in case depolarisation properties of
tissue are sufficiently low, so pathologies like cancer could be detected in a
sooner stage of development. All these parameters make information appear
clearer and so increase image contrast. The election will depend on the
concrete pathological process under study. This Mueller Coherency matrix
method can be applied to a single tissue point, or it can be combined with a
tomographic technique, like Mueller-OCT, so as to obtain a 3D representation
of polarization contrast parameters in pathological tissue. The application of
this analysis to concrete diseases can lead to tissue burn depth estimation
or cancer early detection.
Our previously developed polarization sensitive optical coherence tomography
(PS-OCT) systems were applied to image the retina of patients with macula
and glaucoma related disorders. A transversal scanning time domain system
was used for 2D linear and circumpapillary cross sectional scans, and a fast
spectral domain (SD) system was used for 3D imaging and for generating
retinal birefringence maps in the manner of scanning laser polarimetry (SLP).
More than 70 patient eyes with various diseases (age related macular
degeneration, drusen maculopathy, central serous retinopathy, glaucoma,
etc.) were imaged. Several polarization changing structures were identified:
birefringence was found in the retinal nerve fiber layer (RNFL), Henle’s fiber
layer, the sclera, the lamina cribrosa, and fibrosis tissue. The retinal pigment
epithelium (RPE) scrambles the polarization state of backscattered light (i.e.,
acts as a depolarizing layer). In AMD patients, disturbances of the RPE are
readily visualized by exploiting the depolarization information. Drusen
maculopathy can be distinguished from vitelliform dystrophy by the different
shape of the RPE. In glaucoma patients, areas of abnormally reduced
retardation in the RNFL can be observed. Finally, a comparison with SLP
reveals that cases of “atypical” SLP scans are caused by increased
penetration of the sampling beam to the birefringent sclera. By excluding
light backscattered from the sclera, PS-OCT can generate pseudo-SLP maps
that are free from the atypical birefringence patches.
6627-52, Session 10
Polarization-sensitive Fourier-domain optical
coherence tomography for the imaging the
anterior segment disorder of the eyes
M. Miura, Tokyo Medical Univ. Kasumigaura Hospital (Japan) and Univ.
of Tsukuba (Japan); M. Yamanari, Univ. of Tsukuba (Japan); Y.
Watabnabe, H. Mori, Tokyo Medical Univ. (Japan) and Univ. of Tsukuba
(Japan); T. Iwasaki, Tokyo Medical Univ. (Japan); A. E. Elsner, Indiana
Univ. (USA); K. Kawana, T. Oshika, T. Yatagai, Y. Yasuno, Univ. of
Tsukuba (Japan)
18
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Conference 6628: Diagnostic Optical Spectroscopy
Tuesday-Thursday 19-21 June 2007
Part of Proceedings of SPIE Vol. 6628 Diagnostic Optical Spectroscopy
in Biomedicine IV
6628-11, Poster Session
Multifocal multiphoton microscopy using a novel
field of view zoom scanning protocol
L. Liu, L. Wang, J. Qu, Z. Lin, Z. Fu, H. Niu, Shenzhen Univ. (China)
Multifocal Multiphoton Microscopy (MMM) has become a popular tool in
biomedicine in recent years. It achieves video-rate imaging at high spatial
resolution by employing nonlinear multiphoton excitation and rapidly scanning
an array of high aperture foci across the sample. Many research groups have
reported MMM schemes that use microlens disk, original or cascaded
beamsplitter or diffractive optical element to produce multiple foci
simultaneously. But in such configurations, the microscope objectives are
usually fixed, therefore the scanning systems can work to their full
performance under only certain conditions, which limit their applications. In
order to operate not only in low resolution and large field of view (FOV), but
also in high resolution and small FOV applications, moreover, to make full
use of the capability of each optical element in the system, we propose a
novel FOV zoom scanning protocol and image reconstruction method for
MMM. By combining a pair of galvo mirrors (for fine scanning) and a sample
stage (for coarse scanning or translation), the resolution and FOV of the MMM
system can be changed without changing any optical elements in the system,
including the objective. The spatial resolution of the system can be improved
by using two-photon excitation and small step size in scanning. This protocol
can be used in our simultaneous time- and spectrum-resolved MMM system,
which can provide spectral and lifetime information simultaneously in
fluorescence microscopy for biomedical imaging.
6628-24, Poster Session
Glass based fluorescence reference materials
A. Engel, C. R. Otterman, V. Rupertus, SCHOTT AG (Germany); U.
Resch-Genger, K. Hoffmann, Bundesanstalt für Materialforschung und prüfung (Germany); U. Kynast, Fachhochschule Muenster (Germany)
Fluorescence techniques are known for their high sensitivity and are widely
used as analytical tools and detection methods for product and process
control, material sciences, environmental and bio-technical analysis,
molecular genetics, cell biology, medical diagnostics, and drug screening.
According to DIN/ISO 17025 certified standards have to be used for
fluorescence diagnostics. Even though relative values are available for
fluorescence intensities only.
Therefore reference materials for a quantitative characterization have to be
related directly to the investigated materials. In order to evaluate these figures
it is necessary to calculate absolute values like absorption/excitation cross
section and quantum yield. This can be done for different types of dopands
in various materials like glass, glass ceramics, crystals or nano crystalline
material embedded in polymer matrices.
The chemical, structural and spectroscopic properties of the developed
material will be presented. Based on these facts we will discuss options for
doped glass and glass ceramics with respect to scattering regime, reflection
losses, and stability against blue and UV radiation.
Results on wavelength accuracy and lifetime have been performed. Moreover
intensity patterns and results for homogeneity, isotropy, photo and thermal
stability will be discussed.
In this paper we present first results of these aspects for mainly transparent
glass and glass ceramics doped with various degrees of dopands using ions
of raw earth elements and transition metals. Evolved results are compared
with well known organic pigments of LUMOGEN red embedded in silica and
polyurethane matrices.
Typical employed diagnostic tools are fluorescence (steady state, decay time)
and absorption (remission, absorption) spectroscopy working in different
temperature regimes (10 - 350 K) of the measured samples in order to get a
microscopic view of the relevant physical processes and to prove the
correctness of the obtained data.
This work is funded by BMBF under project number 13N8849
6628-42, Poster Session
Reflectance spectrophotometry as
intraoperative assessment of perfusion in rectal
anastomosis: a feasibility study
A. Karliczek, Martini Hospital (Netherlands) and Groningen Univ.
Medical Ctr. (Netherlands); D. A. Benaron, Spectros Corp. (USA); P.
Baas, A. van der Stoel, Martini Hospital (Netherlands); T. Wiggers,
Groningen Univ. Medical Ctr. (Netherlands); G. M. van Dam, Groningen
Univ. Medical Ctr. (Netherlands) and BioOptical Imaging Ctr. Groningen
(Netherlands)
European Conferences on Biomedical Optics 2007 •
Evaluation of microperfusion in colorectal anastomoses remains a challenging
problem. Intraoperative assessment of anastomotic ischemia might be an
important factor in the prediction, and therefore prevention, of anastomotic
dehiscence. In this study we evaluated the feasibility of tissue oximetry using
shallow-penetrating visible light (visible light spectroscopy[VLS]) to measure
microvascular haemoglobin oxygen saturation (Sto2) in small, thin tissue
volumes in rectal anastomoses in a standardized measurement protocol.
Materials and methods: VLS was evaluated in 11 rectal anastomotic
procedures, all within 20 cm of the anal verge by using a T-Stat(r) Ischemia
Detection system (T-Stat(r) 303) using a catheter probe. Feasibility was defined
as the number of planned measurements performed, stability of the
assessments (expressed as descriptive statistics) and adverse events caused
by the VLS-system or the protocol.
Results: Of 220 planned recordings, 125 (57%) were carried out. Mucosal
recordings showed a high standard deviation (20,4-26,6), whilst serosal
recordings showed less variation (SD 4,2-13,7). After ligation of mesenteric
arteries, a decrease in saturation (9%) of the rectal stump serosa was
observed.
Conclusion: Based on the results of this preliminary study, VLS is an easy to
perform and fast technique for intraoperative real-time assessment of
microperfusion of the serosal surface in colorectal anastomosis. Additional
multicenter studies evaluating VLS as a predictor of anastomotic leakage
are currently being carried out.
6628-43, Poster Session
In vivo measurement of the carotenoid level
using portable resonance Raman spectroscopy
Y. Shao, J. Qu, Y. He, Shenzhen Univ. (China)
Carotenoid is a type of antioxidants that play important roles in the antioxidant
defense system. It is p-electron conjugated carbon-chain molecule and has
polyene structure and optical properties. For ß-carotene and lycopene, their
optical absorptions are strong and occur in broad bands, whose widths are
about 100nm and centered at 450nm and 460nm respectively. They are
believed to act as scavengers for free radicals, singlet oxygen, and other
harmful reactive oxygen species which can lead to premature skin aging,
oxidative cell damage, and even skin cancers. The standard technique for
measuring carotenoid is high-pressure liquid chromatography which involves
using chemicals and is invasive. It works well in serum, but not suitable for
measuring carotenoid in tissue. In this paper, we present a portable resonance
Raman spectroscopy system for in vivo measurement of carotenoid, which
is noninvasive, highly sensitive and compact. A small diode-pumped all solidstate 473nm laser instead of a 488nm argon laser is used to excite carotenoid
in thumb in vivo, and the resonance Raman scattering light intensity is
measured to assess the carotenoid level. Basically, it is difficult to detect the
very weak resonance Raman scattering light because it is overlapping with
the strong fluorescence. Our investigation shows that matching glycerol can
help to reduce tissue scattering and increase optic collecting efficiency. We
demonstrate in our experiments that the employment of optical matching
technology for measuring carotenoid resonance Raman spectra in tissue
can improve the signal-to-noise ratio by 3.9dB.
6628-44, Poster Session
A diffusion approximation model of light
transport in multi-layered skin tissue
M. I. Makropoulou-Loukogiannaki, E. Kaselouris, E. A. Drakaki, A. A.
Serafetinides, National Technical Univ. of Athens (Greece)
In dermatology, biophotonic methods offer high sensitivity and non-invasive
measurements of skin tissue optical properties, in various physiological and
pathological conditions. There are numerous skin diseases, which can be
examined and characterized using diagnostic optical spectroscopy, as the
monitoring of skin aging, diagnosis of benign and malignant cutaneous
lesions, dosimetry in photodynamic therapy (PDT), etc. Several mathematical
models have been used to calculate the tissue optical properties from
experimental measurements and to predict the light propagation in soft
tissues, like skin, based on transport theory or Monte Carlo modeling.
This work analyses the phenomena which are observed experimentally during
the irradiation of skin, such as the absorption, reflectance, scattering,
fluorescence and transmission of laser light. The study was carried out on
animal skin samples, extracted post-mortem. In this work we also tried to
evaluate the utility of diffusion approximation modeling for measuring the
light intensity distribution in the skin samples with cw visible laser beam
(?=632.8 nm). The diffusion theory model was tested for the simulation results
of the spatial light distribution within a five-layer model of animal skin tissue.
We have studied the dependence towards the depth and the radial distance
of the photon density of the incident radiation.
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Conference 6628: Diagnostic Optical Spectroscopy
6628-49, Poster Session
Intracellular protein mass spectroscopy using
mid-infrared laser ionization
K. Awazu, S. Suzuki, Osaka Univ. (Japan)
Large-scale analysis of proteins, which can be regarded as functional
biomolecule, assumes an important role in the life science. A MALDI using
an ultraviolet laser (UV-MALDI) is one of ionization methods without
fragmentation and has achieved conformation analysis of proteins. Recently,
protein analysis has shifted from conformation analysis to functional and
direct one that reserves posttranslational modifications such as the sugar
chain addition and phosphorylation. We have proposed a MALDI using a
mid-infrared tunable laser (IR-MALDI) as a new ionization method. IR-MALDI
is promising because most biomolecules have a specific absorption in midinfrared range, and IR-MALDI is expected to offer; (1) use of various matrices,
(2) use of biomolecules such as water and lipid as the matrix, and (3) supersoft ionization. First, we evaluated the wavelength dependence of ionization
of different matrices using a different frequency generation (DFG) laser, which
can tune the wavelength within a range from 5.5 to 10.0 um. As results,
ionization was specifically occurred at 5.8 um which the C=O vibration
stretching bond in matrix material and mass spectrum was observed. Next,
protein mass spectrum was observed in the culture cells, MIN6, which secrete
insulin, without the conventional cell-preparation processes. We demonstrate
that the IR-MALDI has an advantage over the conventional method (UVMALDI) in direct analysis of intracellular proteins.
6628-50, Poster Session
Time-resolved diffuse optical spectroscopy of
small tissue samples
P. Taroni, D. Comelli, A. Farina, A. Pifferi, Politecnico di Milano (Italy); A.
Kienle, Univ. Ulm (Germany)
Time-resolved transmittance measurements were performed in the
wavelength range of 610 or 700 to 1050 nm on phantom slabs and bone
tissue cubes of different sizes. The data were best fitted with solutions of the
diffusion equation for an infinite slab and for a parallelepiped to investigate
how size and optical properties of the samples affect the results obtained
with two models.
When small samples are considered, the slab model overestimates both
optical coefficients. The error is especially marked for the absorption
coefficient, reaching approximately 0.28 cm-1 for the smallest sample
considered (1 x 1 x 2 cm3), independent of the absorption properties. The
parallelepiped model provides much more accurate estimates of both
absorption and scattering coefficients, with errors that never exceed 14%
and 7%, respectively, even in the worst condition. Monte Carlo simulations
supported the interpretation of the experimental data.
To estimate bone tissue composition, the absorption spectra of bone samples
were best fitted based on the Beer’s law. When absorption data obtained
with the slab model are considered, the fitting procedure is unstable, and the
results are unrealistic. The situation improves significantly when absorption
data obtained with the parallelepiped model are used.
In conclusion, the parallelepiped model performs much better than the infinite
slab model for the estimate of the optical properties and, in particular, of the
absorption coefficient. Thus it can profitably be used to quantify the optical
properties of biological tissue samples, whenever small volumes are involved.
6628-51, Poster Session
Single photon spectrometer for biomedical
application: new developments
S. S. Tudisco, L. L. Lanzanò, F. F. Musumeci, S. S. Privitera, A. A.
Scordino, Instituto Nazionale di Fisica Nucleare (Italy) and Univ. di
Catania (Italy); G. G. Fallica, M. M. Mazzillo, D. D. Sanfilippo, G. G.
Valvo, STMicroelectronics (Italy)
6628-52, Poster Session
Optical spectroscopy of phosphatic urinary
calculi
I. H. Yarynovska, A. I. Bilyi, Ivan Franko National Univ. of L’viv (Ukraine)
Urine is a biological liquid. It is one of the most informing matters for
diagnostics of living organism. Water which enters in its composition, contains
the soluble products of organic and inorganic origin, urea, the element little
bodies of blood such as red corpuscles and leucocytes, and other. Inorganic
connections are salts of oxalate and phosphatic compositions.
Formation of infectious urinary calculi is caused by the nanoprocesses and
European Conferences on Biomedical Optics 2007 •
6628-53, Poster Session
Detection of abnormalities in tissue equivalent
phantoms by multi-probe laser reflectometry
P. P. S., M. Kumaravel, M. Singh, Indian Institute of Technology Madras
(India)
The detection of abnormalities in tissue equivalent phantoms by non-contact
multi-probe laser reflectometer is carried out. The tissue equivalent phantoms,
of optical parameters absorption and scattering coefficients, similar to that
of biological tissues, by mixing measured quantities of paraffin wax with
different wax colors, are prepared. The tissue abnormalities in the form of
phantoms of different optical properties are prepared and embedded in the
tissue phantoms at various locations. The NBI pattern shows distinct variation
as measured from the phantoms with abnormality compared to that of control
phantom. By subtraction of the reconstructed image of the control phantom
the images of the phantoms with abnormality are obtained. From scans of
the subtracted image the location of the abnormality and from the full width
at half maximum (FWHM) the size of the abnormality are obtained. From the
scanned curves the type of abnormality is also determined. This procedure
may prove to be useful in detecting the growth and nature of abnormality in
biological tissues.
6628-54, Poster Session
Application in the surgery planning of brain atlas
of the three-dimension
H. Xiao, F. Dai, X. Chen, South China Normal Univ. (China)
The article introduces the Talairach Daemon brain atlas , Talairach proportional
system in three dimensions, Talairach transformation and the application of
the surgery planning system. Based on this each region linear methods, we
have completed a good system call stereostatic surgery planning. Our
proposed the surgery planning procedure of visual brain atlas of the threedimension is superior to the traditional use of printed stereotactic atlases. It
allows all three orientations to be used for surgery planning and gives the
neurosurgeon flexibility in choosing clearly visible landmarks on the same or
different slices. It provides a accuracy atlas to match in the region of interest
and also gives the neurosurgeon some degree of confidence. The use of this
surgery planning system may improve the definition of the target and have
several advantages over other approaches. We think that the proposed
planning system is a step forward in bringing the atlas and research and
clinical data together within a practical and powerful solution that is fast,
fleible, and affordable.
6628-55, Poster Session
We report on the new developments of SINPHOS project (SINgle PHOton
Spectrometer). The realised device is able to measure simultaneously time
distribution with high accuracy and the wavelength spectrum of photons
coming from a weak source like for instance the Delayed Luminescence of
biological systems.
20
is most common complication accompanying urinary tract infections by
members of the genus Proteus. The major factor involved in stone formation
is the urease produced by these bacteria, which causes local supersaturation
and crystallization of magnesium and calcium phosphates as carbonate
apatite and struvite respectively. This effect may also be enhanced by bacterial
polysaccharides.
The capacity of the apatite nanoparticles to bind to tissues in aqueous liquids,
and the pronounced tendency of NB to form mineralized biofilms, indicate
that NB could affect intracardiac fluid forces. Nanobacteria (NB) appear to
possess at least three qualities, which may cause alone or cooperatively
harm to living systems. These can be related to the mineral apatite itself, a
self-synthesized slime, and possibly a genetic material contained in the
mineral cavity. In case of kidney stones, models predict that the stones are
nucleated by giant solitary NB immobilized within the kidney, where NB find
favorable conditions for growth. The composition of the examples was
identified by luminescent spectroscopy, optical spectroscopy luminescent
microscope. The prosecution is conducted of systematization of the got
results.
Contact probe pressure effects in skin multispectral photoplethysmography
J. Spigulis, L. Gailite, A. Lihachev, Latvijas Univ. (Latvia)
Reflection photoplethysmography (PPG) is a non-invasive method for studies
of the skin blood volume pulsations by detection and analysis of the backscattered optical radiation. Skin blood pumping and transport dynamics can
be monitored this way at different body locations with relatively simple and
convenient PPG contact probes.
New technique for parallel recording of reflection photoplethysmography
signals in broad spectral band (violet to NIR) has been recently developed,
and its potential for assessment of blood microcirculation at various vascular
depths is assessed. PPG signals have been simultaneously detected at laser
wavelengths 405 nm, 532 nm, 645 nm, 807 nm and 1064 nm that have different
skin penetration depths. Various PPG signal responses to changes of fibreoptic contact probe pressure on skin and different shapes of the PPG pulses
originated from the same heartbeat but recorded at different wavelengths
have been observed. This indicates to depth-variety of the skin blood pulsation
dynamics.
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Conference 6628: Diagnostic Optical Spectroscopy
6628-56, Poster Session
6628-60, Poster Session
White-light time-resolved reflectance
spectroscopy for monitoring constituents
concentrations in layered diffusive media
Light-induced autofluorescence of animal skin
used in tissue optical modeling
A. Giusto, C. D’Andrea, L. Spinelli, D. Contini, A. Torricelli, Politecnico di
Milano (Italy); F. Martelli, G. Zaccanti, Univ. degli Studi di Firenze (Italy);
R. Cubeddu, Politecnico di Milano (Italy)
We performed reflectance measurements with a time-resolved white-light
spectroscopy system to monitor concentrations changes in a two-layer liquid
phantom with optical properties similar to human tissues. By varying the
concentrations of three inks with different spectral features, we changed the
absorption coefficient of the upper and lower layer to simulate either
haemodynamics changes in the muscle covered by adipose layer, or functional
brain activation with systemic response in the scalp. Data were analyzed by
a time-resolved spectrally constrained fitting method based on a
homogeneous model of photon diffusion. Although this approach is based
on a homogeneous model and employs a single 2cm source-detector
distance, the technique is able to monitor changes in the lower layer, while it
is scarcely affected by variation in the upper layer. Preliminary in vivo
measurements have been performed on one healthy volunteer to monitor
oxy- and deoxy-haemoglobin changes in the muscle during arterial occlusion
and in the brain during a motor task. Even if the overall sensitivity of the
technique is reduced, in vivo results are in general agreement with the findings
of dedicated system for tissue oximetry.
6628-58, Poster Session
Spectroscopic measurement of adipose tissue
thickness and comparison with ultrasound
imaging
I. Bliznakova, E. G. Borisova, Institute of Electronics (Bulgaria); P.
Troyanova, National Oncological Ctr. (Bulgaria); L. Avramov, Institute of
Electronics (Bulgaria)
Human skin is one of the most investigated subjects using optical
spectroscopy because of its easy access and great diversity of skin
alterations, which give optical properties changes. Light-induced
autofluorescence spectroscopy (LIAFS) is applied for investigation of optical
properties of normal and diseased skin, including skin cancer detection. LIAFS
gives high sensitivity on the early stage of tumor growth, but to achieve better
determination of this condition, investigators must collect spectral information
from huge dataset of patients in vivo or study different tumor models on
animals to obtain objective information for fluorescent properties of every
kind of normal and diseased tissue. Therefore, it is very important to find the
most appropriate and close to the human skin animal model samples from
the point of view of laser-induced fluorescence spectroscopy, which will give
the possibility for easier transfer of data obtained in animal models to medical
diagnostics in humans.
In the current study are presented some initial results for detection of the
autofluorescence signals of the pig and chicken skin in vitro excited in the
region 360-400 nm, and for comparison healthy human skin in vivo was also
detected. Specific features of the spectra measured are discussed and there
are proposed some of the origins of the fluorescence signals obtained. These
experiments are proposed, in order to improve the possibilities of clinical
research and diagnostics for both quality and quantity control of early changes
in human tissues.
6628-61, Poster Session
D. Geraskin, H. Boeth, RheinAhrCampus (Germany); M. Kohl-Bareis,
Univ. of Applied Sciences Koblenz (Germany)
Fluorescence study of bovine serum albumin
and Ti and Sn oxide nanoparticles interactions
Near-infrared spectroscopy (NIRS) is widely applied for applications
monitoring skeletal muscle oxygenation. However, this method is obstructed
by the subcutaneous adipose tissue thickness (ATT) which might vary between
< 1 mm to more than 12 mm. Though diffuse optical imaging can be applied
to measure ATT, the objective here is to get this measure from spectroscopic
data of a single source-detector distance. For the measurement of the optical
lipid signal we used a broad band spatially resolved system (SRS), which is
based on measurements of the wavelength dependence of the attenuation
A for source detector distances r between 29 mm and 39 mm. Ultrasound
images served as an anatomical reference of the lipid layer. The measurements
were taken on 5 different muscle groups of 20 healthy volunteers, each for
left and right limbs, e.g. vastus medialis, vastus lateralis and gastrocnemius
muscle on the leg and ventral forearm muscles and biceps brachii muscle on
the arm. Different analysis strategies were tested for the best calculation of
ATT. There is a good non-linear correlation between optical lipid signal and
ultrasound data, with an overall error in ATT prediction of about 0.5 mm. This
finding is supported experimentally by additional MRI measurements as well
as a multi-layer Monte Carlo (MC) model. Based on this data of the ATT
thickness, a newly developed algorithm which exploits the wavelength
dependence of the slope in attenuation with respect to source-detector
distance and MC simulation for these parameters as a function of absorption
and scattering coefficients delivers a considerably better fit of reflectance
spectra when fitting haemoglobin concentrations. Implications for the
monitoring of muscle oxygen saturation are discussed.
D. M. Togashi, D. McMahon, P. Dunne, J. McManus, A. G. Ryder,
National Univ. of Ireland/Galway (Ireland)
6628-59, Poster Session
Phosphorescence quenching in the vicinity of
gold nanoparticles
M. Ringler, T. Soller, Ludwig-Maximilians-Univ. München (Germany); M.
Wunderlich, Y. Markert, H. Josel, A. Nichtl, K. Kürzinger, Roche
Diagnostics GmbH (Germany); T. A. Klar, J. Feldmann, LudwigMaximilians-Univ. München (Germany)
Gold nanoparticles alter the radiative and nonradiative decay rates of nearby
dye molecules, resulting either in a decreased or increased luminescence
intensity. While the effects of gold nanoparticles on surrounding fluorophores
have been investigated thoroughly, there are no corresponding studies dealing
with the influence of gold nanoparticles on the luminescent properties of
phosphors. Especially for applications in biosensing, phosphors are
particularly suitable, as they allow to cut off autofluorescence.
We have investigated the influence of gold nanoparticles on the radiative
and nonradiative decay rates of two different phosphorescent dyes. The
phosphors are attached to the nanoparticles via a biomolecular recognition
reaction. Time-resolved luminescence spectroscopy reveals an increase of
the radiative as well as the nonradiative rate in all regarded phosphor/gold
nanoparticle hybrid systems. The increase in the radiative rate is outweighed
by the more prominent enhancement in the nonradiatve rate, thus a
luminescence quenching occurs.
European Conferences on Biomedical Optics 2007 •
Nanochemistry offers stimulating opportunities for a wide variety of
applications in the biosciences [1]. Understanding of the interaction of
nanoparticles with biomolecules such as proteins is very important as it can
help better design and fabricate nanocomposites for applications in
diagnostics, drug delivery, and cell imaging monitoring. In this work, the
interaction of Bovine Serum Albumin (BSA) and two types of metal oxide
nanoparticles (titanium and tin) have been studied using the intrinsic
fluorescence of tryptophan residue from the proteins measured by steady
state and time resolved fluorescence techniques. The nanoparticles which
were fabricated using a novel synthetic process have average sizes of ~2 nm
(SnO2) and ~6 nm (estimated for TiO2) and have very high solubilities in a
variety of solvents. The Stern-Volmer plots indicate an effective quenching
process by TiO2 nanoparticles whereas SnO2 nanoparticles have a lower
quenching efficiency for BSA fluorescence. Static quenching is the major
contribution in the overall process which may indicate a high degree of
association between protein and nanoparticles. The difference in BSA
fluorescence quenching efficiency between the two types of nanoparticles
can be explained by the hydrophobicity differences and the thermal stability
of protein-nanoparticle associated species for both materials. References:
[1] - Integrated Nanoparticle-Biomolecule Hybrid Systems: Synthesis,
Properties, and Applications. E. Katz and I. Wilner. Angew. Chem. Int. Ed.
2004, 43, 6042-6108 Acknowledgements: This work was supported by
Science Foundation Ireland under Grant number (02/IN.1/M231).
6628-62, Poster Session
Depth retrieval of a fluorescent inclusion inside
a tissue-simulating phantom using picosecond
time-resolved imaging
R. Bourayou, T. Betz, Charité-Univ. Medicine Berlin (Germany); J. Voigt,
J. Berger, Physikalisch-Technische Bundesanstalt (Germany); J. M.
Steinbrink, Charité-Univ. Medizin Berlin (Germany); R. Macdonald, B.
Ebert, Physikalisch-Technische Bundesanstalt (Germany)
Continuous-wave planar fluorescence imaging allows the detection of small
quantities of fluorophore inside a rodent with high sensitivity and specificity
[Massoud & Ghambir, Genes Dev., 2003]. Still the technique suffers from
limitations regarding quantification and depth retrieval of the source of the
signal, due to the turbidity of the tissue [Klohs et al., Mol. Imaging, 2006].
Time domain measurements provide supplemental information which could
prove useful in depth ranging of a fluorescent inclusion [Liebert et al., NIMG,
2005]. Here we propose two ways of analyzing the distribution of arrival times
of fluorescence photons. These methods are tested on tissue-simulating
phantoms.
A thin cylindrical fluorescent phantom is spanned horizontally at different
depths inside a semi-infinite absorbing turbid liquid also having tissue-like
optical properties. Epi-illumination short pulses are delivered by a ps diode
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Conference 6628: Diagnostic Optical Spectroscopy
laser (? = 758 nm) through a fiber. The fluorescence photons, detected in
reflection geometry, are separated from the excitation photons by a long
pass filter (LP 780nm) and two interference filters (IF Courion 800 nm) before
collection by a time-gated intensified CCD Camera.
From a series of time-resolved fluorescence images, we retrieved the map of
the first moment of the time of flight distribution. The presence of a minimum
in the map is used to locate the fluorescent inclusion in the liquid tissuesimulating semi-infinite phantom, in a better extent as CW measurements
would do. Furthermore, a linear correlation between the depth of the
fluorescence inclusion and the first moment of the above mentioned
distribution was found for depths reaching up to 20 mm.
6628-63, Poster Session
Clinical and pathophysiological aspects of
hyperglycemia by ATR-FTIR spectroscopy
N. S. Eikje, K. Aizawa, T. Sota, Waseda Univ. (Japan)
Attempts were made to non-invasively detect and characterize in vivo
glucose-specific or glucose-related spectral signals by using ATR-FTIR
spectroscopy. Due to known experimentally proved facts that the glucose
components measured by the ATR-FT-Mid-IR are from the secretions on the
skin and glucose components within the body, we collected in vivo ATRFTIR spectra across the inner forearms of healthy and meal-intolerance
subjects, patients with diabetes mellitus. Infrared ATR spectra were recorded
in the wavenumber region from 750 to 4000 cm-1, with closer assessment of
the region between 900 and 1300 cm-1. Despite the difficulty to directly
estimate glucose concentration from the absorbance intensity of known
glucose peaks at 1074, 1108, 1118 and 1137 cm-1 in vitro, there was an
evidence on their in vivo spectral changes due to different glucose
concentrations that were in agreement with glucose detection by portable
glucosemeter in different subject groups. Preliminary results showed that it
was possible to correlate spectral information with the spectroscopic
properties of the glucose molecules in the region 900-1300 cm-1 either
randomly, during an OGTT or a meal-tolerance test, both at individual change
and at time-dependent intervals. Moreover, based on appearance or absence
of glucose-related peak-signals at around 1108, 1118 and 1137 cm-1 we
could clearly observe 2 spectral patterns, for healthy and diabetes subjects
independently on tolerance tests performed. We assume that the latter may
indeed be a sensitive indicator of moderate hyperglycemia and/or of early
glucose intolerance, with further approaches to study pathophysiological and
clinical aspects of diabetes mellitus by IR spectroscopy.
6628-64, Poster Session
Detection of normal and stomach cancer tissue
using auto-fluorescence and Raman
spectroscopy
Y. Yu, Shenyang Ligong Univ. (China); X. Li, Shenyang Ligong Univ.
(China) and Dalian Univ. of Technology (China); D. Wang, Shenyang
Ligong Univ. (China)
Laser-induced fluorescence(LIF) and Raman spectroscopy of serum for
diagnostic stomach cancer was investigated in this paper. The serum sample
is separated in segregator from people’s vein blood and kept in refrigerator
(temperature 4?). Auto-fluorescence and Raman spectroscopy of laser
induced (514.5nm and 488.0nm) was measured by an Ar-ion laser and other
auxiliary instruments to distinguish normal and gastric cancer people. From
the spectroscopy data got by a seris of experiments we found that there
were notable differences between normal and cancer. Three Raman peaks
were consistently observed from normal serum emission using 488.0nm and
514.5nm excitation while there were only two in the spectrocopy of cancer
and the intensity were slighter than the normal’s cases. In order to know the
changes further, three parameters a?ß???were induced. Average value of
diagnosis parameter for normal serum, red shift of fluorescence peak(??) is
less than 12nm, a <1 and ß <0.1 while for the gastric cancer its red shift of
average is bigger than 12nm and the value of a and ß are both oppsite to the
normal. We considered that the parameter ß to be a major way and the
parameter ?? and a an auxiliary way as a criterion and got an accuracy of
85.6% for diagnosis of gastric cancer compared with the result of clinical
diagnosis. These results have important reference values to explore the
method of Raman and LIF for diagnosis of cancer.
cardiovascular complications, e.g. diabetes-related complications [Lutgers,
Diabetes Care; vol 29: 2654-9 (2006)]. In all our previous studies, an excitation
wavelength range from 350 to 410 nm was used to assess AF. The aim of the
present study is to investigate whether the choice of excitation wavelength
within this range plays a role in the recognition of patients with increased risk
of complications. The Excitation Emission Matrix Scanner (EEMS) was used
for this purpose.
Control subjects and groups of patients with DM type 1 and 2, with and
without complications, were included. EEMS-measurements were obtained
in 98 individuals in these five groups. AF was calculated for several peak
excitation wavelengths in the range 355 - 405 nm, by dividing the amount of
measured emission by the amount of reflected excitation.
The results show that, although skin AF gradually changes with excitation
wavelength, the ratio of AF values of the groups remains constant independent
of excitation wavelength. Furthermore, the level of significance between
groups did not change with wavelength.
We conclude that no specific excitation wavelength in the range 355 - 405
nm influences the recognition of increased cardiovascular risk. No other
fluorophores seem to be present that would dictate the use of a specific
wavelength or set of wavelengths. The results confirm the validity of a broad
excitation wavelength range, such as applied in the AGE-Reader.
6628-66, Poster Session
Spectroscopic study of demineralization and
restoration processes in dental enamel
T. N. Sokolova, E. L. Surmenko, Saratov State Technical Univ. (Russia);
V. V. Tuchin, Saratov State Univ. (Russia); A. Kishen, National Univ. of
Singapore (Singapore); Y. V. Chebotarevsky, Saratov State Technical
Univ. (Russia)
The spectroscopic study of dental enamel by LIBS (laser induced breakdown
spectroscopy), FTIR (Fourier transform infrared) and XRD (X-ray diffraction)
are represented. The changes of enamel structure and composition in process
of natural (caries) and artificial demineralization and restoration were studied.
In comparison of sound and carious enamel LIBS showed a decrease of the
content of Ca, P and change of the content of some other macro-and trace
elements (Mn, Na, Fe, Zn etc). The character of the elemental composition
variation was stipulated by the concrete disease. Analysis of FTIR and XRD
spectra of dental samples, subjected to artificial demineralization and
restoration, showed that restoration action reveals more slowly, than
demineralization. And in some cases the damage of crystals after restoration
is more significant than after demineralization.
6628-67, Poster Session
Ocular fundus diagnostics and treatment in
pseudo-transformed light with digital processing
of the image
T. N. Sokolova, Saratov State Technical Univ. (Russia); I. B. Soloveychik,
V. Y. Maximov, Saratov Regional Ophthalmologic Hospital (Russia); E. L.
Surmenko, Saratov State Technical Univ. (Russia)
The developed complex research technique of laser treatment and pre- and
postoperative control of an ocular fundus is described. It is based on selection
of various components of the spectrum from “white” light at which different
objects on the ocular fundus or in a forward piece of an eye become visible.
Smoothly adjusting separate fields of a spectrum we obtain more complete
information in comparison to a traditional ophthalmoscopy. Pots of a retina
and retinal hemorrhages become distinctly visible. The transit of contrast on
pots and their outlet on a surface of a retina is tracked. The laser coagulation
points are sharply highlighted, their regional luminescence is detected.
The series of panretinal laser coagulation of the ocular fundus tissues were
performed for the patient with a diabetic retinopathy. The laser coagulation
was implemented with diode laser (532 nm, «Izumrud», Alcom Medica, SPb.).
The diagnostic and control operations were made in pseudo-transformed
light by the method of pseudo-chromoophthalmoscopy. It allowed
undistinguished pathological changes to be revealed and conditions and
parameters of laser treatment of diseases of a choroid of an eye and retina to
be optimized.
6628-68, Poster Session
6628-65, Poster Session
Excitation emission matrix measurements
support use of a broad excitation range for the
determination of cardiovascular risk from skin
autofluorescence
M. Koetsier, H. L. Lutgers, T. P. Links, A. J. Smit, R. Graaff, Groningen
Univ. Medical Ctr. (Netherlands)
Skin autofluorescence (AF) measured non-invasively at the volar side of the
arm with a prototype of the AGE-Reader (DiagnOptics, The Netherlands),
has shown to be associated with and predictive of the severity of
22
European Conferences on Biomedical Optics 2007 •
Intra-operative probe for brain cancer: feasibility
study
M. Vu Thi, Univ. Paris-Sud II (France)
The present work aims a new medical probe for surgeons devoted to brain
cancers, in particular Glioblastome multiforme. Within the last years, our group
has started the development of a new intra-operative beta imaging probe.
More recently, we took an alternative approach for the same application: a
fluorescence probe. In both cases it’s the purpose to differentiate normal
from tumor brain tissue.
In a first step, we investigate endogenous tissue fluorescence with a dedicated
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Conference 6628: Diagnostic Optical Spectroscopy
epi-fluorescence set-up and by means of fiber optic probes. Relative signal
amplitude, spectral shape and fluorescence lifetime measurements are
foreseen to distinguish normal and cancer tissue. Tissue autofluorescence
are induced with a picosecond laser diode at 375 and 405 nm to analyze
fluorophores like NADH and Porphyrine. The autofluorescence spectra are
recorded in the 420-670 nm range with a low resolution spectrometer; for
lifetime measurements a fast detector (APD, PMT) is used together with a
TCSPC-carte. Intrinsic wavelength- and time-resolutions are 3 nm and 150
ps, respectively. Two different samples have been developed: slices of rat
brain carrying implemented glioma cells and human glioma cell cultures will
be described. Our new detection system has been validated and a first
configuration of our fluorescence probe is described. First results from the
tissue measurements are shown.
6628-69, Poster Session
Pancreatic tissue assessment using
fluorescence and reflectance spectroscopy
M. Chandra, Univ. of Michigan (USA); D. Heidt, D. Simeone, B.
McKenna, J. Scheiman, Univ. of Michigan Medical School (USA); M.
Mycek, Univ. of Michigan (USA)
In this study fluorescence and reflectance spectroscopy were used for the
first time to study freshly excised human pancreatic tumor tissue and human
pancreatic cancer xenografts in nude mice. The measured spectral features
could be associated with expected molecular fluorophores (NAD(P)H and
collagen) and optical scattering and absorption properties. A good
correspondence was seen between the spectra from human adenocarcinoma
and mouse xenografts. The observed differences between the fluorescence
and reflectance properties of normal, pancreatitis and adenocarcinoma tissue
indicate a possible application of multi-modal optical spectroscopy to
differentiating between diseased and normal pancreatic tissue states.
6628-70, Poster Session
Reconstruction of stratum corneum profile of
porcine ear skin after tape stripping using UV/
VIS spectroscopy
A. P. Popov, Univ. of Oulu (Finland); J. Lademann, Humboldt Univ zu
Berlin (Germany); A. V. Priezzhev, M.V. Lomonosov Moscow State Univ.
(Russia); R. A. Myllylä, Univ. of Oulu (Finland)
Tape stripping is a minimally invasive method to reveal in-depth penetration
profiles of substances topically applied onto the surface of skin. Porcine skin
is often used for such studies before application in vivo on humans. In this
paper, we present results of the experiments with porcine skin in vitro (ears
of freshly slaughtered pigs) and compare them with those carried out on
humans in vivo (flexor forearm).
Tape stripping technique introduces consecutive removal of micrometer-thick
cell layers of the stratum corneum (SC) from the same treated skin area using
an adhesive tape. Prerequisite to the substance penetration profile is the
reconstruction of the removed SC by analyzing the amount of corneocytes
(cells of SC) stuck to each tape strip.
Such an analysis is carried out spectroscopically by measuring light
transmittance in UV and visible spectral regions (300-1050 nm) through the
adhesive tape with corneocytes. As we proved experimentally, there is a
linear dependence between the pseudoabsorption (equals to logarithm of
inverse transmittance) and thickness of the corneocytes on tape strips for all
wavelength of the investigated region. Dependence of the cumulative
absorbance of the removed SC on tape strip number can be satisfactory
fitted by exponential function. This relationship allows evaluation of the share
of the removed SC (in %) without complete removal of this layer. All the
obtained results correlate well with those obtained on humans.
6628-01, Session 1
A robust spectral sensor for point-of-care
diagnostics
S. Schönfelder, H. S. Bartos, R. Peters, Boehringer Ingelheim
microParts GmbH (Germany)
Medical diagnostic instrumentation using spectral fingerprint information
enhances sensitivity and specificity. These applications no longer require
expensive analytical instrumentation but dedicated miniaturized sensors. A
microspectrometer as a monolithical spectral sensor made by micro injection
molding has proven sufficient accuracy and robustness in medical diagnostic
devices.
The Microspectrometer is a planar waveguide spectrometer. All optical
elements - entrance slit, self-focusing grating, mirror, light traps - are realized
in a single molded part. The monolithic design allows a reliable and durable
calibration during production and therefore no recalibration in the application
is needed. The high degree of integration ensures a remarkable reduction of
weight and overall dimensions of the final product. This design in combination
with selected materials guarantees for an excellent stability and insensitivity
European Conferences on Biomedical Optics 2007 •
against environmental effects such as to mechanical shock and vibration or
large temperature shifts of more than 100 K.
The fabrication is based on micro injection molding, physical vapor deposition
(PVD) and active optical assembly processes. This guarantees for an excellent
inter-instrument agreement and thus a transferable calibration in the final
application of the devices. The combination of the optical set up with state
of the art Silicon- and InGaAs detector technology ensures good sensitivity
and signal-to-noise performance in the UV/VIS and in the NIR wavelength
range.
The presentation will introduce the Microspectrometer in different diagnostic
concepts like non-invasive measurements, reagent-less in-vitro
measurements, fluorescence measurements and classic in vitro absorbance
measurements. Common performance criteria will be demonstrated with
typical applications like hand-held Point-of-Care instruments, single- and
multi-channel sensors. The specific detection principles of these examples,
optical sampling configurations, dynamic ranges and detection limits are
presented.
6628-02, Session 1
Spectroscopic imaging using acousto-optic
tuneable filters
M. Bouhifd, M. P. Whelan, European Commission (Italy)
We report on a novel hyper-spectral fluorescence imaging filter-module based
on an acousto-optic tuneable filter (AOTF). The AOTF functions as a full-field
tuneable bandpass filter which offers fast continuous or random access tuning
with high filtering efficiency. Due to the diffractive nature of the device, the
unfiltered zero-order and the filtered first-order images are geometrically
separated. The module developed exploits this feature to simultaneously
route both the transmitted white-light image and the filtered fluorescence
image to two separate cameras. Incorporation of prisms in the optical paths
and careful design of the relay optics in the filter module has overcome a
number of aberrations inherent to imaging through AOTFs, leading to excellent
spatial resolution. A number of practical uses of this technique, both for in
vivo auto-fluorescence endoscopy [1] and in vitro fluorescence microscopy
[2] were reported. We will describe the operational principle and design of
two recently improved prototype instruments for fluorescence-based
diagnostics and will demonstrate their performance by presenting challenging
hyper-spectral fluorescence imaging applications.
[1] Mounir Bouhifd, Maurice P Whelan, Marc Aprahamian “Fluorescence
imaging spectroscopy utilising acousto-optic tuneable filters” Proc. SPIE Int.
Soc. Opt. Eng. 5826, 185-193 (2005)
[2] Mounir Bouhifd, Maurice P. Whelan, Marc Aprahamian “Use of acoustooptic tuneable filters for imaging fluorescence spectroscopy applications in
vivo and in vitro” Proc. SPIE Int. Soc. Opt. Eng. 5692, 11-20 (2005)
6628-03, Session 1
Human maxillary sinus monitoring using tunable
diode laser spectroscopy
L. Persson, M. Andersson, T. Svensson, M. Cassel-Engquist, K.
Svanberg, S. Svanberg, Lund Univ. (Sweden)
We demonstrate a novel non-intrusive technique based on tunable diode
laser absorption spectroscopy to investigate the human maxillary sinuses in
vivo. The technique relies on the fact that free gases have much sharper
absorption features (typical a few GHz) than the surrounding tissue. Molecular
oxygen was detected at 760 nm on 10 volunteers of which one had constantly
recurring sinus problems. The light was launched fiber-optically out into the
mouth and the multiply scattered light was detected externally by a handheld
probe. Expected measured levels were simulated by the Monte Carlo concept
and implemented in the Advanced Systems Analysis Program (ASAPTM)
software. A good agreement between the experiments and the simulations
was obtained. A significant oxygen absorption imprint difference could be
observed between volunteers with widely different anamnesis regarding
maxillary sinus status. Control measurements through the hand and through
the cheek below the cheekbone were also performed to investigate any
possible oxygen offset in the setup. These provided a consistently nondetectable signal level. Gas exchange between the maxillary sinuses and
the nasal cavity was also successfully demonstrated by using inhaled nitrogen
as a displacement gas. The results suggest that a clinical trial together with
an ear-nose-throat (ENT) clinic should be carried out to investigate the clinical
use of the new technique.
6628-04, Session 1
Spatially-resolved in-vivo measurement system
for estimating the optical properties of tissue in
the wavelength range 1000-1700nm
P. Hjalmarsson, S. N. Thennadil, Newcastle Univ. (United Kingdom)
For non-invasive estimation of optical properties (i.e. determination of the
absorption and the reduced scattering coefficients) of turbid media such as
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Conference 6628: Diagnostic Optical Spectroscopy
tissue, spatially resolved diffuse reflectance spectroscopy is one of most
used technique. So far this has only been done for wavelengths covered by
CCD-detectors (about 350-1050nm). The NIR region beyond 1050nm i.e.
the second and first overtone regions, has absorption peaks of interest e.g.
for tissue the glucose peak at around 1250nm and 1600nm. Thus for noninvasive medical diagnostics applications, a spatially resolved measurement
system capable of estimating optical properties in this region will be very
useful. Until now optical properties of tissue in this region have only been
estimated using in vitro methods e.g. using an integrating sphere set-up.
In this paper we describe a spatially resolved system that will extend the
region up to 1700nm by using a TE cooled 320x256 pixel InGaAS detector, a
white light source and a probe that consists of 9x200micron fibres spanning
0.3 to 2.7mm from the source. Across the 320 pixels 680nm will be dispersed
giving a resolution of 2.125nm/pixel and a resolving power of about 14nm.
The system is validated using tissue-like phantoms. Since tissue has a high
concentration of water which leads to high absorption after 950nm, the diffuse
approximation cannot be used to extract the optical properties from the
spatially resolved measurements. Instead, different techniques, based around
Monte Carlo simulations of diffuse reflectance profiles, have been used to
calculate the optical properties for different wavelengths. The performance
of these techniques are compared and extensions and modifications to
improve their performance are discussed.
6628-05, Session 2
Monitoring cellular metabolic pathways by
wavelength- and time-resolved intracellular
autofluorescence
Y. Wu, W. Zheng, J. Y. Qu, Hong Kong Univ. of Science and Technology
(Hong Kong China)
Most human cancers originate in epithelium. The elevated metabolic rate in
cancer cells provides a contrast mechanism for the diagnosis of early cancer
lesions. In the past two decades, autofluorescence spectroscopy has been
widely explored as a noninvasive technique to detect the precancerous
development in epithelial tissue. The epithelial fluorescence is mainly
determined by the intrinsic fluorophores of the reduced form of nicotinamide
adenine dinucleotide (NADH) and oxidized form of flavin adenine dinucleotide
(FAD). It is well known that NADH and FAD are metabolic cofactors that act
as electron donors and acceptors in the metabolic pathways including
glycolysis, pyruvate decarboxylation, citric acid cycle and oxidative
phosphorylation. Thus, the reduction-oxidation (redox) state of the cells can
be sensed by the ratio of NADH fluorescence over FAD fluorescence. In
addition, NADH and FAD bind with a variety of proteins (enzymes) in the
reactions of metabolic pathways. Consequently, the ratio of free NADH over
protein-bound NADH can also produce the information on cell metabolic
state. In this study, we investigated the methods to monitor cellular
metabolism based on the ratio of NADH over FAD fluorescence and the ratio
of free NADH over protein-bound NADH fluorescence, respectively. The
signals of free NADH, protein-bound NADH and FAD were isolated from the
intracellular autofluorescence using wavelength- and time-resolved
fluorescence spectroscopy.
We instrumented a time-resolved confocal fluorescence spectroscopy system
utilizing the multi-channel time-correlated single photon counting (TCSPC)
technique. The excitation source was the second harmonic generation of a
femtosecond Ti:sapphire laser covering the wavelength range from 365-435
nm. The samples used in the measurements were the monolayered cell
cultures. The cell lines were established from the normal and cancer
ectocervical cells. The cell metabolism was manipulated by using the
mitochondrial inhibitor (NaCN) and uncoupler (CCCP). It was found that NADH
and FAD fluorescence measured from the cell cultures reached to a balanced
level with the excitation wavelength at 405nm. The fluorescence in the
wavelength bands from 440 - 480 nm and 530 - 570 nm were dominated by
the NADH and FAD signals, respectively. The ratio of the fluorescence in the
bands could be used for quick and accurate sensing of redox ratio [1]. With
the excitation wavelength at 365 nm, the intracellular autofluorescence was
dominated by NADH signal. The fluorescence time decay could be well
described by a dual-exponential function, consisting of a short-lifetime
component (?1~ 0.40 - 0.47 ns) and a long-lifetime component (?2 ~ 3.3 4.0 ns). Spectral analysis of the spectra associated with the short- and longlifetime components revealed that the long-lifetime component carried the
information of protein-bound NADH and short-lifetime component was mainly
determined by free NADH with certain interference from bound NADH. The
ratio of the amplitudes of two lifetime components was found to be a sensitive
indicator of the cellular metabolism [2]. However, the sensitivity decreased
with the increasing of excitation wavelength from 365 to 405nm, possibly
due to the interference from free/bound FAD fluorescence. Overall, the results
demonstrated that the wavelength- and time-resolved intracellular
autofluorescence can be used to monitor the cellular metabolic pathways
and differentiate the normal cells from the cancer cells.
24
European Conferences on Biomedical Optics 2007 •
6628-06, Session 2
Spectral and time-resolved studies on ocular
structures
D. Schweitzer, Friedrich-Schiller-Univ. Jena (Germany); S. Jentsch,
Fachhochschule Jena (Germany); S. Schenke, C. U. Biskup, FriedrichSchiller-Univ. Jena (Germany); E. R. Gaillard, Northern Illinois Univ.
(USA); M. Hammer, Friedrich-Schiller-Univ. Jena (Germany)
Several ocular diseases are caused by alterations in metabolic pathways.
There is a possibility for studying metabolic processes by the autofluorescence of endogenous fluorophores. Such fluorophores are e.g. the
redox pairs of co-enzymes NADH-NAD and FADH2-FAD which change the
fluorescence properties depending on oxygen concentration. An other
fluorophor is the ageing pigment lipofuscin, which accumulates in retinal
pigment epithelium in age - related macular degeneration. In sclerotic
processes and also in glaucoma, components of connective tissue collagen
and elastin are detectable. Tryptophan and kynurenin are of interest in cataract
formation. PP IX is an endogenous fluorophor in hem synthesis. Advanced
glycation end-products accumulate in Diabetes.
In 10 porcine eyes, the cornea, chamber water, lens, vitreous, neural retina,
pigment epithelium, choroid, and sclera were separated and the absorption,
excitation, and emission spectra were measured. The fluorescence lifetime
was determined using a home-build lifetime-laser-scanner-ophthalmoscope.
The spectra of ocular tissues were compared with spectra of pure
fluorophores, expected in tissue. An optical fundus section of a human donor
was investigated. Furthermore, a set up will be explained for detection of
fluorescence spectra of the living human fundus avoiding the lens
fluorescence.
Autofluorescence is detectable from all ocular structures. A discrimination of
ocular structures is quite difficult according to the spectra of fluorescence.
The lifetime of auto-fluorescence allows separation between connective
tissues and neural retina as well as retinal pigment epithelium. Fluorescence
spectra of porcine and human eyes are comparable only to a certain degree.
The human macular fluorescence spectrum is dominated by lipofuscin.
6628-07, Session 2
Multi-spectral FLIM of tissue autofluorescence
W. Becker, V. Katsoulidou, A. Bergmann, Becker & Hickl GmbH
(Germany)
We present a fluorescence lifetime imaging technique with simultaneous
spectral and temporal resolution. The method is based on a multi-dimensional
TCSPC (time-correlated single photon counting) technique. The sample is
scanned with a high-repetition-rate laser beam. The fluorescence light is
detected through a descanned confocal beam path. A grating spreads the
light into a spectrum. The spectrum is projected on the cathode of a 16channel multi-anode PMT. For every detected photon, the electronics
determines the time of the photon in the pulse period of the excitation laser,
the PMT channel number, and the position of the laser beam in the scanning
area. The recording process builds up a four-dimensional photon distribution
over these parameters. The technique delivers a near-ideal counting efficiency
and a time-resolution essentially limited by the transit time-spread in the
PMT. We demonstrate the performance of the technique for autofluorescence
imaging of tissue.
6628-08, Session 2
Multiphoton imaging and fluorescence lifetime
studies on unstained cells and tissue at
cryogenic conditions
M. Stark, D. Dörr, A. Ehlers, D. Sauer, Fraunhofer-Institut für
Biomedizinische Technik (Germany); R. Bückle, S. Martin, F. Ehrhart, J.
Baunach, A. Katsen-Globa, H. Zimmermann, JenLab GmbH (Germany);
K. König, Fraunhofer-Institut für Biomedizinische Technik (Germany)
Fluorescence lifetime relates to characteristic material properties on the
molecular level. The time-resolved information is not only relevant in
compositional analysis but provides additional control e.g. in drug delivery.
However, statistical significant analysis is hampered by the lack of dedicated
analysis procedures.
Current analysis of Fluorescence Lifetime Imaging Microscopy (FLIM) data
relies on model-based (multi-)exponential fitting of fluorescence decay curves.
As exponential fitting is rather sensitive to noise, the quality of such an analysis
strongly depends on a-priori knowledge and experience of the user.
Here we propose a methodology based on classification schemes. Classes
of similar fluorescence decay curves are constructed. A suited set of
parameters describes these prototypes of fluorescence decay, among others
allowing identification of second harmonic generation, noise dominated
regions and irregular/non-exponential decay. Additionally, the data is related
to morphological information (e.g. prominent features on cells and tissue),
as well parameterized. Both, the classified fluorescence decay as well as the
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Conference 6628: Diagnostic Optical Spectroscopy
morphological data are not specific for one scene only (i.e. for one particular
image), but parameterize the image content in a generalized manner. Data
gathered on several images of the same object can be merged into one
common ensemble to obtain a significant and automated sample
characterization.
To highlight performance and to discuss limitations, we present the analysis
of FLIM data obtained on images of plant and human tissue.
The first generation of fluorescence bronchoscopes was based on regular
optics (rigid or fiber-based), equipped with a filtered external 3 CCD color
camera that allow autofluorescence detection as well as conventional white
light bronchoscopy. The newly developed generation integrates a chip-ontip technology, enabling to perform fluorescence videobronchoscopy. A
clinical study is currently performed and preliminary results will be presented
in this report.
6628-09, Session 2
6628-13, Session 3
Intrinsic optical signals of brains in rats during
loss of tissue viability: effect of brain
temperature
Multiple fluorophore-analysis (MFA) for
qualitative tissue diagnosis in the oral cavity
S. Kawauchi, S. Sato, H. Ooigawa, H. Nawashiro, M. Kikuchi, National
Defense Medical College (Japan)
To investigate the changes in intrinsic optical signals (IOSs) during loss of
tissue viability for brains, we performed simultaneous measurement of light
absorption due to the redox states of cytochrome c oxidase and light
scattering, which reflects morphological characteristics of cells in the tissue,
for rat brains after blood removal by saline infusion. To measure IOSs, we
first examined an isosbestic wavelength of the redox states of cytochtome c
oxidase for each rat. We then measured diffuse reflectance intensity at the
isosbestic wavelength as a scattering signal, while diffuse reflectance intensity
at 800 nm was detected to monitor the reduction of CuA in cytochrome c
oxidase. When infusion was performed with saline at 30°C, the scattering
signal showed a drastic, triphasic change at 100 s after starting infusion;
during this scattering change, the reduction of CuA started and proceeded
rapidly. The start time of triphasic scattering change as well as the start time
of the reduction of CuA was extended for more than 2 min by lowering infusion
temperature from 30 to 23°C and we found that there was a linear correlation
between these two start times. These results suggest that tissue metabolic
activity can be maintained for longer time by keeping the brain at lower
temperature, and triphasic scattering change can be used as an optical signal
indicating the reduction of CuA in cytochrome c oxidase, and hence the loss
of tissue viability for brain.
6628-10, Session 2
Sensing metabolic activity in tissue engineered
constructs
M. Chandra, R. H. Wilson, W. Lo, K. Vishwanath, K. Izumi, S. Feinberg,
M. Mycek, Univ. of Michigan (USA)
Simulations and experiments were executed to optimize non-invasive optical
sensing of metabolic activity in tissue engineered oral mucosa constructs.
Fluorescence measurements (at 355nm excitation) and Monte Carlo
simulations (at 355nm and 450nm excitation) were employed to design fiber
probe and excitation wavelengths that would potentially increase the ability
of fluorescence spectroscopy to sense cellular changes in the constructs.
6628-12, Session 3
Detection of early bronchial cancer by
autofluorescence bronchoscopy: from
spectroscopic studies to videoendoscopy
B. Lovisa, T. Gabrecht, École Polytechnique Fédérale de Lausanne
(Switzerland); B. Weber, Richard Wolf GmbH (Germany); H. van den
Bergh, G. A. Wagnieres, École Polytechnique Fédérale de Lausanne
(Switzerland)
Autofluorescence (AF) bronchoscopy, which can still be improved, in particular
in terms of specificity, has been shown to be a highly sensitive tool for the
detection of early bronchial cancers. Several endoscopic imaging systems
exploit the spectral and intensity contrast of AF between healthy and (pre)neoplastic bronchial tissues.
Over the past years, this technology has been developed in our group from
fundamental spectroscopic studies to clinical imaging applications. The initial
spectroscopic measurements were performed with a spectrally and intensity
calibrated optical fiber-based spectrofluorometer. We observed a range of
excitation wavelengths going from 350 nm to 480 nm with a specially designed
probe to avoid spectral distortion observed with “point” measurement
systems. This study showed a significant decrease in fluorescence intensity
in (pre-)neoplastic lesions versus normal tissue especially in the green region
of the emission spectrum, whereas the red region remains more or less
constant. Additionally, the best contrast was achieved with excitation around
410 nm. Following these observations, a new fluorescence imaging apparatus
was designed to detect precancerous lesions in the tracheo-bronchial tree.
This device was optimized regarding excitation wavelengths, backscattered
light (blue and red) and autofluorescence spectral domains, and implemented
as a commercial device. The light source was a filtered Xenon lamp, producing
light with specific intensities in selected parts (blue-violet, red) of the spectrum.
European Conferences on Biomedical Optics 2007 •
R. Pauli, C. Betz, M. Havel, R. Sroka, H. G. Stepp, A. Leunig, LudwigMaximilians-Univ. München (Germany)
Introduction: Diagnosis of tumours in the oral cavity is usually achieved via
surgical tissue biopsy. The rate of early stage tumors discovered this way is
disappointing. A non-invasive optical method might contribute to earlier stage
detection and enhance therapeutic outcome.
Materials & Methods: Multi-spectral analysis (MFA) was performed on a total
of 19 patients with suspicious lesions of the oral cavity and 7 healthy
volunteers. Using a mercury vapour lamp as a light source and a spectrometer
as detector, excitation and detection of endogenous fluorophores (tryptophan,
NADH, FAD) was achieved using corresponding filter sets (for excitation:
296 nm, 350 nm, 465 nm) in an automated system. As applicator, a branched
fiberoptic bundle was used. By including simultaneously recorded white light
remission spectra into the analysis, it was possible to calculate “intrinsic”
fluorescence spectra. Subsequently, the histopathological results of the
lesions were compared to the spectroscopic findings.
Results: Significant differences between (pre-)malignant and normal oral
mucosa were obtained for the evaluated spectra at 350 nm and 465 nm
excitation, but not for 296 nm excitation. The mucosa of the healthy volunteers
showed a similar spectral pattern as the non-cancerous control areas in
tumour patients. The calculation of “intrinsic” spectra considerably improved
the significance level.
Conclusions: With regards to the results in this pilot study, MFA might serve
as a helpful tool in early diagnosis of cancer in the oral cavity. It seems
suggestive to combine this method with suitable screening techniques such
as autofluorescence imaging.
6628-14, Session 3
Reflectance spectrophotometry as
intraoperative assessment of microperfusion in
esophageal anastomosis: a feasibility study
A. Karliczek, Groningen Univ. Medical Ctr. (Netherlands) and Martini
Hospital (Netherlands); D. A. Benaron, Spectros Corp. (USA); P. Baas, A.
van der Stoel, Martini Hospital (Netherlands); T. Wiggers, J. Plukker, G.
M. van Dam, Groningen Univ. Medical Ctr. (Netherlands)
Following esophageal resection, the most important complication and cause
of death is anastomotic leakage. Ischemia might be an important underlying
cause of anastomotic leakage, as the esophagus is reconstructed with a
conduit constructed from the stomach, removed from its anatomical position.
Furthermore the circulation is compromised by resection of the affected part
of the esophagus, and as such, the vascular supply from the upper stump of
the esophagus is compromised in cervical anastomoses. In this study the
technical feasibility of a intraoperative device, developed to measure tissue
microperfusion by reflectance spectrophotometry (T-Stat(r) Ischemia Detection
system), also known as Visible Light reflectance Spectrometry (VLS), is
assessed.
Ten patients who underwent esophageal resections for malignancies were
included. Feasibility was defined as the number of planned measurements
performed, stability of the assessments (expressed as descriptive statistics)
and number of adverse events caused by the VLS-system or the protocol.
The measurements were recorded at 71,2% of the predefined time-points
during a surgical procedure described in the protocol. The standard deviation
of the saturated hemoglobin value (Sto2) were less variable in serosal
recordings (3,6-10,5%). Two patients showed anastomotic leakage in the
postoperative course. In these patients a saturation drop of 18,3% in the
gastric conduit was measured compared to 1,7% in non-anastomotic leakage.
CONCLUSION: Reflectance spectrometry measurements are easy and safe
to perform during esophageal resections and show a small variability in
measurements. In this feasibility study a decrease in gastric serosal saturation
was demonstrated in patients with an anastomotic leakage. Further
multicenter studies are in progress to validate the value of VLS and StO2 as
a predictor of anastomotic leakage.
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Conference 6628: Diagnostic Optical Spectroscopy
6628-15, Session 3
6628-17, Session 3
FTIR biochemical imaging of the prostate: an in
vitro proof of concept study
Variation of skin autofluorescence with age and
gender in humans
M. Isabelle, Gloucestershire Hospitals NHS Foundation Trust (United
Kingdom); J. J. Aning, Gloucestershire Royal Hospital (United Kingdom); H. W. Gilbert, A. W. S. Ritchie, Gloucestershire Hospitals NHS
Foundation Trust (United Kingdom); N. Stone, Gloucestershire Royal
Hospital (United Kingdom)
R. Graaff, H. L. Lutgers, A. M. Van Roon, M. Koetsier, T. P. Links,
Groningen Univ. Medical Ctr. (Netherlands); H. J. G. Bilo, Isala Clinics,
Zwolle (Netherlands); A. J. Smit, Groningen Univ. Medical Ctr. (Netherlands)
Introduction
Prostate cancer is a biologically heterogeneous disease with considerable
variation in clinical aggressiveness. Conventional histological analysis is
subjective and gives limited predictive information regarding prostate cancer
progression. Fourier Transform Infrared (FTIR) microspectroscopy is a powerful
bioanalytical technique that uses infrared light to interrogate biological tissues.
The aim of this study was to evaluate the potential of FTIR to biochemically
map whole transverse prostate sections, to correlate the morphology with
the biochemistry, to establish a diagnostic algorithm and to assess
reproducibility of the technique whilst evaluating the effect of tissue
preparation on the FTIR biochemical analysis.
Methods
Selected 10 micron, wax embedded transverse prostate sections, from 5
prospectively consented patients who underwent radical nerve sparing
prostatectomy were analyzed in their entirety, using FTIR. The FTIR microspectrometer (Perkin Elmer Spectrum Spotlight System (r)) analyzed the
sections in transmission mode. Spectral maps were constructed and
multivariate analysis applied to the spectral dataset. Cluster analysis of the
spectra was also performed. This was used to create a false colour image
map. The resultant maps were directly correlated with the pathological
interpretation of the same sections after hematoxylin and eosin staining.
Results
Over 100,000 spectra were obtained from each section. FTIR imaging was
able to reproducibly discriminate between prostate cancer, benign prostatic
hyperplasia (BPH), prostatic calculi, prostatic intraepithelial neoplasia (PIN)
and the embedding medium with sensitivities and specificities of greater
than 90%. Detailed biochemical information about the composition of the
aforementioned pathologies was elicited and could be demonstrated visually
using cluster analysis.
Conclusion
FTIR is a useful technology which has demonstrated significant promise as a
future classification tool for prostate cancer. The technique’s greatest strength
is the extra objective biochemical information that it is capable of delivering.
This may with further work enable the prediction of prostate cancer biological
activity based on biochemical markers, therefore making a patient specific
treatment approach possible. Continuing work in refining the spectral model
is in progress and further results will be presented.
6628-16, Session 3
Cardiac tissue characterization via optical
spectroscopy techniques
B. Lin, D. L. Matthews, Univ. of California/Davis (USA); S. G. Demos,
Lawrence Livermore National Lab. (USA)
Cardiac tissue lesions may result from disease or therapeutic intervention
such as RF ablation to treat atrial fibrillation. Current methods for tissue
evaluation and treatment include the utilization of various catheter types
capable of reaching different heart compartments. Determining tissue
characteristics by incorporating fiber optic technology into these catheters
may provide a method for physiological, anatomical, and biochemical
evaluation of cardiac disease, or monitor catheter-based therapeutic
procedures in real time. The focus of this work is to understand how NIR
light propagates through cardiac tissue using imaging or point measurement
approaches towards developing methods to assess tissue or lesion depth.
Specifically, we investigate light scattering of porcine cardiac tissue as a
function of tissue thickness. We first explore the change in light scattering
intensity as a function of tissue thickness within homogeneous samples in a
polarization sensitive imaging arrangement using various NIR spectral bands.
Our results indicate an increase of scattering intensity as thickness increases
up to about 3 mm. When using a dual fiber configuration (to inject light into
the sample and collect backscattered light) we found that the spectral profiles
change as a function of thickness for up to about 4 mm, presumably due to
the change in optical properties of the tissue as a function of wavelength.
These results suggest that optical spectroscopy methods can provide the
means for tissue thickness assessment up to about 4 mm in depth. This
approach may also be applied to evaluate the thickness of a lesion located
over normal tissue.
26
European Conferences on Biomedical Optics 2007 •
Several clinical studies have shown that skin autofluorescence (AF) is a
valuable prognostic marker for cardiovascualar disease and other chronic
complications in diabetes mellitus and other diseases with increased
cardiovascular risk. Skin biopsies showed correlations with several skin AGEs
(Advanced Glycation Endproducts). Skin autofluorescence is determined by
dividing the amount of measured emission by the amount of reflected
excitation, using blacklight with peak wavelength around 370 nm. The aim of
this study is to describe the variation of AF with age and gender of normal
subjects.
AF values were collected from 199 healthy subjects of various age (16-90
years), who had been established not to have a history of diabetes,
cardiovascular events, or renal disease. Skin autofluorescence was
determined with a prototype of the AGE-Reader (DiagnOptics, The
Netherlands), by measuring at three positions at the volar side of the lower
arm.
The results were grouped per decade. Mean AF values increased with
age from 1.68 (20-30 yrs) to 2.71 (70-80 yrs). A parabolic fit showed an
improved description of age-dependency compared to a linear fit. Mean
values per decade were obtained for the male and female gender. In all but
the last decade, the mean values for women were higher than those for men.
Our finding of increased skin aging in women compared to men was also
noted by Koehler et al [Opt. Lett. 31 (2006), 2979-81], using multiphoton
laser scanning tomography. It is concluded that the value of autofluorescence
in the determination of cardiovascular risk can be improved by correcting
the results for age and gender.
6628-18, Session 3
Analysis of breast tissue calcifications using
FTIR spectroscopy
R. N. Baker, N. Shepherd, Gloucestershire Royal Hospital (United
Kingdom); K. D. Rogers, Cranfield Univ. (United Kingdom); N. Stone,
Gloucestershire Royal Hospital (United Kingdom)
Breast calcifications can be found in benign and malignant lesions and the
composition of these calcifications can indicate the possible disease state.
Calcium oxalate (dihydrate) (COD) is found to be associated with benign
lesions, whereas calcium hydroxyapatite (HAP) is found mainly in malignant
tissue. As current practices such as mammography and histopathology
examine the morphology of the specimen, they can not reliably distinguish
between the two types of calcification, which frequently are the only
mammographic features that indicate the presence of a cancerous lesion.
FTIR spectral maps were carried out on paraffinized sections of breast tissue
of different pathology types containing known calcification. The biopsies were
taken from biological archives of patients that had undergone breast biopsy
for mammographically suspicious lesions. Areas of tissue were mapped that
contained suspected calcification, using a 6.25 micrometer step size with a
resolution of 4cm-1. The calcification was detected using white light images
of the tissue section and taking point spectra of areas suspected.
The morphology and chemical composition of breast calcifications has been
analysed and correlated with tissue pathology. Calcifications appear both
chemically different (shown by differences in carbonate content) and
structurally different (from the width of the phosphate bands). As the two
types of calcifications appear to be associated with different kinds of breast
lesion, the differentiation of these calcifications by spectroscopic techniques
may have positive implications in early diagnosis if the techniques can be
applied in vivo, and spectroscopy of paraffin sections enables biochemical
information to accompany histopathology of the sample.
6628-19, Session 3
Optical spectroscopy for therapeutic guidance
in breast conserving therapy
M. D. Keller, S. K. Majumder, Vanderbilt Univ. (USA); M. C. Kelley,
Vanderbilt Univ. Medical Ctr. (USA); A. Mahadevan-Jansen, Vanderbilt
Univ. (USA)
Most women with early breast cancer have the option of breast conserving
therapy, which involves the complete removal of the primary breast lesion (a
lumpectomy) with tumor-free margins, followed by radiotherapy. Since the
presence of tumor at or near the margin is strongly correlated with the risk of
local tumor recurrence, there is a need to develop a non-invasive, real-time
tool that can differentiate normal breast tissue from tumor at the margins to
assure complete removal. Our previous studies have demonstrated the ability
of combined autofluorescence and diffuse reflectance spectroscopy to
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Conference 6628: Diagnostic Optical Spectroscopy
differentiate normal from non-normal breast tissue ex vivo with 91% sensitivity
and 93% specificity. Using a portable, combined fluorescence and reflectance
spectroscopy system, we took measurements from each of the six surfaces
of the tissue mass immediately following removal during lumpectomies for
30 patients. The measurement sites were marked by a surgical suture already
routinely used by the surgeon to delineate specimen orientation, and these
sites were investigated via punch or shave biopsies by a surgical pathologist
to determine whether the margins were positive (tumor present) or negative.
This histopathological diagnosis served as the gold standard in evaluating
the performance of our diagnostic algorithm previously developed, and these
diagnostic results will be presented. Since clear margins of 1mm or greater
are desired, we have also begun work on making these spectroscopy
measurements in a depth-resolved manner, the validation of which will also
be presented.
6628-20, Session 3
Detecting skin malignancy using elastic light
scattering spectroscopy
M. Canpolat, A. Akman, A. Ciftcioglu, E. Alpsoy, Akdeniz Univ. (Turkey)
We have used elastic light scattering spectroscopy to differentiate between
malign and benign skin lesions. The system consists of a UV spectrometer, a
single optical fiber probe and a laptop. The single optical fiber probe is
basically a 1x2 optical fiber coupler with a fiber core diameter of 100 ?m. The
single optical fiber probe was used for both delivery and detection of white
light to tissue and from the tissue. The single optical fiber probe received
singly scattered photons rather than diffused photons in tissue. Therefore
the spectra are correlated with morphological alterations of the cells. It has
been shown that spectra of malign skin lesions are different than spectra of
benign skin lesions. While slopes of the spectra taken on benign lesions or
normal skin tissues are positive, slopes of the spectra taken on malign skin
lesions tissues are negative. In vivo experiments were conducted on 20 lesions
from 18 patients (11 men with mean age of 68 9 and women mean age of 52
20) applied to the Department of Dermatology and Venerology. Before the
biopsy, spectra are taken on the lesion and adjacent (approximately 1 cm
distant) normal-appearing skin. Spectra of the normal skin are used as a
control group. The spectra are correlated to the pathology results with
sensitivity and specificity of 82% and 89%, respectively. Due to small diameter
of fiber probe and limited number of sampling (10-15), some positive cases
are missed, which is lowered the sensitivity of the system. The results are
promising and could suggest that the system may be able to detect malignant
skin lesion non-invasively and in real time.
6628-21, Session 4
New method to detect caries via fluorescence
J. Eberhart, Dürr Dental GmbH & Co. KG (Germany); M. Frentzen, Univ.
Bonn (Germany); M. Thoms, Dürr Dental GmbH & Co. KG (Germany)
and Univ. of Erlangen (Germany)
Caries - a common and widespread infectious disease - has to be detected
as early as possible. Based on the need for an easy and handy tool for
preventing invasive treatment needs we developed a new optical device.
The so-called porphyrins, catabolic products of oral pathogenic bacteria can
be visualized by the new fluorescence camera system. The fluorophores are
excitated by using GaN-diodes at a wavelength of 405 nm. The diseased
hard dental tissue is fluoresces in the red spectral range and furthermore the
healthy shows a green autofluorescence.
To prove the reliability of this fluorescence camera system, freshly extracted
teeth were examined. Three different methods of analysis were verified and
compared to give information about the lesions (sensitivity & selectivity): The
extent of the fluorescence area, the integral of the red/green ratio of the
lesion and the maximum red/green ratio in the area of interest. Histological
sections of the teeth served as reference. In addition, the camera was
compared to a tip probe sensor already available on the market.
In total, our results showed that regarding the three different algorithms of
analysis, the maximum of the red/green ratio is a very good method to evaluate
carious lesions. The sound tissue, enamel caries and dentin caries could be
clearly distinguished. This new fluorescence camera is a handy, efficient and
fast device in order to detect hidden lesions. The optical fluorescence camera
seems to be superior to the tip probe sensor. Further studies are required.
6628-22, Session 4
Polarization optical spectroscopy: the technique
for puncture diagnosis
V. A. Kamensky, N. M. Shakhova, P. D. Agrba, A. Mjakov, Institute of
Applied Physics (Russia)
We propose to realize an endoscopic all-fiber clinical device for polarized
reflectance spectroscopy based on polarization-maintaining fiber. This
promising method of extraction is based on depolarization properties of
underlying stroma. Biological tissue is illuminated by radiation of fixed
polarization, whereas the scattered radiation is received in two orthogonal
European Conferences on Biomedical Optics 2007 •
polarizations. As epithelial cells almost do not make a depolarization impact
on the incident radiation, it reaches stroma without changes in polarization,
where it is depolarized completely and returns to the receiving system. Thus,
it is possible to separate scattering from stroma and from epithelium. This
technique is interesting for medical diagnosis because the size of the probe
is determined by the size of the polarization-maintaining fiber with safety
shell (about 800 mcm), which allows one to use for endoscopic examination
endoscopes with small-size operating channels (bronchoscopy, laryngoscopy,
arthroscopy)
Results of testing the above device in model media and the first data of
clinical investigations are presented.The PRS results were analyzed and
compared with data obtained using the cross-polarisating optical coherence
tomography technique
Investigations in vivo were carried out on the uterine cervix with benign and
malignant alterations 7 female patients were examined. PRS technique
confirm a possibility of differentiating neoplastic changes by the depolarization
ratio and spectrum.
6628-23, Session 4
Combined fiber optical-thermal sensor for
noninvasive monitoring of blood and human
tissue through diffuse scattering and metabolic
parameters
V. A. Saetchnikov, E. A. Tcherniavskaia, Belarusian State Univ. (Belarus);
G. Schweiger, Ruhr Univ. Bochum (Germany)
A method of noninvasive monitoring of human tissue and blood components
based on optical diffuse scattering spectroscopy combined with metabolic
heat measurements has been developed. A compact integrated fiber optical
and thermal sensor for different applications has been developed.
The sensor pickup measures thermal generation, heat balance, blood flow
rate, hemoglobin and it’s derivative concentrations as well as environment
conditions. Temperature measurements: surface cutaneous tissue thermal
radiation, ambient room temperature and background radiation temperature
are used to measure conduction, convection, and radiation of heat from the
human body. Blood flow rate in the body is estimated by monitoring the
change in temperature between the contact and adjacent thermal detectors.
Optical measurements based on diffuse spectroscopy generate the values
for hemoglobin and it’s derivative concentrations.
The calibration and measurement are performed independently. Multivariate
statistical analysis involving the variables from sensor signals, polynomials
from various variables, regression analysis of individual patients, and cluster
analysis of patients group are performed.
Clinical testing of developed sensor for different application is being
performed. Optical signal monitored the blood pulsation and under optimal
path through the tissue - respiration rhythm. Localized reflectance data
correlate with hemoglobin and it’s derivative concentrations. Diffuse scattering
signal with thermal data can monitor the change of glucose concentration.
Further developments of the technology which are under progress now are
the following: clinical studies to further characterize the performance of this
technology and development of compact and low cost sensor device for
home diagnostics.
6628-25, Session 5
Optical pharmacokinetics measurement of
photosensitising drug concentrations for
photodynamic therapy
M. R. Austwick, J. Woodhams, C. Elliot-Laize, V. Chalau, A. J.
MacRobert, Univ. College London (United Kingdom); I. J. Bigio, Boston
Univ. (USA); S. G. Bown, Univ. College London (United Kingdom)
Measuring the concentration of a photosensitising drug (PS) non-invasively
in tissues could provide substantial benefits for photodynamic therapy (PDT).
The aim of this study was to assess the use of Elastic Scattering Spectroscopy
(ESS) for Optical Pharmacokinetics (OP) (measuring the tissue concentration
of the PS Aluminium Disulphonated Phthalocyanine (AlS2Pc) in vivo) to see
if this helped predict the extent of PDT necrosis.
AlS2Pc was given intravenously to Wistar rats (0.1-5mg/kg), 1-24 hours prior
to OP measurements in the liver, colon, skin, muscle, oral mucosa and
stomach. For comparison, AlS2Pc in these tissues was measured using
spectrofluorimetry after alkaline extraction. In a separate group of animals,
AlS2Pc PDT was also performed on the liver and colon (670nm, 50J at 100mW
via a bare cleaved 400µm fibre) where OP measurements were taken just
prior to light delivery in the oral mucosa and the target organ. These animals
were recovered and the size of PDT necrosis was measured at 3 days. OP
data was correlated with ex vivo chemical extraction and with the extent of
PDT necrosis to see if the variation in PDT effect was due to differences in
tissue drug concentration.
AlS2Pc tissue levels were assessed from the OP spectra by analysis of the
height and area under curve of the absorption peak. All OP results correlated
well with chemical extraction. Spectral analysis method was refined to
eliminate the need to take a reference measurement on unsensitised tissue.
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Conference 6628: Diagnostic Optical Spectroscopy
This project is supported by the Network for Translational Research: Optical
Imaging (NTROI).
6628-27, Session 5
Fluorescence based fast diagnostics platform
for the direct and indirect immunodiagnostic
analysis methods
R. Mannila, VTT Optical Instruments (Finland); T. Pulli, H. K. Saari, K.
Tappura, VTT Information Technology (Finland); J. Tuppurainen, I.
Vikholm-Lundin, H. Valimaki, VTT Elektroniikka (Finland); A. Niskanen,
Ani Biotech Oy (Finland)
The need to develop simple bioanalytical systems for home, sports training,
healthcare centres, and in instant tests of intoxicants by the authorities is
expected to grow considerably. FASTDIAG project tries to meet these
challenges. The objective of the patented (Ref. FI116261B) remote diagnostics
system is to be as flexible as possible so that new tests can be easily added
to it. The direct and indirect immunodiagnostic analysis methods based on
fluorescence are studied. In the FRET test, a bound analyte must not be
separated from a free one for the measurement of fluorescence, and thus
the test is quick to perform. Consequently it can replace the existing, slower
instant drug tests based on lateral flow.
The results obtained with the direct fluorescence test reader prototype will
be presented for lateral flow tests based on a colour or fluorescence detection.
The reader has a wireless Bluetooth connection. VTT and Ani Biotech Oy
(ref. www.anibiotech.fi) have developed the BIOCARD Duplo(tm) reader
product based on the prototype.
The first phase of FASTDIAG included the optimization of homogeneous FRET
assays for morphine and THC to be used as model tests for the FRET test
reader prototype. The first results obtained with the FRET reader will be
presented for the morphine and THC in saliva. The sensitivity of the current
Morphine test is clearly adequate but the THC sensitivity is on the limit and
optimization is still needed.
6628-28, Session 5
FT-infrared spectroscopic studies of lymphoma,
lymphoid, and myeloid leukemia cell lines
J. Babrah, R. Lush, A. Rye, K. McCarthy, Gloucestershire Hospitals
NHS Foundation Trust (United Kingdom); C. Bessant, Cranfield Univ.
(United Kingdom); N. Stone, Gloucestershire Hospitals NHS Foundation
Trust (United Kingdom)
This study aims to present the application of FT-IR spectroscopy as a novel
method to characterise differences that distinguish leukaemia and lymphoma
cell line types. This is based on objective spectral measurements of major
cellular biochemical constituents and mathematical-statistical procedures.
Lymphoma (Karpas), Lymphoid (REH and ACV) and Myeloid (HL60 and
Meg01) cell lines were examined by FT-IR spectroscopy. The hyperspectral
IR image maps and white light images of each cell line were obtained and
MATLAB was utilised to create a classification model using multivariate
statistical analysis for the evaluation of spectral maps.
Spectra collected at different sites were averaged for each examined cell
line. Results have shown spectral differences in the 4000 to 720 cm-1 spectral
region. Bands in the averaged spectra for the cell line were assigned to the
major biochemical constituents including; proteins, fatty acids, carbohydrates
and nucleic acids. A classification model created using multivariate statistical
analysis was constructed to identify the significant differences in the spectra
across each map. This resulted in the clustering of cell line populations,
indicating distinct bio-molecular differences.
The combination of FT-IR spectroscopy and multivariate statistical analysis
provides an important insight into the fundamental spectral differences
between the cell lines, which differ according to the cellular biochemical
composition. These spectral differences can serve as potential biomarkers
for the differentiation of leukaemia and lymphoma cells. Consequently these
differences could be used as the basis for developing a spectral method for
the detection and identification of haematological malignancies.
methods from the view point of safety. Or the analysis with small quantities
of materials could be possible if the quantities of materials are acceptable. A
non-contact and non-destructive quality control method has been required.
Recently, the use of Fourier Transform Infrared spectroscopy (FT-IR) has been
used to monitor biochemical changes in cells, and has gained considerable
importance. The changes in the cells and tissues, which are subtle and often
not obvious in the histpathological studies, are shown to be well resolved
using FT-IR. Moreover, although most techniques designed to detect one or
a few changes, FT-IR is possible to identify the changes in the levels of various
cellular biochemicals simultaneously under in vivo and in vitro conditions.
The objective of this study is to establish the infrared spectroscopy of tissue
specific progenitor cell differentiations as a quality control of cell sources for
regenerative medicine. In the present study, as a basic study, we examine
the adipose differentiation kinetics of preadipose cells (3T3-L1) and the
osteoblast differentiation kinetics of mesenchymal stem cells (Kusa-A1) to
analyze the infrared absorption spectra.
6628-30, Session 5
Alignment techniques for preparation of proteincontaining surfactant nematic cells
M. M. Omelchenko, Institute of Physical Optics (Ukraine)
Lyotropic surfactant liquid crystals are used as orienting matrices for biological
molecules [1]. Application of high magnetic field usually employed to obtain
uniform orientation of lyotropic liquid crystals is inconvenient at optical
characterization of liquid crystals, which requires well-aligned sandwich-type
transparent cells. Numerous techniques to align thermotropic liquid crystals
in a cell are available for years. Some of them are industrially exploited. Uniform
alignment of lyotropic liquid crystals in a cell between glass substrates is
hard to achieve. Only recently alignment techniques for preparation of
uniformly oriented lyotropic chromonic nematics (other name
chromonematics) have been suggested [2]. However at present there are no
reliable techniques for uniform alignment of surfactant nematics. We have
developed alignment techniques for preparation of uniformly oriented
homeotropic and planar cells of lyotropic surfactant nematics (surfonematics).
Well-aligned cells allowed us to measure dispersion of birefringence for pure
surfonematics as well as to characterize optically surfonematic cells doped
with hemoglobin molecules. We find high orientation order of hemoglobin
molecules in surfonematic matrix. Birefringence, light absorption, dichroism
dispersion and scalar order parameter are measured and analyzed. Optical
characterization of aligned protein-containing surfonematic cells is proposed
as a tool for detection of pathological structural transformations in biological
macromolecules.
6628-31, Session 5
Spectral analysis of esophagus cancer using
fluorescence and Raman spectroscopy
D. Wang, Shenyang Ligong Univ. (China)
In this paper, laser induced human serum Raman spectra of esophagus cancer
are measured. The spectra in serum differences between normal people and
esophagus cancer patients are analyzed. The model was set up from more
than 1500 samples. And 151 samples were used to test this algorithm of the
model prospectively. The serum spectra were excited by laser of the
wavelength 488.0 nm and 514.5 nm. The apparent differences of autofluorescence and Raman spectroscopy were observed for patients compared
to the normal: the majority of the fluorescence spectra did not have violent
alteration, but three Raman peaks had disappeared or very weak. We present
three parameters here. ? ? value (red shift of fluorescence peak) and a-value
(rate of fluorescence intensity) also provide the reference for future research.
And I-value (intensity of Raman peak) will decrease with progression of the
tumor. The results of spectral analysis are accordance with the clinical
diagnosis. These results have important reference values to explore the
method of laser spectrum diagnosis.
6628-57, Session 5
Study of antiangiogenic drugs by fluorescence
imaging and spectroscopy of a contrast agent in
mice
6628-29, Session 5
Analysis of tissue specific progenitor cell
differentiation using FT-IR
K. Ishii, A. Kimura, T. Kushibiki, K. Awazu, Osaka Univ. (Japan)
G. Valentini, C. D’Andrea, R. Ferrari, A. Pifferi, R. Cubeddu, Politecnico
di Milano (Italy); D. Caronia, M. Martinelli, R. Giavazzi, Istituto di
Ricerche Farmacologiche Mario Negri (Italy)
Tissue specific progenitor cells and its differentiations have got a lot of
attentions in regenerative medicine. The process of differentiations, the
formation of tissues, has become better understood by the study using a lot
of cell types progressively. These studies of cells and tissue dynamics at
molecular levels are carried out through various approaches like histochemical
methods, application of molecular biology and immunology. However, in case
of using regenerative sources (cells, tissues and biomaterials etc.) clinically,
they are measured and quality-controlled by non-contact and non-destructive
We used fluorescence imaging and spectroscopy with Indocyanine Green
contrast agent to study the effectiveness of antiangionenic drugs. To this
purpose, the volume of the active vasculature in different tumor models
implanted in mice was assessed by means of a low noise fluorescence
imaging setup and by a photon counting system working in transmission
geometry. Using a first tumor model (carcinoma MDA-MB-435) we observed
that mice treated with a Vascular Disrupting Agent (ZD6126) showed a
reduction in fluorescence emission with respect to control mice. This was a
28
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Conference 6628: Diagnostic Optical Spectroscopy
clear indication of the vascular shutdown that took place in tumors. The
effectiveness was also confirmed by histological sections.
Then we considered a second tumor model (carcinoma 1A9-VS1)
overexpressing the Vascular Endotelial Growth Factor (VEGF121), which is
used by tumor cells to promote angiogenesis. We measured the Indocyanine
Green fluorescence in mice treated with an antioangiogenic drug (AvastinTM)
and in control mice. In tumors of treated mice we observed an ICG emission
lower than the one detected in control mice. This demonstrated that VEGF
was effectively blocked by the treatment with Avastin.
ICG fluorescence provides a simple and reliable way to assess the
effectiveness of vascular targeting therapies. Measurements of the
fluorescence signal can be repeated every 24 hours, thus allowing oncologists
to perform longitudinal studies on the same animals.
6628-32, Session 6
Object localization within turbid slab media
using time-resolved transillumination contrast
functions: a finite element approach
V. M. Piron, J. L’Huillier, École Nationale Supérieure d’Arts et Métiers
(France)
In the last few years, the propagation of diffuse photons in scattering media
has become an important field of interest. This is mainly due to the possibility
offered by the low absorption of light in the range 700 to 900nm [1]. Indeed,
this property leads to a potential deep penetration. But a non negligible
limitation appears: the scattering processes that strongly reduce both the
contrast and the resolution.
In this paper, the time-dependent light propagation in highly scattering media
containing an inclusion is solved by means of a finite element method, tacking
into account Robin type air-tissue boundary conditions. This study is devoted
to the depth localization of a tumor enclosed into a breast tissue-like slab.
The tissue is modeled by a rectangular meshed domain that mimics a breast
compressed between two transparent plates. Cartesian coordinates are used
in order to solve the time-dependent diffusion approximation. A short laser
pulse of 1ps is considered. The transillumination technique is able to detect
laterally the object when the source and detector are moved together on the
same axis [2]. In order to perform the localization of the inclusion in this
study, the optical properties of the object and the slab are varying, and different
size of the object are tested. Knowing the lateral position of the inclusion, we
determine interesting temporal contrast functions based on the mean time
of flight of photons. These functions allow to localize axially the inclusion
using the high scattering processes.
To conclude, our study demonstrates the possibility to detect laterally and
axially a tumor enclosed in a breast tissue.
[1] D. A. Boas, A. H. Brooks, E. L. Miller, C. A. DiMarzio, M. Kilmer, R. J.
Gaudette, and Q. Zhang, “Imaging the body with Diffuse Optical Tomography,”
IEEE Signal Processing Magazine, 18, 57-75, 2001.
[2] V. Piron and J.-P. L’Huillier, “Detection of heterogeneities embedded within
turbid slab media using time- and frequency-domain methods: application
to the mammography,” Lasers in Medical Science, 21, 67-73, 2006.
6628-33, Session 6
Semi-analytical method for rapid calculation of
time-resolved reflectance from bi-layered tissue
models
R. H. Wilson, K. Vishwanath, M. Mycek, Univ. of Michigan (USA)
A semi-analytical technique (“PI-scaling”) combined Monte Carlo (MC)
simulation, absorption scaling, and path integrals (PI) to rapidly reconstruct
time-resolved reflectance from the surface of bi-layered epithelial tissue
models. Initial comparisons to forward MC simulations indicated that the PIscaling method was accurate to better than 10% for several tissue models in
which the optical properties of the top layer did not greatly influence the
time-resolved reflectance. The PI-scaling method was then applied to a skin
model to demonstrate its potential for clinical relevance.
6628-34, Session 6
Computational analysis of light scattering from
collagen fiber networks
D. Arifler, Eastern Mediterranean Univ. (Cyprus); I. Pavlova, The Univ. of
Texas/Austin (USA); A. Gillenwater, The Univ. of Texas M.D. Anderson
Cancer Ctr. (USA); R. R. Richards-Kortum, Rice Univ. (USA)
Neoplastic progression in epithelial tissues is accompanied by structural and
morphological changes in the stromal collagen matrix. Analysis of the influence
of neoplastic changes on stromal scattering properties can improve our ability
to extract diagnostic information from optical signals. Since collagen fibers
in the stroma underlying the epithelium form an extremely intricate network,
analysis of the light scattering properties of these fibers is challenging. We
used the Finite-Difference Time-Domain (FDTD) method, a popular
European Conferences on Biomedical Optics 2007 •
computational technique for full-vector solution of complex problems in
electromagnetics, to establish a relationship between structural properties
of collagen fiber networks and light scattering, and to analyze how neoplastic
changes alter stromal scattering properties. To create realistic collagen
network models, we acquired optical sections from the stroma of fresh normal
and neoplastic oral cavity biopsies using fluorescence confocal microscopy.
These optical sections were then processed to construct three-dimensional
collagen networks of different sizes as FDTD model input. Image analysis
revealed that volume fraction of collagen fibers in the stroma decreases with
neoplastic progression, and statistical texture features computed suggest
that fibers tend to be more disconnected in neoplastic stroma. The FDTD
modeling results showed that neoplastic fiber networks have smaller
scattering cross-sections compared to normal networks of the same size,
whereas high-angle scattering probabilities tend to be higher for neoplastic
networks. These results provide valuable insight into the micro-optical
properties of normal and neoplastic stroma. Characterization of stromal
scattering is expected to provide a basis to better interpret spectroscopic
optical signals and to develop more reliable computational models to describe
photon propagation in epithelial tissues.
6628-35, Session 6
An in vitro study on skin cancer phantoms to
test diffuse reflectance spectroscopy’s ability to
detect depth and thickness variations at several
collecting to excitation fiber separations
M. Amouroux, Ctr. de Recherche en Automatique de Nancy (France)
and Ctr. Alexis Vautrin (CAV) (France); G. Diaz Ayil, E. Pery, W. W.
Blondel, F. H. Guillemin, Ctr. de Recherche en Automatique de Nancy
(France)
Introduction
Microsurgery is used to remove skin tumours. Beforehand dermatologists
need to evaluate safety boundaries around the visible tumour which are
calculated thanks to indices that depend on tumour’s thickness and depth.
In order to avoid several surgeries (invasive thickness and depth measurement
followed by full resection up to safety boundaries) this study aims at
investigating Diffuse Reflectance Spectroscopy (DRS) as a non-invasive tool
to evaluate thickness and depth. We used different collecting to excitation
fibre separations (CEFS) to test DRS’s sensitivity to depth and thickness
variations of diffusive and absorbing layers in multi-layer skin phantoms.
Materials and methods
Phantoms were made of gelatine and Intralipids to achieve epidermis and
dermis scattering coefficients in the visible wavelength bandwidth. Absorbing
material was added to another layer mimicking a layer of cancer cells that
was made thin or thick depending on cancer stage. We chose to locate that
layer at different depths of the epidermis. For each phantom we tested diferent
CEFS as each one is supposed to give information from a specific depth.
Numerical simulation (Monte-Carlo) was associated to compare to
experimental data and test further CEFS.
Results and Discussion
Our first results show that DRS is significantly sensitive to different top layer’s
thicknesses when that layer is absorbing and diffusive but not when it is only
diffusive. DRS signals are also significantly different when an absorbing layer
is put underneath the epidermis but not when the epidermis gets too thick.
So far we haven’t been able to detect a most relevant CEFS.
6628-36, Session 6
Physiological spectroscopic imaging for
diagnosis of skin cancer
K. P. Nielsen, A. Bhandari, B. Hamre, L. Zhao, PhotoSense AS (Norway);
G. A. Ryzhikov, M. S. Biryulina, Geminali AS (Norway); J. J. Stamnes,
PhotoSense AS (Norway); K. H. Stamnes, Balter Inc. (USA); L. Akslen,
L. Rustad, Helse Bergen Haukeland Univ. Hospital (Norway)
By combining diffuse reflectance spectroscopy with an accurate radiative
transfer model, we have found that it is possible to retrieve up to 6 of the
parameters that describe the physiological state of the skin, including the
melanin content and the blood content. In this study we have investigated
the diagnostic potential of the retrieved parameters for optical imaging of
pigmented skin cancer. A clinical study (n = 150) was performed at Haukeland
University Hospital in Bergen, Norway that included lesions with the following
clinical diagnoses: Certain benign, slightly irregular, uncertain case (50%/
50%), suspicious malignant lesion, and certain malignant lesion. For our
optical imaging method
the diagnostic specificity is 97%. Thus, it appears, from this preliminary
study, that malignant lesions may be well separated from the other lesions
with only a few false positives.
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Conference 6628: Diagnostic Optical Spectroscopy
6628-37, Session 6
Improvements in Alzheimer’s disease diagnosis
using principle components analysis (PCA) in
combination with Raman spectroscopy
J. K. J. Archer, C. D. Sudworth, The Univ. of Liverpool (United Kingdom); D. M. Mann, Univ. of Manchester (United Kingdom); R. A. Black,
The Univ. of Liverpool (United Kingdom); N. Stone, Gloucestershire
Royal Hospital (United Kingdom)
In the UK dementia currently affects 1 in 20 people under the age of 65; this
figure dramatically increases to 1 in 5 amongst people under the age of 80.
Alzheimer’s disease is the most common form of dementia, affecting over
55% of sufferers. Alarmingly, the population of UK is ageing and it is
anticipated that by the year 2050 over 1.6 million people will suffer from the
disease.
There is currently no definitive diagnostic test for Alzheimer’s disease: the
criteria describes a rigorous physical, psychiatric and neurological
examination that amounts to nothing more than a process of elimination and
no more than a 90% confidence level in the diagnosis. The only definitive
means to diagnosing this disease is available at post mortem, where it is
identified by the presence of two structural biomarkers: senile plaques and
neurofibrillary tangles. The pre-cursors to each of these markers is the
excessive presence of two proteins, ß-amyloid and tau, both of which break
down and accumulate, forming the markers and killing off the neuron.
Principal Components Analysis (PCA) has been demonstrated as a potential
tool in aiding the identification of the Raman spectra taken from ethically
approved known Alzheimer’s disease, Huntington’s disease and control brain
tissues, taken from both the frontal and occipital lobes. Initial models have
now been expanded to and refined in order to classify and unknown disease
status brain tissues. Emphasis on the detected differences has opened up
the expansion of the study to further tissues excluding the brain in the pursuit
of a minimally invasive technique for diagnosing Alzheimer’s disease. This
work demonstrates the improved statistical analysis which identifies additional
optimum principal components, and introduces values of sensitivity and
specificity.
(without absorbent) varies smoothly. An example of the case is
photodynamical diagnostics of malignant tumors, here the turbid medium is
biological tissue, and the absorbent is photosensitizer. We considered the
task with irradiation of media by cylindrical beam of white light and with
registration of reflected light spectra at some distance from the initial beam.
As the measured quantity we consider relative change of spectrum near
absorption peak.
Based on this mathematical model an experimental setup and program code
for determination of absolute concentration of admixtures in turbid media
were developed. This complex was applied to determine the concentration
of photosensitezer Radachlorin in tissue equivalent media. Results were quite
successful.
6628-40, Session 6
Surface-enhanced Raman scattering (SERS) in
single gold nanoparticle dimers
M. Ringler, T. A. Klar, A. Schwemer, J. Stehr, Ludwig-Maximilians-Univ.
München (Germany); A. Nichtl, K. Kürzinger, Roche Diagnostics GmbH
(Germany); G. Raschke, Ludwig-Maximilians-Univ. München (Germany);
R. T. Phillips, Univ. of Cambridge (United Kingdom); J. Feldmann,
Ludwig-Maximilians-Univ. München (Germany)
We have used protein-ligand interaction to assemble gold nanoparticle dimers,
which have a well-defined SERS hot spot in the inter-particle gap. Surfaceenhanced Raman scattering spectra from individual protein-linked gold
nanoparticle dimers were measured, while at the same time the inter-particle
geometry was monitored through Rayleigh scattering spectroscopy of the
coupled particle plasmon. The Raman emission and Rayleigh scattering
spectra are strongly correlated. Raman emission from the dimer hot spot
can only be excited when the polarization of the Raman laser beam is parallel
to the dimer axis. SERS spectra fluctuate both in shape and amplitude. We
discuss possible explanations of these fluctuations.
6628-38, Session 6
Reflection spectroscopy for assessment of the
kinetics of bilirubin and hemoglobin in bruises
B. Stam, J. de Wit, Univ. van Amsterdam (Netherlands); L. L.
Randeberg, Norwegian Univ. of Science and Technology (Norway); M.
C. G. Aalders, Univ. van Amsterdam (Netherlands)
Background: One of the factors that may indicate child abuse is the presence
of several bruises at different stages of healing on the victim’s body. Besides
for recognizing abuse, aging is also important to identify the perpetrator(s),
to determine whether multiple episodes of trauma occurred, and to ensure
the child’s safety. Methods based on the comparison with a standard color
chart are currently the most widely used in practice, despite the acknowledged
inaccuracy. The amount of blood, size and location of the involved area and
the time after inflicting the injury account for the appearance of the bruise.
By measuring the optical reflectance spectrum of the bruise, its individual
components can be determined. By looking at the spatiotemporal behavior
of hemoglobin and bilirubin, the age can be determined.
Materials and Methods: This study will include healthy volunteers and children
with bruises of known age and cause. Injuries will be documented using
digital photography, reflection spectroscopy (350-900 nm), hyperspectral
imaging and morphological imaging. Data will be evaluated using image
analysis, optical transport theory and models for biological processes. These
will include the diffusion of blood and bilirubin through the skin, enzyme
controlled conversion of blood to bilirubin etc.
Results: The data analysis will be focused on the influence of the thickness
of the skin on the predicted age and on differences between children and
adults.
Conclusion: Further investigations will be required to be able to fully classify
bruises in children, however, preliminary results show that an improvement
on the currently gold standard techniques is possible with reflectance
spectroscopy.
6628-39, Session 6
Mathematical model and method for
determination of absolute concentration of
admixtures in turbid media using diffuse
reflectance spectroscopy
A. V. Lappa, K. V. Dmitriev, Chelyabinsk State Univ. (Russia)
In the framework of kinetic approach to light transport in turbid media a
closed equation system is obtained for the task of distant determination of
small concentration of light absorbing admixtures in media by means of diffuse
reflectance spectroscopy. It is supposed that there is a wavelength region
where the absorbent has a narrow absorption peak and medium absorbance
30
European Conferences on Biomedical Optics 2007 •
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Conference 6629: Diffuse Optical Imaging in Tissue
Tuesday-Thursday 19-21 June 2007
Part of Proceedings of SPIE Vol. 6629 Diffuse Optical Imaging of Tissue
6629-23, Poster Session
Functional imaging of autoregulation
R. L. Barbour, SUNY/Downstate Medical Ctr. (USA) and NIRx Medical
Technologies, LLC (USA); Y. Pei, NIRx Medical Technologies, LLC
(USA); M. Farber, SUNY/Downstate Medical Ctr. (USA); H. L. Graber,
SUNY/Downstate Medical Ctr. (USA) and NIRx Medical Technologies,
LLC (USA); Y. Xu, D. Sreedharan, SUNY/Downstate Medical Ctr. (USA);
C. H. Schmitz, NIRx Medical Technologies, LLC (USA); G. T. Voelbel, G.
R. Wylie, J. Lengenfelder, J. DeLuca, Kessler Medical Rehabilitation
Research and Education Corp. (USA)
We present a novel approach of using functional Diffuse Optical Tomography
(fDOT) for studying vascular autoregulation, i.e., the process whereby, through
various feedback mechanisms, tissues self-regulate their environment to
maintain homeostasis. The particulars of these influence virtually all aspects
of intermediary metabolism, and disruptions lead to a multitude of disease
states (e.g., autonomic, vascular, endocrine, etc.). Despite this general
understanding, strategies well suited to explore this rich phenomenology
have not yet been developed.
Key to our approach is appreciation for the importance of studying contrast
variations that are tied to feedback mechanisms. Furthermore, it is critical to
adopt specific attributes related to data collection and processing, specifically:
(i) acquisition speeds fast compared to the relevant phenomenology, and (ii)
organization of the data in a manner consistent with the feedback processes
themselves.
In our analysis, we delineate the process of vascular autoregulation into six
hemodynamic states that are experimentally definable using fDOT. Using a
vector (oxyHb, deoxyHb, totalHb), in which each element can indicate the
relative increase (+) or decrease (-) of concentration of the corresponding Hb
class, we identify three principal elements: states of oxygen balance [1: (-,-,) and 4: (+,+,+)]; uncompensated imbalance [2: (-,+,-) and 5: (+,-,+)]; and
compensated imbalance [3: (-,+,+) and 6: (+,-,-)]. While the existence of
these permutations is generally appreciated, the juxtapositions of opposite
algebraic signs indicates that summation of the six states, which is the usual
practice, will render individual features undetectable.
The considered approach suggests that physiological triggers and related
factors can now be studied in ways previously not possible. Examples for
the peripheral vasculature and for neuroimaging will be shown.
using a mechanical indentor, using diffuse side-lighting and a CCD videocapture device. Using the blue colour plane of the image and polarisation
filters, it is possible to examine the surface topography only, and track the
decay of the imprint over time.
In this paper, two algorithms are discussed for the extraction of information
on the skin’s displacement: thresholding involves isolating and measuring
the imprinted area of the skin in two dimensions while another approach
uses curve-fitting to the greyscale profile of the imprint. Both methods lead
to a characteristic decay curve for the subject, which is in turn analysed
using a simple viscoelastic model.
6629-52, Poster Session
Filtering effect to improving the reconstructed
image quality of diffuse optical imaging
M. Pan, Tung Nan Institute of Technology (Taiwan); C. Chen, L. Chen,
M. Pan, National Central Univ. (Taiwan)
Diffuse optical tomography (DOT) using diffuse light, red or near-infrared (NIR)
light, is in an attempt to image the interior of human tissues such as breasts,
arms, etc. However, the NIR imaging suffers from low resolution due to the
diffusive nature of the scattered light, which results into poor reconstructed
image quality. Thus, the effort to improving the image quality remains in
progress. In this article, first of all, theoretical analysis is investigated where
several parts are included as follows. Varied filers are discussed such as
filtering using wavelet, high pass filter and so on; then, filtered image
reconstruction in the DC domain is studied where image reconstruction
incorporates an filtering process.
The numerical simulation to reconstruct tomographic images of optical
properties was performed using 16 by 16 sources/detectors combination
and the initial guess with a homogenous background for all cases where
finite element method was implemented where results reveal that several
inclusions (tumors) can be well defined separately; finally, result comparison
will be demonstrated to show high resolution quality with the optimal filtering
process. Subsequently, resolution of separation between inclusions is
discussed with this optimal filter. It is anticipated that filtered image
reconstruction algorithm based on the frequency domain system will be
developed to precisely quantify the diffusion images in the near future.
Keywords: Diffuse optical tomography (DOT), filtered image reconstruction,
optical property.
6629-50, Poster Session
6629-53, Poster Session
High frequency oscillations in brain
hemodynamic response
A. Akin, Bogaziçi Univ. (Turkey); H. Bolay, Gazi Univ. (Turkey)
Tight autoregulation of vessel tone guarantees proper delivery of nutrients to
the tissues. This regulation is maintained at a more delicate level in the brain
since any decrease in the supply of glucose and oxygen to neuronal tissues
might lead to unrecoverable injury. Functional near infrared spectroscopy
has been proposed as a new tool to monitor the cerebrovascular response
during cognitive activity. We have observed that during a Stroop task three
distinct oscillatory patterns govern the control of the cerebrovascular
reactivity: very low frequency (0.02-0.05 Hz), low frequency (0.08-0.12 Hz)
and high frequency (0.12-0.18 Hz). High frequency oscillations have been
shown to be related to stress level of the subjects. Our findings indicate that
as the stress level is increased so does the energy of the high frequency
component indicating a higher stimulation from the autonomic nervous
system.
6629-51, Poster Session
Analysis of skin recovery from mechanical
indentation using diffuse lighting and digital
imaging
N. T. Clancy, M. J. Leahy, Univ. of Limerick (Ireland); G. E. Nilsson, C.
Anderson, Linköpings Univ. (Sweden)
Skin behaves as a viscoelastic material, having mechanical properties
composed of elastic and fluid components. Upon indentation, the fibres are
stretched and fluid displaced from the compressed region. The rate of recovery
from this imprint is therefore dependent on the hydration and elasticity of the
skin. A reliable measurement could be applied to the assessment of clinical
conditions such as oedema, rare genetic disorders such as cutis laxa (elastin
gene mutation) and the evaluation of the ‘effective age’ of skin in vivo.
This paper describes a new approach to the non-invasive indentation
technique and a novel method of analysis. A non-contact method of
measuring the relative displacement of the skin after indentation has been
developed that has advantages over techniques involving mechanical contact
(ultrasound or linear variable differential transformers). Here, a method is
proposed which tracks the skin’s recovery optically from an initial strain made
European Conferences on Biomedical Optics 2007 •
Time-gated, intensified CCD camera for imaging
of a non-homogenous medium at null sourcedetector separation
P. L. Sawosz, M. Kacprzak, A. Liebert, R. Maniewski, Institute of
Biocybernetics and Biomedical Engineering (Poland)
The intensified, time-gated CCD camera (LaVision, Germany) was applied
for imaging of a non-homogenous, liquid phantoms simulating human tissue.
We measured spatial distribution of diffusely reflected photons in reflectance
geometry at null source-detector separation. The surface of the phantom
was scanned by laser beam generated by picosecond, near-infrared, diode
laser BHL-600 (Becker&Hickl, Germany) at wavelength of 780nm over the
grid of 5 by 5 points separated by distance of 1 cm. We measured distributions
of reflectance for each position of laser beam for two different time windows.
Both time windows were significantly delayed versus laser pulse and the
early photons were skipped. The observed late photons, which penetrated
deeply in the optically turbid phantom allowed us to image absorbing inclusion
(10mm diameter black ball) located at the depth of 15mm. After summing
the obtained images for each of the two time windows the immersed nonhomogeneity can be easily localized.
We conclude that the method based on imaging at null source-detector
separation distance for late time windows may be applied in development of
brain oxygenation imaging system.
6629-54, Poster Session
Development of a computer vision binocular
system for non-contact small animal model skin
cancer tumour imaging
D. S. Gorpas, M. Kyriazi, K. Politopoulos, D. M. Yova, National Technical
Univ. of Athens (Greece)
This paper describes the development of a novel gauging computer vision
system for murine non-melanoma skin cancer tumours volume imaging. The
system utilizes binocular stereo vision, enhanced through the use of telecentric
lenses. These lenses optically compromise for the distortion factors and
provide orthographic projection, leading to parallax free image acquisition.
Furthermore, z-axis translation is possible without the need of repeating
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Conference 6629: Diffuse Optical Imaging in Tissue
system calibration. In order to improve the resolution of the system, a
structured light projector, with 450 nm dominant wavelength, was used to
illuminate the target with a custom pattern. Calibration was performed under
photogrammetric fashion, by inspecting a three-dimensional calibration
object, highlighting the almost linearity of the camera models due to the
telecentric lenses. Robust image processing algorithms granted accurate
segmentation, feature recognition, labeling and correlation between the stereo
pairs. Under these premises, the well known “matching” problem was resolved
successfully and geometrical interpolation provided an accurate threedimensional reconstruction of the tumour volume. Through back-projection
of the calibration object the resolution of the system was calculated up to
0.04 mm. The system was applied to measure the induced geometrical
alterations of the tumour after PDT by using the Fosgel photosensitizer, excited
by a laser diode emitting at 652 nm. The measurement of the volume induced
alterations after each PDT treatment and up to the final tumour shrinkage is
critical, to compare PDT efficacy between different protocols. The accuracy
and robustness of the system will provide an objective criterion to support
the visual inspection of PDT end point.
rotationally symmetric phase functions. This simplification is appropriate for
measurements of scattering media which have spherical scatterers, like clouds
or milk, or scattering media which consists of aligned scatterers with random
directions. Contrarily to this, one finds many types of aligned microstructures
in biological tissue like in muscle, tendon, tooth and various other. A great
amount of scattering is caused by this aligned microstructures und the
resulting phase functions are not rotational symmetric and depend on the
incident angle of the radiation. It can be shown that the optical properties of
biological tissue, acquired without taking respect to the microstructure, may
have errors of more than 50 %. To measure the asymmetric phase function
of tissues, a fully automated two axis goniometrical system, which is capable
of the measurement of the phase function in almost the whole solid angle,
was developed. The measurement of the phase function of tissue with aligned
microstructures is only possible for thin slabs. A method was developed to
correct the geometrical errors due to the slab geometry. We will present
goniometric measurements of the phase function of phantom media and
measurements of biological tissue with aligned microstructures.
6629-60, Poster Session
6629-56, Poster Session
Three dimensional near infrared tomography of
the breast
M. E. Eames, Univ. of Exeter (United Kingdom); B. W. Pogue, Dartmouth
College (USA); H. Dehghani, Univ. of Exeter (United Kingdom)
Near-Infared (NIR) Diffuse Optical Tomography (DOT) is a non-invasive imaging
technique which is used to obtain functional and physiological images of
soft tissue, such as the female breast specifically for the detection and
characterization of breast cancer. The vast majority of the work to date has
been limited to two dimensional (2D) models. The results from the 2D models
have provided valuable insight into tissue function and physiology which has
enabled a better understanding of tumor development and treatment.
Although the 2D image reconstruction approach is fast and computationally
efficient, it has limitations as it does not correctly represent the volume under
investigation. The 2D models therefore do not provide the most accurate
model for image reconstruction. 3D modeling and image reconstruction is
becoming more accessible through the development of sophisticated
numerical models and computationally fast algorithms. This work will
demonstrate the inaccuracies and errors seen when using 2D models versus
3D models and highlights the need to utilize correct and realistic modeling
and image reconstruction techniques. A robust and general method will be
presented which reconstructs more qualitatively and quantitatively accurate
3D optical images. Clinical results will be presented to demonstrate the clinical
importance of 3D image reconstruction on optical tomography.
6629-57, Poster Session
Monitoring muscle metabolic indexes by timedomain near infrared spectroscopy during knee
flex-extension induced by functional electrical
stimulation
An instrument for small animal 3D-diffuse and
fluorescence optical imaging
P. Poulet, Univ. Louis Pasteur (France)
Time-resolved optical methods with diffuse near infrared photons were used
to image the optical properties of tissues and their inner fluorescent probe
distribution. The assembled scanner uses picosecond laser diodes at four
wavelengths in a sequential mode, an eight-anode MCP-photomultiplier tube
and time-correlated single photon counting techniques. A simple and reliable
scanning unit, placed at some distance from the animal was conceived. The
scanning of the animal uses a non contact, circular fan-beam geometry with
seven output positions times sixteen input positions. A holographic technique
was added to the tomography set-up in order to record the coordinates of
the animal body surface, as required for the reconstruction process. Optical
absorption and reduced scattering images as well as fluorescence emission
images were computed from temporal profiles of diffuse photons. This method
should improve the spatial resolution and the quantification of fluorescence
signals. We used the diffusion approximation of the radiation transport
equation and the finite element method to solve the forward problem. The
scanner and its performances are presented, together with absorption,
scattering and fluorescent images obtained with it.
6629-61, Poster Session
Spatial resolved diffuse reflection as a tool for
determination of size and embedding depth of
blood vessels
A. V. Bykov, M.V. Lomonosov Moscow State Univ. (Russia) and Univ. of
Oulu (Finland); A. V. Priezzhev, M.V. Lomonosov Moscow State Univ.
(Russia); R. A. Myllylä, Univ. of Oulu (Finland)
A time-domain fNIRS multichannel system was used in a sustained attention
protocol (continuous performance test) to study activation of the prefrontal
cortex. Preliminary results on volounteers show significant activation
(decrease in deoxy-hemoglobin and increase in oxy-hemoglobin) in both left
and right prefrontal cortex.
In this work, we analyze the feasibility of spatial resolved diffuse reflection
measurement method for determination of shape, characteristic size and
embedding depth of a nonhomogeneity mimicking a cylindrical blood vessel
embedded into a scattering medium mimicking skin. For this purpose the 2D
spatial resolved reflection maps of intensity resulting from CW illumination of
the phantom with an NIR pencil beam with normal incidence were simulated
with Monte Carlo method. The optical properties of the nonhomogeneity
medium are chosen close to those of the human blood considered as a
suspension of nonaggregating erythrocytes with hematocrit of 35 %. The
optical properties of the medium surrounding the nonhomogeneity correspond
to those of intralipid 2 %, which mimics the human skin. The sensitivity of
the obtained signals (spatially resolved diffuse reflectance measured at
different source-detector separations in 2D) to a variation of the
nonhomogeneity location was analyzed. It is shown that in the case of a
cylindrical nonhomogeneity, due to the strong absorption and scattering
properties of the medium enclosed therein, a decrease in the reflected
radiation is maximal when measured directly over the embedded cylinder.
This feature makes the technique potentially useful for imaging and sizing
blood vessels. It is also shown that the image blur increases linearly with an
increase in the cylindrical nonhomogeneity embedding depth. This feature
can be used for determining the latter. The optimal position for the laser
probe yielding the highest image quality of the cylindrical nonhomogeneity
was found. The numerical simulations were performed with the supercomputer
MVS-15000BM.
6629-59, Poster Session
6629-62, Poster Session
Measurement of the phase function of phantoms
and biological media with a 2 axis goniometer
Optical tomography of small tissue volumes with
the ERT: frequency-domain sensitivity analysis
R. Michels, A. Kienle, Univ. Ulm (Germany)
X. Gu, Columbia Univ. (USA); U. Netz, J. Beuthan, Charité-Univ.
Medicine Berlin (Germany); A. H. Hielscher, Columbia Univ. (USA)
A. Torricelli, D. Contini, L. Spinelli, R. Cubeddu, Politecnico di Milano
(Italy); F. Molteni, Ctr. di riabilitazione Villa Beretta, Ospedale Valduce
(Italy); S. Ferrante, A. Pedrocchi, G. Ferrigno, Politecnico di Milano (Italy)
A time-domain NIRS multichannel system was used to monitor hemodynamic
changes in the muscle of volunteers and hemiplegic patients during functional
electrical stimulation for rehabilitation purposes.
6629-58, Poster Session
Continuous performance test assessed with
time-domain functional near infrared
spectroscopy
A. Torricelli, D. Contini, L. Spinelli, M. Caffini, M. Butti, G. Baselli, A. M.
Bianchi, Politecnico di Milano (Italy); A. Bardoni, IRCCS E. Medea (Italy);
S. Cerutti, R. Cubeddu, Politecnico di Milano (Italy)
The common solutions of the transport equation simplify the complexity of
the scattering media. A widely used approximation of the transport equation
of light in scattering media is the (isotropic) diffusion theory. The scattering
media are approximated as a homogeneous distribution of scatterers with
32
European Conferences on Biomedical Optics 2007 •
Optical tomography of small imaging domains holds great promise as the
signal-to-noise
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Conference 6629: Diffuse Optical Imaging in Tissue
levels are usually high and the spatial resolutions are much better than that
of large imaging domains. Emerging applications range from imaging of joint
diseases in human fingers to monitoring tumor growth or brain activity in
small animals. In these cases, the diameter of tissue under investigation is
typically smaller than 30mm, and the optical path length is only a few
scattering mean-free path. It is well known that under these conditions the
widely applied diffusion approximation to the equation of radiative transfer
(ERT) is of limited applicability. To accurately model the light propagation in
these small domains, the ERT has to be solved directly. In the study at hand,
we perform numerical and experimental sensitivity analyses for the imaging
problem in small-volume optical tomography with the frequency-domain ERT
as light propagation model. Varying the optical properties, tissue geometries,
and contrast, we show that source modulation frequencies in the range of
300-600MHz yield maximal signal-to-noise ratios and therefore maximal
sensitivity to tissue inhomogeneities. These results will be useful in designed
experiments and optical tomographic imaging system that probe small tissue
volumes.
6629-66, Poster Session
Approach to estimating low contrast inclusion
with a priori guidance
6629-63, Poster Session
Transmission RF diffuse optical tomography
instrument for human breast imaging
M. Pan, C. Chen, L. Chen, National Central Univ. (Taiwan); M. Pan, Tung
Nan Institute of Technology (Taiwan)
K. Lee, S. D. Konecky, A. Corlu, R. Choe, T. Durduran, A. G. Yodh, Univ.
of Pennsylvania (USA)
In this paper, we describe a novel clinical breast diffuse optical tomography
(DOT) instrument for CW and RF data acquisition in transmission geometry.
It is designed to be able to acquire massive data in a short amount of time
available for patient measurement, by using a 209-channel galvo-based fast
optical switch and a fast electron-multiplying CCD. In addition to CW
measurement, RF measurement was made possible by using a electro-optic
modulator for source modulation and a gain-modulated image intensifier for
detection. The patient bed was made to be both comfortable to the patient
and efficient for measurement, by using a top part of a commercial breast
biopsy table and making a separate support mechanism for the patient table
and breast box.
A series of preliminary results will be shown, including its CW performance in
resolution and a fast heterodyne RF measurement. In order to deal with big
number of data, a linear reconstruction algorithm that exploits separability of
the inverse problem in Fourier domain is used for fast and memory-load-free
reconstruction.
6629-64, Poster Session
Correction of dead-time related distortions in
time-correlated single photon counting at high
count rates
H. Wabnitz, Physikalisch-Technische Bundesanstalt (Germany); M.
Möller, Hochschule für Technik und Wirtschaft des Saarlandes
(Germany); W. Becker, Becker & Hickl GmbH (Germany); R. Macdonald,
Physikalisch-Technische Bundesanstalt (Germany)
Its high sensitivity favors time-correlated single photon counting (TCSPC)
for picosecond time-resolved in vivo optical imaging and spectroscopy. When
recording images or time series, distributions of times of flight photons have
to be acquired with good signal-to-noise ratio within a few tens of milliseconds.
High count rates ranging up to several MHz are thus mandatory, and today’s
TCSPC technology is capable of processing them. However, if the count
rate is on the order of the reciprocal dead time of the TCSPC electronics, a
noticeable fraction of single photon events is lost during dead time periods.
Consequences are a decrease in differential sensitivity and distortions in the
time-of-flight histograms. In contrast to the classic pile-up effect, no remedy
has been reported yet for the inter-pulse pile-up effect, i.e. the typical situation
that the dead time ends within one of the signal periods following the detection
of a photon. We present a method to quantitatively correct measured timeof-flight distributions for the time-independent counting loss as well as for
shape distortions. The algorithm requires knowledge of the dead time as a
function of the start-stop interval which is obtained from a calibration
measurement. We demonstrate the effect of the correction on in-vivo
measurements performed with our time-domain brain imager. For small signal
changes as observed in functional activation studies the overall counting
loss is the major effect. Signals with large changes in count rate, e.g. during
fluorescence recording of a dye bolus, are additionally subject to shape
distortions of the time-of-flight histograms.
6629-65, Poster Session
A new cerebral hemorrhage auto-segment
mechanism
T. Shen, Beijing Institute of Technology (China)
This paper presents a novel method for CT cerebral hemorrhage (CH) image
automatic segmentation. This mechanism uses expert system which model
human knowledge about the cerebral hemorrhage (CH) automatic
segmentation problem. The algorithm uses a series of special steps and
European Conferences on Biomedical Optics 2007 •
extracts some easy ignored CH features. Then by using these features, a
decision tree will be established for the cerebral hemorrhage (CH) judgment.
By statistic results of mass real cerebral hemorrhage images, some cerebral
hemorrhage (CH) features have been found, such as region area, region CT
number, region smoothness and some statistic cerebral hemorrhage region
relationship. For extracting these cerebral hemorrhage (CH) features, a seven
steps’ extracting mechanism will ensure that we could get these cerebral
hemorrhage (CH) features correctly and efficiently. There are about
preprocessing, region growth, analyze, judgment and so on. Using these
cerebral hemorrhage (CH) features, a decision tree which models the human
knowledge about the cerebral hemorrhage (CH) automatic segmentation
problem has been built. It will ensure the rationality and accuracy of the
algorithm. At the end, for verifying the correctness and reasonable of the
automatic segmentation results, we will use mass of real cerebral hemorrhage
(CH) images for the testing, and could get satisfied results.
Abstract
Diffuse optical tomography (DOT) for noninvasive tissue monitoring have been
in use for nearly two decades, mainly estimating in near-infrared (NIR) light
range (650~950 nm) the distribution of the optical properties or their changes
within a tissue volume, and then relating them to spatial variation of the
physiological status. The NIR imaging, however, suffers from low resolution
due to the diffusive nature of the scattered light; there are compelling reasons
for merging high-resolution structural information from other imaging
modalities with the functional information attainable with NIR DOT. In this
article, slight variation of the inclusion (tumor) in low contrast of optical
properties is estimated and investigated. We present that an initial study of
using a structural a priori knowledge in NIR tomography where absorption
image reconstruction of the tested phantom is well defined with the aid of a
structural a priori knowledge obtained from other imaging modalities. This is
advantageous compared to either modality alone. As well, the reconstructed
optical absorption coefficient is achieved more accurate near to be exact
value with incorporating the empirical updating information being proportional
to the off-boundary distance but not size of inclusion against the background.
Numerical simulation is demonstrated on varied sizes, locations and contrast
of the inclusion. With the comparison between with or without a priori and
empirical updating information, it is found that the reconstructed optical
properties are more accurate than near-infrared imaging alone.
Keywords: Diffuse optical tomography (DOT), near-infrared (NIR), structural
a priori knowledge, empirical updating information, image reconstruction.
6629-67, Poster Session
Fluorescence lifetime imaging through turbid
media reconstructed in the Fourier domain using
time gated imaging data
V. Y. Soloviev, Univ. College London (United Kingdom); K. Tahir, J. A.
McGinty, D. S. Elson, M. A. A. Neil, A. Sardini, J. V. Hajnal, Imperial
College London (United Kingdom); S. R. Arridge, Univ. College London
(United Kingdom); P. M. W. French, Imperial College London (United
Kingdom)
This work presents the reconstruction of the quantum yield and the lifetime
distribution in highly scattering phantoms from experimental time gated
imaging data.
Most reported work to date concerning fluorescence lifetime imaging (FLIM)
in turbid media has utilized frequency domain lifetime imaging instrumentation.
In this work we present an instrument that exploits wide-field time gated
detection to acquire temporally and spatially resolved data sets of transmitted
light. The time-gated imaging approach provides high temporal resolution
and considerable flexibility in reconstruction. The temporal dataset is Fourier
transformed for subsequent reconstruction in the Fourier domain, which
provides considerable advantages with respect to computational load in the
time domain. The reconstructed complex valued function contains all required
information for recovering the quantum yield and lifetime distribution in a
sample for frequencies from d.c. up to GHz.
In this work the telegraph equation (TE) is utilized for modeling the light
transport in turbid media due to its better accuracy at high frequencies than
the well-known diffusion approximation. We have applied this technique to
the reconstruction of the lifetime distribution of phantoms incorporating tubes
filled with Rhodamine 6G embedded inside a highly scattering slab. Relatively
accurate fluorescence lifetime reconstruction demonstrates the effectiveness
and the potential of this proposed technique. We have also undertaken
experiments using phantoms incorporating GFP and have developed a new
technique to account for background autofluorescence in the scattering
medium.
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Conference 6629: Diffuse Optical Imaging in Tissue
6629-68, Poster Session
Near infra-red imaging through a scattering
medium using the NOISE technique
A. M. Cuddihy, B. M. Hennelly, R. O’Neill, C. Markham, National Univ. of
Ireland/Maynooth (Ireland)
Experimental work has been carried out to extend a technique introduced by
Rosen et al. [1], namely non-invasive optical imaging by speckle ensemble
(NOISE), to non-invasively image a structure embedded beneath a 2.5mm
thick layer of biological tissue (bacon). Rosen’s method uses a microlens
array (MLA) and a coherent light source in transmission mode (figure 1). This
setup has been enhanced by use of a more powerful laser source (75mW
HeNe) and a higher resolution camera (2048?2048). Image reconstruction is
achieved by averaging individual images from selected microlenses, thus
reducing the speckle noise created due to the tissue layers. Experimental
observation at 632nm has allowed us to quantify limitations on the depth
beneath the tissue at which the object can be probed. These results suggest
that the underlying object’s structure cannot be reconstructed using this
method unless the object is very close to the front tissue’s surface. Optimal
system configuration will be discussed in this work. Current efforts are now
concentrating on extending the method to the near infrared (NIR) region, as
it is well known that tissue offers reduced absorption and scattering at NIR
wavelengths. A rotating glass diffuser has also been introduced to the system
and this has been effective in further reducing the speckle noise, and thus
enhancing image quality. Results relating to observations at 632nm and first
results at 785nm will be presented.
[1] Non-invasive optical imaging by speckle ensemble, J. Rosen and
Abookasis D., Optics Letters, 29(3), 2004.
6629-01, Session 1
Wavelet-based model reduction applied to
fluorescence diffuse optical tomography
A. Frassati, A. DaSilva, J. Dinten, Lab. d’Electronique de Technologie de
l’Information (France); D. Georges, Institut National Polytechnique de
Grenoble (France)
Fluorescence diffuse optical tomography is becoming a powerful tool for the
investigation of molecular events in small animal studies for new therapeutics
developments.
Here, the stress is put on the mathematical problem of the tomography, that
can be formulate in terms of an estimation of physical parameters appearing
as a set of Partial Differential Equations (PDEs). The Finite Element Method
has been chosen here to resolve the diffusion equation because it has no
restriction considering the geometry or the homogeneity of the system. It is
nonetheless well-known to be time and memory consuming, mainly because
of the large dimensions of the involved matrices.
Our principal objective is to reduce the model in order to speed up the model
computation. For that, a new method based on a multiresolution technique
that uses a wavelet decomposition is chosen. All the matrices appearing in
the discretized version of the PDEs are projected onto an orthonormal wavelet
basis, leading to more sparse matrices. The inversion of these matrices is
then easier to carry out, and the computation time is obviously reduced.
With the first order resolution, this compression leads to the reduction of a
factor 22 of the initial dimension. The inversion of the matrices is approximately
4 times faster than without the wavelet projection. With the second order
resolution, the factor is 42, and the inversion is ~7 times faster.
A validation study on a phantom was conducted to evaluate the feasibility of
this reduction method, and its performance in computation time reduction.
6629-02, Session 1
Digital signal processor based dynamic optical
tomography imaging system
A. H. Hielscher, J. M. Lasker, J. M. Masciotti, Columbia Univ. (USA); C.
Schmitz, SUNY/Downstate Medical Ctr. (USA); Y. Li, A. Bur, C. J. Fong,
Columbia Univ. (USA)
In this paper, we introduce a prototype dynamic optical tomography system
that is, unlike currently available analogue instrumentation, based on digital
data-acquisition and filtering techniques. We outlined design considerations
and engineering issues concerning the realization of such a system with up
to 4 wavelength and 64 detector channels. At the core of this dual-wavelength,
continuous wave instrument is a digital signal processor (DSP) that collects,
collates, processes, and filters the digitized data set. The processor is also
responsible for managing the system timing and the imaging routines, which
can acquire real-time data rates as high as 140Hz. Many of the synchronouslytimed processes are controlled by a complex programmable logic device
(CPLD) that is also used in conjunction with the DSP to orchestrate data
flow. The operation of the system is implemented through a comprehensive
graphical user interface designed with the LabVIEW software which
seamlessly integrates automated calibration, data acquisition, data
organization, and signal post-processing. Performance analysis demonstrates
34
European Conferences on Biomedical Optics 2007 •
very low system noise (~6pW RMS noise equivalent power), excellent signal
precision (<0.25%) and system stability (<1.2% over 40 min). Linearity was
confirmed over the entire dynamic range (~178dB). Experiments on tissue
phantoms show that dynamic behavior can accurately be captured and spatial
location can be correctly extracted using this system. Quantification and
analysis of instrument performance demonstrates that making use of precision
circuitry in a digital detection environment greatly enhances the system
functionality as compare to previous presented analogue instruments.
6629-03, Session 1
Speckle pattern characterization by circular
statistics
M. C. Péron, E. Deléchelle, Univ. Paris 12 Val-de-Marne (France); S.
Guyot, École Polytechnique (France)
It is well known that the interactions between coherent monochromatic
radiation and a scattering liquid medium induce a speckle phenomenon.
The spatial and temporal statistics of this speckle are employed to analyze
many applications in laser imaging. The direct exposure of a photographic
film, without a lens to the backscattered radiation, gives a speckle pattern.
The main problem lies in the determination of those parameters which can
efficiently characterize this pattern. In this paper, we present a circular statistic
approach to differentiate media.
The estimation of the local phase and the local amplitude is an important
step in many signal and image processing tasks. A second crucial task in
image processing is the estimation of the local orientation. In 2-dimensional
space one can replace the Hilbert transform by the Riesz transform. The
analytic image constructed using the Riesz transform allows the estimation
of the local orientation, local phase and local amplitude at the same time.
The Riesz transform yields efficient estimates of local orientation of the field.
To address this problem, we used the direction difference (or direction
gradient). The statistical properties of the set of differences contain information
about the correlations of neighbouring local direction values. Using a new
family of symmetric distributions, named psi-distribution, we show that the
circular statistics of local direction gradient, obtained from orientation
estimation, is related with the scattering length of the medium. Our study
allows with the simply parameters extract from the psi-distribution to
characterize the speckle pattern from the scattering length. When the
scattering length increases, the diffusion and consequently the anisotropy,
decrease.
6629-04, Session 1
Phantom study on contrast mechanisms in timedomain fluorescence imaging
O. Steinkellner, D. Grosenick, A. J. Hagen, Physikalisch-Technische
Bundesanstalt (Germany); R. Ziegler, T. Nielsen, Philips Research Labs.
(Germany); R. Macdonald, H. H. Rinneberg, Physikalisch-Technische
Bundesanstalt (Germany)
We have experimentally and theoretically studied fluorescence imaging of
phantoms simulating a tumor-bearing female breast slightly compressed
between two parallel glass plates. Results were obtained on a rectangular
glass cuvette of thickness of 60 mm filled with a diffusely scattering and
weakly absorbing liquid the optical properties of which were similar to those
of breast tissue in the near infrared spectral range, and containing a
tricarbocyanine-based fluorescent dye (SIDAG) at various concentrations.
Spherical objects positioned at selected locations within the phantom were
filled with background medium of the same or enhanced intrinsic absorption
and increased dye concentration using ratios of dye enrichment between
about 2:1 and 10:1 compared to the background medium. Short (fs-) laser
pulses (730 nm) were injected at a large number of scan positions and times
of arrival of transmitted or remitted laser and fluorescence photons were
measured by time-correlated single photon counting. In addition,
transmittance was measured in cw mode using an EMCCD camera for
imaging. Normalized transillumination images at the laser and fluorescence
wavelength were generated from raw data, their contrast and contrast to
noise ratios determined and simulated using the diffraction of diffuse photon
density waves as forward model. From cw and Fourier-transformed timedomain data, taken at the laser and fluorescence wavelength total absorption
and dye concentration were reconstructed within the Rytov and (normalized)
Born approximations. Our study serves to optimize performance of a timedomain fluorescence mammograph based on parallel plane geometry.
6629-05, Session 2
Evaluation of the image reconstruction
algorithm for near infrared topography by virtual
head phantom
H. Kawaguchi, E. Okada, Keio Univ. (Japan)
Near infrared topography is a quite simple technique to obtain the images of
brain functions and has been applied to the measurements of the brain activity
in various fields. However, it has been pointed out that several technical
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Conference 6629: Diffuse Optical Imaging in Tissue
problems have remained in its accuracy of position, size and so on. We have
constructed the virtual head phantom, of which structure was obtained from
MRI slices of an adult head, to evaluate the image reconstruction algorithm
and the arrangement of fibre-probes under the condition close to the
experiment for a real adult head. In this study, we proposed the image
reconstruction algorithm using the prearranged spatial sensitivity profile to
improve the accuracy of near-infrared topography and the algorithm was
evaluated by the virtual head phantom. The spatial sensitivity profiles used
in the proposed algorithm were estimated by the simplified five layered slab
model of an adult head. The change in intensity detected by source-detector
pairs caused by the brain activation was calculated by the virtual head
phantom and the conventional mapping method and the proposed algorithm
were adopted to obtain the topographic images. When the conventional
mapping method was used for the image reconstruction, the distribution
and the contrast of the activated region in the images obviously depend on
the relative position between the activated region in the gray matter and the
probe-set on the scalp. This problem was greatly improved by using the
proposed algorithm though the prearranged spatial sensitivity profile used in
the algorithm was not completely the same as the actual spatial sensitivity
profile in the virtual head phantom.
method relies on the difference in bulk tissue attenuation of two spectral
bands within the broad fluorescence spectrum, characteristic for all
fluorescent substances. The detected intensity ratio between the two spectral
bands is then dependent on the propagation distance for the fluorescence.
Comparison of the detected fluorescent intensity ratio with a mathematical
forward model yields a prior information weight map outlining the most
probable fluorophore position within the medium. The method is verified with
experimental data using a simple setup consisting of a slab-shaped tissue
phantom with a cylinder fluorescent inclusion. Further the ability to render a
priori information when multiple fluorophores are present is investigated.
To assess the reconstruction performance using the prior weight map,
rendered by the presented method, the normalized Born approach is
implemented. The use of prior information results in a faster convergence as
well as a more accurate assessment of the fluorophore position.
6629-09, Session 2
Wavelength optimization in multispectral diffuse
optical tomography considering uncertainties in
absorption spectra
6629-06, Session 2
B. Brendel, T. Nielsen, Philips Research Labs. (Germany)
Near-surface sensitivity suppression way for
diffuse reflective optical tomography: simulation
and a phantom study
Diffuse optical tomography using continuous wave light at a single wavelength
can not distinguish between scattering and absorption. This can be overcome
by using data from several wavelengths together with a model of the
wavelength dependency of scattering and absorption. The absorption is
described as the sum of contributions of different tissue chromophores, i.e.
the model is based on the knowledge of the chromophore absorption spectra.
Corlu et.al. presented a method to select the optimal laser wavelengths in a
way that 1.) scattering and absorption can be separated, and 2.) the
contributions of the different chromophores can be identified.
In this work we present an extension of the method of Corlu, which includes
uncertainties of the underlying chromophore absorption spectra in the
wavelength selection process. This is required, because the deviations
between the published absorption spectra of e.g. water, hemoglobin and fat
are substantial.
We present the results of the new method for measurements with 5 and 6
wavelengths in the range of 650 nm to 930 nm and compare it to the results
of Corlu.
The effect of the different wavelength sets are demonstrated on reconstructed
images using different combinations of published chromophore absorption
spectra. The comparison of these images illustrates the importance of taking
spectra uncertainties into account and shows advantages of the new
wavelength sets: less cross talk between different chromophores and less
artifacts.
K. Fukuda, Tokyo Metropolitan College of Industrial Technology (Japan);
M. Fujii, Sophia Univ. (Japan)
Diffuse reflective optical measurement is an attractive method for monitoring
the oxygen consumption of living tissue such as brain and muscle [1]. This
usually uses light sources and detectors, which are separated with individual
apertures in each, on the surface to obtain topographic image. Detected
light contains considerable amount of light diffused and reflected in the nearsurface region, thus the measurement is sensitive to the skin blood flow and
variation in intervening tissue thickness. To avoid this, we propose a new
method in which plural pairs of light source and detector shearing the same
aperture are arranged on the surface to selectively detect the diffuse reflective
light in the near-surface region. By subtracting it from conventional diffuse
reflective light detected at the other apertures, the near-surface sensitivity is
suppressed and the targeted tissue sensitivity is measured accurately.
The intensity and the dependence of sensitivity on the absorption position
were simulated. The sensitivity detected at the source-detector sharing
aperture was significantly high at the near-surface region and applicable for
suppression. The near-surface sensitivity was reduced by more than 90%
with keeping the targeted sensitivity (3% deterioration).
Experiments with a phantom were performed. Light (785 nm) modulated at
1kHz was provided to the object through an optical fiber bundle.
Measurement with a pair of fiber bundles shows that the near-surface
sensitivity can successfully suppressed by our method.
[1] Maki et al., Med. Phys. 22, 1997-2005 (1995).
6629-07, Session 2
Optimized determination of absorption changes
from moments of time-of-flight distributions for
a two-layer tissue model
A. Liebert, Institute of Biocybernetics and Biomedical Engineering
(Poland); H. Wabnitz, C. Elster, Physikalisch-Technische Bundesanstalt
(Germany)
Novel method for depth-resolved brain
functional imaging by time-domain NIRS
D. Contini, L. Spinelli, A. Torricelli, A. Pifferi, R. Cubeddu, Politecnico di
Milano (Italy)
A novel approach to improve depth selectivity based on time-domain contrast
functions is presented. The method was tested with Monte Carlo simulations
showing sensitivity to absorption changes of deep inclusions and improved
rejection of superficial effects. Preliminary in-vivo measurements were
performed on healthy volunteer during a Valsalva maneuver and during finger
tapping discriminating brain cortex activation from hemodynamic changes
associated to systemic effects in the scalp.
6629-08, Session 2
Spatial a priori information in fluorescence
molecular tomography by use of spectrally
resolved fluorescence emission
J. Axelsson, J. Svensson, S. Andersson-Engels, Lunds Tekniska
Högskola (Sweden)
Fluorescence molecular tomography (FMT) has evolved, within the field of
molecular imaging, to become a powerful modality to localize and quantify
fluorescent agents in turbid media. The inherent ill-posedness in image
reconstruction algorithms is a major problem since it renders multiple nonunique solutions to the reconstruction problem. To reduce the ill-posedness
prior spatial information about the fluorophore position can be used. In order
to achieve a priori information about the inclusion position, we present a
method based only on the multispectral fluorescence of the fluorophore. The
European Conferences on Biomedical Optics 2007 •
6629-10, Session 2
Time-resolved near-infrared spectroscopy allows for depth-selective
determination of absorption changes in the adult human head which facilitates
separation between cerebral and extracerebral responses to stimulation. For
the analysis of measurements we recently focused on moments of
distributions of times of flight of photons (DTOF) and found that from multidistance time-resolved measurements depth-resolved absorption profiles can
be derived. However, for brain imaging such multi-distance approach is not
feasible.
Therefore the present work aims at finding which combinations of moments
and distances are optimal for reconstruction of absorption changes in a twolayered tissue model corresponding to extra- and intracerebral compartments.
To this end we calculated the uncertainty of absorption changes in both layers
due to photon statistics. This calculation relies on the uncertainty matrix of
the moments under consideration (attenuation, mean time of fight, variance)
which is composed of the variances and covariances of these quantities.
The analysis of propagation of uncertainties was carried out using sensitivity
factors obtained from Monte-Carlo simulations and with photon noise in
moments estimated for realistic measurement conditions. The results show,
as expected, that the uncertainty of the absorption change in the deeper
(superficial) layer increases (decreases) with the thickness of the superficial
layer. It was also confirmed that the usage of higher order moments
(particularly variance of the DTOF) leads to a decreased uncertainty of the
change in the absorption coefficient in the deeper layer.
Results of this study may be useful in designing optimal near infrared
spectroscopy instruments used in brain oxygenation studies in adults.
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Conference 6629: Diffuse Optical Imaging in Tissue
6629-11, Session 2
6629-14, Session 3
Depth selective diffuse optical computed
topography: simulations and phantom
experiments
Detection and characterization of an optical
inhomogeneity by diffuse photon-pairs density
wave in a multiple-scattering medium
M. Fujii, A. Kawanaka, K. Nakayama, Sophia Univ. (Japan)
L. Yu, National Yang-Ming Univ. (Taiwan); J. Wu, L. Su, National Central
Univ. (Taiwan); C. Chen, Y. Chan, National Yang-Ming Univ. (Taiwan); C.
Chou, National Yang-Ming Univ. (Taiwan) and National Central Univ.
(Taiwan)
Diffuse optical topography has great features as a noninvasive metnod which
can provide 2D location information of cortical activity. However it cannot
distinguish the depth where activation occurs. Because regions just beneath
optodes have extremely high sensitivity, skin circulation change near or
beneath optode strongly degrades the reliability of topogram. We propose a
new image reconstruction algorithm, which can suppress undesirable effect
of skin circulation without using time-resolved technique. The proposed
method consists of two operations. One is the filtering which extracts target
signals from the observation data contaminated by disturbing signals, and
the other is 2-D visualizing procedure which back-projects the filtered data
onto the imaging plane. Computer simulation demonstrated the beautiful
performance of the proposed algorithm. We also developed a prototype of
depth selective diffuse optical topography system, and performed phantom
experiments. 10 light source(780nm) fibers and 8 detecting fibers were
arranged on the side wall of plastic box which contains dye-diluted aqueous
suspension of polystyrene particles adjusted to have typical optical
coefficients of human cortex. Two small absorption bodies made of black
acrylic resin, A(2x2x2mm3) and B(1.15x1.15x2mm3), were submerged in the
phantom container as the target and the disturbing body. The body A was
moved in the plane 10mm distant from the optodes surface, and the body B
was moved in the plane 2mm distant from the optodes. Experimental results
also showed that the proposed method dramatically suppressed the influence
of disturbing body in the shallow plane with minimal degradation of the target
signal.
6629-12, Session 3
Assessment of collagen absorption and related
potential diagnostic applications
P. Taroni, A. Giusto, A. Pifferi, Politecnico di Milano (Italy); N. S. Shah,
Univ. of California/Irvine (USA); L. Spinelli, A. Torricelli, R. Cubeddu,
Politecnico di Milano (Italy)
The sensitivity to collagen could be useful for diagnostic purposes, as collagen
seems to be involved in the development of breast cancer. Moreover, collagen
content is expected to be related to breast density (i.e. breast parenchymal
pattern) and its quantification could allow the classification of breast type.
Thus we have measured the absorption properties of collagen from 610 to
1040 nm. Absorption spectra of breast from healthy volunteers were then
interpreted adding collagen to the other absorbers previously considered
(i.e. oxy- and deoxyhemoglobin, water, and lipids). A significant amount of
collagen, depending on breast type, is estimated to be present and seems to
correlate with breast type. Moreover, adding collagen to the fitting procedure
affects remarkably the estimated values of blood content and oxygenation.
We have also upgraded our time-resolved multi-wavelength optical
mammograph, adding a long wavelength (1060 nm) to improve the spectral
information and, in particular, the sensitivity to collagen. Breast measurements
on volunteers have recently started.
Conventionally, the detection and characterization of an optical inhomogeneity
embedded in turbid media is dependent on the perturbation of diffuse photon
density wave (DPDW), and it is mainly determined by the signal-to-noise ratio
(SNR) of detected signal. In this study, we proposed the diffuse photon-pairs
density wave1 (DPPDW) to detect and characterize an optical inhomogeneity
in a multiple scattering medium. The results and SNR analysis on detection
and characterization of an inhomogeneity by DPPDW are demonstrated and
discussed. DPPDW is composed of polarized photon-pairs which experience
multiple scattering events. Polarized photon-pair are highly correlated and
their temporal frequencies are slightly different. Thus the optical heterodyne
signal of polarized photon-pair can be detected. We anticipate that the
propagating properties of DPPDW on the degree of spatial coherence (DOC)
and the degree of polarization (DOP) might result in a significant improvement
on the sensitivity of detecting perturbation in multiple scattering media.
6629-15, Session 3
Depth-resolution by continuous-wave imaging
E. B. Aksel, A. Akin, Bogaziçi Univ. (Turkey)
Potential of depth resolution of continuos-wave (CW) illumination in diffuse
optical imaging is explained. It is known both experimentally and numerically
that in CW measurements photons traversing a homogenous, semi-infinite,
highly scattering medium between a source and a detector located on the
surface of the medium follow paths that the volume interrogated resembles a
banana-shape. Also is known that, sensitivity profile of photon propagation in
CW measurements is non-uniformly distributed in depth, reaching a maximum
at a certain value depending on geometry, source-detector separation, and
optical properties of the medium. The presence of an inclusion with a higher
absorption coefficient with respect to that of the background in a homogeneous
medium can be estimated by increasing time-rate-of-photon-injection into the
medium. The inclusion is assumed to be at a depth between the optode pair
such that distances to optodes are the same. An increment in the time-rateof-photon-injection will give different detection slopes depending on the depth
of the inclusion, because the number of photons which is blocked by the
inclusion is high if it resides at a depth where the sensitivity profile has a higher
value. In this work, preliminary results of Monte-Simulation of light propagation
show that measuring slopes of increase in detected light intensity for different
interoptode distances are different for extreme case of screen between optodes
blocking all photons below a certain depth. Specification of inclusion with this
method may enable us to make predictions about the depth and optical
properties of the inclusion to be used as a priori information to be used image
reconstruction in diffuse optical tomography that may be integrated imaging
systems.
6629-16, Session 3
6629-13, Session 3
Influence of cell shape on the optical properties
of human erythrocytes
CW and time domain procedures for accurate
calibration of optical properties of liquid diffusive
media at NIR wavelengths
M. C. Meinke, Charité-Univ. Medizin Berlin (Germany); M. Friebel, Laserund Medizin-Technologie GmbH, Berlin (Germany); G. J. Müller, CharitéUniv. Medizin Berlin (Germany)
F. Martelli, Univ. degli Studi di Firenze (Italy); L. Spinelli, A. Farina, A.
Pifferi, A. Torricelli, R. Cubeddu, Politecnico di Milano (Italy); G. Zaccanti,
Univ. degli Studi di Firenze (Italy)
The characteristic changes in the cell shape of red blood cells (RBCs) can be
linked to various changes in both shear rate and osmolarity. The influence of
shear rate and osmolarity was investigated on the optical parameters:
absorption coefficient µa, scattering coefficient µs, and effective scattering
phase function of blood in the spectral range from 250 nm to 1100 nm.
Integrating sphere measurements of light transmittance and reflectance in
combination with inverse Monte-Carlo simulations were carried out for
different wall shear rates between 0 and 1000 s-1 and osmolarity variations
from 225 to 400 mosmol/L. Changes in shear rate and osmolarity have a
significant influence on the optical parameters which can in part be explained
by changes in the complex refractive index, cell shape and organization.
Spherical forms of RBCs induced by low osmolarity show reduced scattering
effects compared to the normal RBC biconcave disks shape. Spinocytes,
induced by high osmolarity, show the highest scattering behavior. Randomly
oriented cells exhibited maximum µa and µs values whereas cell alignment
and elongation at high shear rates lead to an asymptotical decrease. Moreover
a relationship was found to exist between the observed effects and the
hemoglobin absorption. In the paper there will be a discussion as to whether
optical parameters can be used to obtain information about the cell shape.
In spite of many progresses achieved both with theories and with experiments
in studying light propagation through diffusive media, a reliable method for
accurate measurements of the optical properties of diffusive media at NIR
wavelengths is, in our opinion, still missing. It is therefore difficult to create a
reference diffusive medium. We describe two methods in the CW and time
domain to calibrate the reduced scattering coefficient, m’s, of a liquid diffusive
medium and the absorption coefficient, ma, of an absorber with a standard
error smaller than 2% for both the coefficients. In this study Intralipid diluted
in water has been used as diffuser and Indian ink as absorber. The CW method
is based on multidistance measurements of fluence into an infinite medium
illuminated by a steady state laser diode. The optical properties are retrieved
with simple inversion procedures exploiting the knowledge of the absorption
of the liquid into which the diffuser and the absorber are dispersed. The time
domain method is based on time resolved measurements of transmittance
through a scattering cell illuminated by a mode-locked picosend Ti:sapphire
laser. The absorption coefficient is retrieved by the logarithmic ratio of two
measurements at different concentration of absorber, while m’s is retrieved by
the logarithmic ratio of two measurements at different source-detector
distances. The inversion procedures used to invert both CW and time domain
measurements are based on linear regressions. The asymmetry factor of the
scattering function of Intralipid and the single scattering albedo of Indian ink
have been also determined by measurements of collimated transmittance.
36
European Conferences on Biomedical Optics 2007 •
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Conference 6629: Diffuse Optical Imaging in Tissue
6629-17, Session 3
6629-20, Session 3
Determination of the optical properties of turbid
media by measurement of the spatially and
spectrally resolved reflectance
Time-resolved measurement of the scattered
light with an interferometric method based on
the use of a camera
M. Pilz, A. Kienle, Univ. Ulm (Germany)
D. Ettori, K. Zarychta, E. Tinet, S. Avrillier, J. Tualle, Ctr. National de la
Recherche Scientifique (France)
The absorption and reduced scattering coefficients determine the radial
dependence of the diffuse reflectance that is due to a point source. A system
consisting of a HeNe laser source and a CCD camera was built for making
remote measurements of spatially resolved diffuse reflectance. First, liquid
tissue phantoms were made of Intralipid and trypan blue. The absorption
and the reduced scattering coefficients are linear with the Intralipid and trypan
blue concentrations. A programme code was developed to determine the
optical properties of the tissue phantoms by fitting the measured data with
the solution of the diffusion equation. In addition, solid tissue phantoms were
made of agar, Intralipid and ink. Here a fiber-optic-based system was used
to measure perpendicular-incidence reflectance. The white light from a Xenon
lamp was coupled into the illumination fiber of the probe. A multiple fiberoptic detector in contact with the phantom surface at varying distances from
the source was used. The intensity signal was measured with twelve low
noise photodiodes. This allowed to determine the optical properties.
Measurement data both for semi-infinite and for parallelepipeded and
cylindrical tissue phantoms were compared with results of the diffusion theory.
6629-18, Session 3
Light attenuation through turbid slabs calculated
by solutions of the Maxwell equations
J. Schäfer, A. Kienle, F. K. Forster, Institut für Lasertechnologien in der
Medizin und Messtechnik (Germany); A. Strey, Univ. Ulm (Germany)
Two-dimensional light propagation in dentin slabs has been examined. Dentin
consists of long parallel oriented cylindrical tubules, hence a two-dimensional
light propagation model can be used. With the help of an analytical solution
of the scattering by two parallel cylinders a large dependency of the scattering
properties on the cylinder position was found. When
one cylinder lies behind the other relative to the orientation of the incident
light wave, the total scattering cross section gets much smaller.
The impact of this effect on scattering by dentin slabs containing multiple
cylindrical structures was studied. For this purpose we used the twodimensional finite-difference time-domain (FDTD) method. An appropriate
simulation model for dentin was designed. With a self-developed parallel
2D-FDTD code scattering parameters for different tissue thicknesses and
cylinder concentrations have been computed. Dentin models with randomly
distributed cylinders have been created and multiple simulation runs have
been performed for each configuration to acquire averaged results. We
examined a large decrease of the scattering cross section per scatterer when
concentration or thickness increased. We state that this behaviour is caused
by interference and cannot be explained with a theoretical model neglecting
interference effects.
6629-19, Session 3
Path-length correction for the haemoglobinconcentration measurement using the skull
cranial window by multi-spectral imaging
analysis
K. Sakaguchi, S. Furukawa, Keio Univ. (Japan); T. Katsura, K. Yamazaki,
H. Kawaguchi, A. Maki, Hitachi, Ltd. (Japan); E. Okada, Keio Univ.
(Japan)
Optical imaging of exposed cortex to measure the concentration change in
haemoglobin has been applied to the basic researches on the haemodynamic
responses caused by the brain activity. Recently, the simplified cranial window,
which is transparent thinned skull, is more commonly used for optical imaging
of the cortex. In this study, we analyse the difference between the spectral
images of the cortex through the thinned skull and those of the exposed
cortex in experimental measurements. The concentration changes in oxyhaemoglobin and deoxy-haemoglobin in the brain cortex of guinea pigs
associated with brain activation are measured from the multi-spectral images
of the cortex at 510, 540, 560 and 580 nm wavelengths. The cortical tissue is
observed through the thinned skull and the skull thickness is varied from 50
to 150 µm. The wavelength dependence of the optical path length estimated
from the multi-spectral images by the proposed method. The amplitude of
the change in reflectance decreases with an increase in skull thickness. The
wavelength dependence of optical path length in the cortical tissue with the
thinned skull is almost the same as that of the exposed cortex when the skull
thickness is less than 120 µm. Although the skull thickness affects the
sensitivity of the change in haemoglobin concentration, the influence of skull
thickness on the wavelength dependence of the optical path length can be
ignored when the skull thickness is approximately less than 100 µm.
European Conferences on Biomedical Optics 2007 •
We have already demonstrated the potentiality of interferometry to perform
time-resolved measurements of the light scattered by a tissue: the fluctuations
of the speckle pattern, linked to a wavelength-modulation of the source, are
registered, and the time-resolved average intensity can then be numerically
obtained from these data. Competitive results were obtained with a simple
photodiode as detector.
Such a method can be cheaper and more accessible for biomedical
applications, but it is not its unique interest: this method allows to perform
Diffusing Wave Spectroscopy (DWS) with selected photon pathlengths; for
instance, we have shown that we can improve the spatial resolution in
transillumination imaging of a dynamic heterogeneity through the selection
of short photon transit times. Therefore such a method can offer interesting
applications, in mammography for instance.
A way to improve the signal to noise ratio of this method can consist in
multiplying the number of detectors. That’s the reason why we decide to
consider the use of a high speed camera, that can reach a rate of 1000
frames per second. We will present the first results obtained with this new
system: the performances will be discussed, and compared to our previous
setup. Potential applications in imaging and time-resolved DWS will be
considered and discussed.
6629-21, Session 3
Determination of the optical properties of
anisotropic biological media using isotropic and
anisotropic diffusion models
A. Kienle, Univ. Ulm (Germany); C. Wetzel, Institut für Lasertechnologien
in der Medizin und Messtechnik (Germany); A. L. Bassi, D. Comelli, P.
Taroni, A. Pifferi, Politecnico di Milano (Italy)
We investigated the anisotropic light propagation in biological tissue in the
steady-state and time domains. Monte Carlo simulations performed for tissue
that consists of aligned cylindrical and of spherical scatterers show that the
steady-state and time-resolved reflectance depends strongly on the
measurement direction relative to the alignment of the cylinder axis. We
examined the determination of the optical properties using an isotropic
diffusion model and found that in the time domain, in contrast to steadystate
spatially-resolved reflectance measurements, the obtained absorption
coefficient does not depend on the measurement direction and is close to
the true value. Contrarily, the derived reduced scattering coefficient depends
strongly on the measurement direction in both domains. Measurements of
the steady-state and time-resolved reflectance from bovine tendon confirmed
the theoretical findings.
In addition, we compared the results obtained from Monte Carlo simulations
with the solutions of the anisotropic diffusion theory in the steady-state and
time domains. In contrast to the literature, we found that the anisotropic
diffusion equation in many cases is not a valid approximation to the anisotropic
light propagation even
in the diffusive regime. Finally, we investigated the determination of the optical
properties from spatially-resolved and time-resolved
transmittance using the anisotropic diffusion theory.
6629-22, Session 4
Imaging of metabolic and vascular reactivity in
joints with dynamic optical tomography
A. H. Hielscher, J. M. Lasker, C. J. Fong, E. Dwyer, Columbia Univ.
(USA)
Dynamic optical tomography is increasingly applied to clinically relevant areas
such as brain and cancer imaging. In this approach, some external stimulus
is applied and changes in relevant physiological parameters, e.g. oxy or deoxyhemoglobin concentrations, are determined. The advantage of this approach
is that the pre-stimulus state can be used as a reference or baseline against
which the changes can be calibrated. Here we present the first application of
this method to the problem of characterizing joint diseases, especially effects
of rheumatoid arthritis (RA) in the proximal- interphalangeal (PIP) finger joints.
Using a dual-wavelength tomographic imaging system together with
previously implemented model-based iterative image reconstruction schemes,
we have performed dynamic imaging studies on 6 healthy volunteers and 8
patients diagnosed with RA.
Image-series obtained from patients afflicted with varying degrees of
rheumatoid arthritis and healthy patients, demonstrate complex but promising
results. We observed numerous differences between healthy and afflicted
joints in both the temporal profiles of the detected light intensities, and of the
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Conference 6629: Diffuse Optical Imaging in Tissue
resulting reconstructed images of the spatially-dependent optical and
hemodynamic properties. Our analysis of the images seems to be in
agreement with other vascular studies that have shown an enhanced vascular
supply feeding the rheumatoid joint. Additionally, the dynamic hemoglobin
characteristics support existing works that show a rise in oxygen consumption
and metabolic activity in rheumatoid arthritis. Overall our study confirms our
hypothesis that differences in the vascular reactivity exist between affected
and unaffected joints, which can be used in clinical assessment of RA.
6629-24, Session 4
Non-invasive, depth-selective recovery of
fluorescence signals from the adult human head
by time-domain measurements
J. M. Steinbrink, Charité-Univ. Medizin Berlin (Germany); H. Wabnitz, A.
Jelzow, Physikalisch-Technische Bundesanstalt (Germany); H. Obrig,
Charité-Univ. Medizin Berlin (Germany); R. Macdonald, PhysikalischTechnische Bundesanstalt (Germany)
Non-invasive detection of fluorescent probes in small animals has opened
new opportunities for studying brain pathologies [Ntziachristos et. al., Nat.
Med., 2002]. Very recently, it has been shown that the fluorescence of a nonspecific dye can also be detected non-invasively from the human brain [Liebert
et. al., Neuroimage, 2006] at considerable low detection limits of 0.01
micromole/l [Steinbrink et. al., J. Neurodeg. Disease, submitted], opening
the possibility of non-invasive molecular imaging in the human brain. Besides
a molecular approach, non-specific dyes can be used to monitor cerebral
perfusion at the bed-side. However, the current challenge for both
applications is to separate between the disturbing extra-cerebral fluorescence
and the intra-cerebral fluorochrome distribution of interest.
In this contribution we propose a novel algorithm for determining the
fluorochrome distribution in the brain from time-domain measurements. The
algorithm is based on a linear equation linking the fluorochrome concentrations
in the extra- and the intra-cerebral compartment to the measured distribution
of arrival times of fluorescence photons via time-dependent sensitivity factors.
These sensitivity factors can be derived from simulations or in-vivo
measurements. For the latter case bolus injection experiments in healthy
volunteers using indocyanine green (ICG) as a tracer are analysed.
Assumptions on the time course of the tracer concentrations in the brain and
in the scalp allow for the determination of the time-dependent sensitivity
factors. Based on these sensitivities, the time course of the tracer
concentration in the brain as well as in the extracerebral tissue is retrieved.
6629-25, Session 4
Algorithms for muscle oxygenation monitoring
corrected for adipose tissue thickness
D. Geraskin, Univ. of Applied Sciences Koblenz (Germany); P. Platen, J.
Franke, Ruhr Univ. Bochum (Germany); M. Kohl-Bareis, Univ. of Applied
Sciences Koblenz (Germany)
The measurement of skeletal muscle oxygenation by NIRS methods is
obstructed by the subcutaneous adipose tissue which might vary between <
1 mm to more than 12 mm in thickness. A new algorithm is developed to
minimize the large scattering effect of this lipid layer on the calculation of
muscle haemoglobin / myoglobin concentrations. First, we demonstrate by
comparison with ultrasound imaging that the optical lipid signal peaking at
930 nm is a good predictor of the adipose tissue thickness (ATT).
Second, the algorithm is based on measurements of the wavelength
dependence of the slope DA/Dr of attenuation A with respect to source
detector distance r and Monte Carlo simulations which estimate the muscle
absorption coefficient based on this slope and the additional information of
the ATT. Third, we illustrate the influence of the wavelength dependent
transport scattering coefficient of the new algorithm by using the solution of
the diffusion equation for a two-layered turbid medium. This method is tested
on experimental data measured on the vastus lateralis muscle of volunteers
during an incremental cycling exercise under normal and hypoxic conditions
(corresponding to 0, 2000 and 4000 m altitude). The experimental setup uses
broad band detection between 700 and 1000 nm at six source-detector
distances. We demonstrate that the description of the experimental data as
judged by the residual spectrum is significantly improved and the calculated
changes in oxygen saturation are markedly different when the ATT correction
is included.
6629-26, Session 4
Assessment of muscle vascular disease with
diffuse light
G. Yu, T. Durduran, C. Zhou, G. Lech, R. Choe, E. R. Mohler, A. G. Yodh,
Univ. of Pennsylvania (USA)
Peripheral arterial disease (PAD) affects an estimated 25% of the elderly
population in North America. Non-invasive characterization of blood flow,
oxygenation and oxygen consumption in skeletal muscles has important
application for understanding vascular conditions and to screen and assess
38
European Conferences on Biomedical Optics 2007 •
PAD treament. In order to determine dynamic blood flow and oxygenation
simultaneously in the deep microcirculation, we have developed a hybrid
diffuse optical probe combining two qualitatively different methodologies:
(1) Diffuse Correlation Spectroscopy(DCS) for measurement of blood flow
and (2) Diffuse reflectance Spectroscopy (DRS) for measurement of tissue
oxygenation. The experimental protocols used in this study include two
parts: three-minute arterial cuff occlusion of leg (220mmHg) and one-minute
plantar flexion exercise. Nine healthy subjects, ages between 24-34
(28.4±3.0), and nine male PVD patient ages between 55-75 (63±6) were
measured. The mean ABI of the healthy subjects is 1.01±0.02, considered
normal, whereas the ABI of the patients is in the range of 0.35-0.85. Using
the hybrid optical instrument we were able to distinguish features
differentiating normal and diseased muscle responses. For example, the
increases of blood flow (355.8 ± 82.6%) and oxygen consumption (416.8 ±
118.3%) in PAD patients (9 legs) during exercise were lower than those (473.7
± 138.6%, 694.5 ± 176.5%) in nine healthy controls and the recovery times
of StO2 and rBF after cuff occlusion were significantly longer than those of
controls. These results indicate that diseased tissues have weaker capability
of oxygen delivery and consumption, and need longer time for recovery.
6629-27, Session 4
fDOT imaging of vascular autoregulation in
healthy and TBI subjects
H. L. Graber, SUNY/Downstate Medical Ctr. (USA) and NIRx Medical
Technologies, LLC (USA); M. Farber, D. Sreedharan, SUNY/Downstate
Medical Ctr. (USA); Y. Pei, NIRx Medical Technologies, LLC (USA); Y. Xu,
SUNY/Downstate Medical Ctr. (USA) and NIRx Medical Technologies,
LLC (USA); C. H. Schmitz, NIRx Medical Technologies, LLC (USA); G. T.
Voelbel, G. R. Wylie, J. Lengenfelder, J. DeLuca, Kessler Medical
Rehabilitation Research and Education Corp. (USA); R. L. Barbour,
SUNY/Downstate Medical Ctr. (USA) and NIRx Medical Technologies,
LLC (USA)
Because vascular autoregulatory mechanisms are strongly influenced by the
properties of feedback mechanisms, transitions from states of homeostatic
balance to imbalance, and back, can be expected to follow a specific
sequence, the dynamics of which will be sensitive to the particular stimulus
and its duration. This suggests that damage to tissue caused by disease or
trauma could produce alterations in the pathways/dynamics leading from or
to states of homeostatic balance (see, also, the accompanying report by
Barbour et al.). Here we report method validation findings and present results
from functional neuroimaging studies on healthy subjects and those with
documented traumatic brain injury (TBI).
Validation Studies: Under normal circumstances, the vascular response to
oxygen debt is vasodilation, not vasoconstriction, suggesting an underlying
directionality to autoregulatory state transitions. Studies performed in the
resting forearm confirm that the preferred directionality is at least 2:1 in favor
of the expected direction, increasing to \>10:1 with provocation. Backward
transitions can be induced, given specific stimuli (e.g., use of carbogen).
When (computer-generated) white noise or experimentally measured noise
is substituted for the physiologic time series, this bias is eliminated.
Brain Imaging Studies: We have analyzed data from healthy subjects and
those with documented TBI, at rest, while undergoing an N-back study, and
during tests of verbal fluency. Comparison of state transitions suggests that
event-related transitions and those seen at rest can be markedly different in
subjects with TBI. Also different are the amplitudes of responses within a
particular state. A comparison of these results to those obtained from
integration of Hb states (usual approach) is presented.
6629-28, Session 5
Modeling of influence of frontal sinus on NIRS
signal of brain activation
D. Yamamoto, Keio Univ. (Japan)
In the brain activation measurements by near infrared spectroscopy (NIRS),
the partial optical path length, which is an index of the sensitivity of the NIRS
signal to brain activation, is strongly affected by the thickness and the structure
of the superficial tissues. In this study, we investigated the influence of the
frontal sinus on the NIRS signal of the brain activity. The NIRS signal caused
by the calculation task was measured to observe the difference in light
propagation caused by the presence of the frontal sinus. The source-detector
spacing was 32 mm and 16 mm to detect the change in the tissue absorption
in the deep and shallow regions. The source-detector pairs at spacing of 32
mm detect the brain activation on both the sides of the prefrontal region
caused by the calculation task. In the case of source-detector spacing of 16
mm, the source-detector pairs on the right side without the frontal sinus
scarcely detected the NIRS signal whereas those on the left side with the
frontal sinus detected the signal caused by the calculation task. The light
propagation in a simplified head model including the frontal sinus was
predicted by Monte Carlo simulation to investigate the influence of the frontal
sinus on the partial optical path length in the brain. The frontal sinus strongly
affects the light propagation in the head and the partial optical path length
for small source-detector separation is increased by the presence of a frontal
sinus.
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Conference 6629: Diffuse Optical Imaging in Tissue
6629-29, Session 5
Optical tomographic imaging of activation of the
infant auditory cortex using perturbation Monte
Carlo with anatomical a priori information
J. K. Heiskala, Helsinki Univ. of Technology (Finland) and Consultant
(Finland) and Univ. of Helsinki (Finland); K. M. Kotilahti, L. T. Lipiäinen, P.
J. Hiltunen, Helsinki Univ. of Technology (Finland) and Univ. of Helsinki
(Finland); P. E. Grant, Massachusetts General Hospital (USA) and
Consultant (USA); I. T. Nissilä, Massachusetts General Hospital (USA)
We study the use of the perturbation Monte Carlo (pMC) method with
anatomical a priori information for reconstructing cerebral blood volume
and oxygen saturation changes from near-infrared optical imaging
measurements of infants.
Monte Carlo methods can simulate the propagation of near-infrared radiation
in tissue accurately and are capable of correctly modeling the effect of lowscattering tissues such as the cerebrospinal fluid. The use of the pMC method
for solving for changes in optical properties of tissue has been successfully
demonstrated.
We propose a novel 3D voxel-based pMC method which can make use of
time or frequency domain data. Using time and frequency domain data may
allow a more accurate reconstruction of the hemodynamic changes than
previously reported pMC methods which only use intensity data. The voxelbased implementation of the pMC method allows the tissue model to have
an arbitrary geometry. We demonstrate the use of an MR imaging based
infant head model for localizing brain activations.
Digitized landmark points on the head of the infant are used to warp the
computerized head model to correspond to the head of the infant being
imaged.
We study the usefulness of our method by comparing its performance in
reconstructing simulated brain activation to that of standard reconstruction
methods relying on the diffusion approximation of the radiative transfer
equation and pMC reconstruction using only intensity data. Finally we
demonstrate the use of our method by reconstructing brain activation from
real measurement data obtained with our frequency-domain optical imaging
instrument.
6629-30, Session 5
Cerebral oxygenation monitoring during cardiac
bypass surgery in infants with broad band
spatially resolved spectroscopy
J. Soschiniski, Univ. of Applied Sciences Koblenz (Germany); U. Fischer,
Univ. zu Köln (Germany); D. Geraskin, Univ. of Applied Sciences
Koblenz (Germany); U. Mehlhorn, G. Bennink, Univ. zu Köln (Germany);
M. Kohl-Bareis, Univ. of Applied Sciences Koblenz (Germany)
Neurological impairments following cardio-pulmonary bypass (CPB) during
open heart surgery can result from microembolism and ischaemia. Here we
present results from monitoring cerebral hemodynamics during CPB with
near infrared spatially resolved broadband spectroscopy. In particular, the
study has the objective (a) to monitor oxy- and deoxy-hemoglobin
concentrations (oxy-Hb, deoxy-Hb) and their changes as well as oxygen
saturation during CPB surgery and (b) to develop and test algorithms for the
calculation of these parameters from broad band spectroscopy. For this
purpose a detection system was designed based on an especially designed
lens imaging spectrograph with optimised sensitivity of recorded reflectance
spectra for wavelengths between 600 and 1000 nm. The high f/#-number of
1:1.2 of the system results in about a factor of 10 higher light throughput
combined with a lower astigmatism and crosstalk between channels when
compared with commercial mirror spectrometers (f/# = 1:4). For both
hemispheres two independent channels each with three source-detector
distances (r = 25 - 45 mm) were used resulting in six spectra. The broad
band approach allows to investigate the influence of the wavelength range
on the calculated haemoglobin concentrations and their changes and oxygen
saturation when the attenuation A(lambda) and its slope dA(lambda)/dr are
evaluated. Furthermore, the different depth sensitivities of these measurement
parameters are estimated from Monte Carlo simulations and exploited for an
optimization of the cerebral signals. It is demonstrated that the system does
record cerebral oxygenation parameters during CPB in infants. In particular,
the correlation of haemoglobin concentrations with blood supply (flow,
pressure) by the heart-lung machine and the significant decreases in oxygen
saturation during cardiac arrest is discussed.
6629-31, Session 5
Comparison of various methods to enhance
depth selectivity in time-domain brain imaging
H. Wabnitz, Physikalisch-Technische Bundesanstalt (Germany); A.
Liebert, Institute of Biocybernetics and Biomedical Engineering
(Poland); D. Contini, L. Spinelli, A. Torricelli, Politecnico di Milano (Italy)
time-resolved diffuse reflectance solves this problem and allows to achieve
depth localization of absorption changes. The present work aims at optimizing
time-domain brain imaging by comparing different approaches to discriminate
between cerebral and extracerebral signals. In particular, changes in moments
of the measured time-of flight distribution of photons were compared to
contrast based on the ratio of photon counts in late and early time windows.
By using a perturbation approach, 3D sensitivities to absorption changes
were calculated. Furthermore, Monte-Carlo simulations were employed to
model a focal activation at various depths. To compare the discrimination
potential of the different quantities, the ratios of changes upon deep and
superficial activation were calculated. With time windows of optimized position
and width, a higher contrast ratio can be achieved compared to moments.
However, moments and time windows differ with respect to the influence of
the instrumental response function (IRF). As expected, the relative changes
in integral, mean time of flight and variance did not alter upon convolution of
the simulated distribution with a typical experimental IRF, whereas the relative
contrast for time windows dropped considerably. Finally, examples of the
application of both approaches to in-vivo experiments with functional
stimulation are shown. In conclusion, both, moments and ratios of time
windows are suited to isolate cerebral signals, however, the selection of the
optimum quantity depends on the particular experimental situation.
6629-32, Session 5
Time-resolved non-contact diffuse optical
tomography measurements with ultra-fast timecorrelated single photon counting avalanche
photodiodes
Y. Bérubé-Lauzière, V. Robichaud, É. Lapointe, Univ. de Sherbrooke
(Canada)
Time-resolved (TR) diffuse optical tomography (DOT) systems using timecorrelated single photon counting (TCSPC) developed thus far use exclusively
photomultiplier tubes (PMTs). Temporal resolutions of 200ps are achieved
with moderate cost PMTs (~5k$). MCP-PMTs were until recently the best
detectors for TCSPC in terms of temporal resolution (30ps), but are much
more expensive (~25k$) and easy to damage. Our objective is to demonstrate
the viability of using APDs in TR DOT, in particular in a non-contact DOT
scanner we are currently developing for small animal optical fluorescence
molecular imaging. Recent advances in the design and fabrication of ultrafast APDs for TCSPC has made available detectors with temporal resolutions
as high as for MCP-PMTs, but at about 1/5th of the price. APDs are much
more compact than PMTs, can incorporate all the electronics and cooling on
a single chip, do not require high voltage supplies, and are not damaged,
even by ambient light levels. We demonstrate that with appropriate optical
design of the detection channel, photon count rates with APDs can be as
high as with PMTs despite the APD small sensitive area, with the additional
benefit of a better temporal resolution and less dark counts. We present
TCSPC fluorescent tomographic measurements we have performed on
representative diffusing and absorbing media with an APD to support our
approach and compare these with measurements made under the same
experimental conditions with a PMT. This is to our knowledge, the first
implementation of non-contact TR fluorescence DOT measurements with
APDs.
6629-33, Session 5
Transient tissue dynamics in the stimulated
human brain measured by time-resolved
diffusing-wave spectroscopy
T. Gisler, J. Li, F. Jaillon, G. Dietsche, T. Elbert, B. Rockstroh, G. Maret,
Univ. Konstanz (Germany)
Diffusing-wave spectroscopy (DWS), the extension of quasi-elastic light
scattering to highly turbid media, was recently introduced as a non-invasive
optical method for the detection of human brain function through the intact
scalp and skull [1, 2]. Measurements on the sensorimotor cortex showed a
marked hemispheric asymmetry in the tissue dynamics measured by DWS
following motor stimulation.
In the present contribution we present a multi-speckle detection setup which
allows to measure DWS autocorrelation functions with an integration time of
26ms. This method allows to follow transient scatterer dynamics in the human
cortex following activation such as by motor and visual stimuli. Motor
stimulation data show a strong coupling of the DWS decay rate with the
NIRS signal and very strong variations during the pulsation cycle, indicative
of strong coupling of the observed scatterer dynamics with pulsation. We
analyze the shapes of the DWS autocorrelation function at different phases
of the pulsation cycle and discuss the possible origins of the strong coupling
of the DWS signal to pulsation.
1. Durduran, T., et al., Opt. Lett., 2004. 29: p. 1766-1768.
2. Li, J., et al., J. Biomed. Opt., 2005. 10: p. 044002-1-12.
A key problem in optical imaging of the adult brain is the elimination of the
influence of superficial, systemic effects from the measured signals. Measuring
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Conference 6629: Diffuse Optical Imaging in Tissue
6629-34, Session 6
Time-of-flight non-contact fluorescence diffuse
optical tomography with numerical constant
fraction discrimination
Y. Bérubé-Lauzière, V. Robichaud, Univ. de Sherbrooke (Canada)
Localizing fluorescent inclusions in 3D in thick turbid media non-invasively
by optical imaging techniques is the subject of fluorescence diffuse optical
tomography (FDOT). Fluorescence imaging through large tissue volumes
paves the way to optical molecular imaging in-vivo which is of great interest
for the medical and pharmaceutical communities, such as for breast cancer
detection and small animal imaging for drug discovery. In our work, we perform
the measurements in a non-contact manner with the medium, thereby
implementing non-contact FDOT which is a necessity as we target small
animal imaging. We have recently introduced a numerical constant fraction
discrimination (NCFD) technique [SPIE Optics East ’06] for processing timeresolved optical signals to extract, in a stable manner, the arrival time of early
photons emitted by a fluorescent inclusion embedded in a scattering medium.
We have demonstrated that these arrival times correlate quasi-linearly with
inclusion depth. Here, we go one step further by exploiting this arrival time
vs depth relationship for inferring the 3D position of the inclusion by way of a
novel spherical wavefront intersection algorithm, thus enabling true direct
time of flight DOT for the first time. Our approach does not rely on intensity
information. Our experimental set-up uses several detectors arranged in a
multi-view ring configuration. We give experimental results showing the
capability of our approach to localize a 3mm diameter spherical inclusion
filled with indocyanine green (ICG) embedded in a cylindrical thick turbid
medium (5cm in diameter) with absorption and scattering properties
representative of biological tissues.
6629-35, Session 6
Double labeling optical fluorescence
tomography for rodents using a multiwavelength
scheme
R. Bourayou, Charité-Univ. Medicine Berlin (Germany); J. M. Steinbrink,
Charité-Univ. Medizin Berlin (Germany); J. Klohs, R. Cordell, P.
Bahmani, A. Wunder, U. Lindauer, Charité-Univ. Medicine Berlin
(Germany); F. Lehmann, Dyomics GmbH (Germany); A. Villringer, U.
Dirnagl, Charité-Univ. Medicine Berlin (Germany)
Multiple labeling is of current use in fluorescence microscopy and resolves
structural and chemical information of the tissue [Wang XF & Herman B,
“Fluorescence Imaging Spectroscopy and Microscopy”, 1996, J. Wiley &
Sons, New York]. The interpretation of in-vivo images taken on animal models
is on the contrary intricate [Klohs et. al. Mol. Img. 2006], particularly when
multiple dyes are used and quantification is of relevance. For most
applications, the distribution of the excitation and fluorescence photons inside
the tissue is different for each fluorophore; Optical Fluorescence Tomography
inherently models these quantities and already proved successful for a single
dye [Ntziachristos et al., European Radiology, vol. 13, pp. 195-208, 2003].
We show here the extension of the technique for murine model investigations
where tagging with multiple fluorescent NIR probes is wished.
Using a fiber-based fluorescence tomograph, we show the possibility to
reconstruct the bio-distribution of two dyes simultaneously. Measurements
and associated tomographic reconstructions are carried out on a tissuesimulating phantom. The setup uses laser diodes and optical filters, so the
choice of the fluorescing substances conditions the usefulness of the
wavelengths for the excitation and for the detection, but also the relevance
of cross-talk effects. We chose a label needing a red excitation (DY682-BSA
Dyomics, Jena, Germany) and indo-cyanine green (ICG, Pulsion, Munich,
Germany) in order to minimize the unwished superimposed auto-fluorescence
signal. What’s more, by changing the combination of the excitation
wavelengths, the amount of cross-talk in the sensitivity matrix can be varied
in order to allow the performance comparison of optical fluorescence
tomography with independent and partially dependent dataset.
6629-36, Session 6
360° free space fluorescence molecular
tomography using silhouette surface
reconstruction
T. R. Lasser, Munich Univ. of Technology (Germany); N. Deliolanis, A.
Soubret, Massachussetts General Hospital (USA); J. Ripoll, Foundation
for Research and Technology (Greece); V. Ntziachristos,
Massachussetts General Hospital (USA)
Complete projection (360°) free-space fluorescence tomography of opaque
media is poised to enable highly performing three-dimensional imaging
through entire small animals in-vivo. This approach can lead to a new
generation of Fluorescence Molecular Tomography (FMT) systems since it
allows high spatial sampling of photon fields propagating through tissue at
any projection, employing non-constricted animal surfaces.
Key features of this development is the implementation of non-contact
40
European Conferences on Biomedical Optics 2007 •
illumination, for example by using beam scanning techniques for light delivery
on the tissue surface and direct non-contact imaging with CCD cameras.
Similarly, the development of free-space geometries, i.e. implementations
that do not utilize immersion of the animal in matching fluids are essential for
obtaining appropriate experimental simplicity and avoid unnecessary diffusion
through scattering matching media.
To facilitate these developments it is important to retrieve the threedimensional surface and a common coordinate system for the illumination
system, the detection system and the animal. Herein, we employ a volume
carving method to capture three-dimensional surfaces of diffusive objects
from its silhouettes and register the captured surface in the geometry of an
FMT 360°-projection acquisition system to obtain three-dimensional
fluorescence image reconstructions. Using experimental measurements we
evaluate the accuracy of the surface capture procedure by reconstructing
the surfaces of phantoms of known dimensions and demonstrate how this
surface extraction method can be utilized in an FMT inversion scheme. We
then employ this methodology to characterize the animal movement of
anaesthetized animals and study the effects of animal movement on the FMT
reconstructed image quality.
6629-37, Session 6
Whole body in vivo examination of small animals
by simultaneous X-Rays/optical tomography:
comparison between the reconstructions
obtained with different types of fluorescent
labels
A. Da Silva, T. Bordy, M. Debourdeau, J. Dinten, P. Peltie, P. Rizo, Lab.
d’Electronique de Technologie de l’Information (France)
Small animal diffuse optical tomography is an appealing tool for the
investigation of molecular events in cancer research and drug developments.
The combination of the functional information brought by an optical system
and the anatomical information delivered by X-Rays enables i) a fast
multimodality animal examination; ii) the correlation between the
biodistribution of the molecular probes and the morphology of the animal; iii)
a more accurate optical data reconstructions by using the anatomy of the
animal as a constrain in the reconstructions.
A small animal multimodality tomographer for the coregistration of
fluorescence optical signals and X-rays measurements is used in the present
study. The optical system is composed with a CW laser and a CCD camera
coupled with an appropriate combination of filters for the fluorescence
detection. The animal is placed inside a transparent tube filled with an index
matching fluid. The X-ray generator and detector have been positioned
perpendicularly to the optical chain.
Original optical calibration techniques have been developed in order to control
at any time the alignment between the incident beam, the axis of the cylinder
and the focus plan of the CCD. Specific developments have also been handled
for obtaining the geometry correlation between optical and X-rays data
reconstructions.
This experimental setup is used in the present work for a in vivo biological
study conducted on mice bearing tumors in the lungs, and tagged with
different kinds of near infrared optical probes (targeting probes such as
Transferin-AlexaFluor 750 or activatable such as RAFT-(cRGD)4-Alexa700).
6629-38, Session 6
Time-domain fluorescence diffuse optical
tomography in heterogeneous media
S. Fortier, F. Leblond, ART Advanced Research Technologies Inc.
(Canada)
In the last few years, the importance of fluorescence diffuse optical
tomography (FDOT) has been growing in the molecular imaging field. The
goal of FDOT is the recovery of the location and concentration of fluorescent
probes embedded in specimens such as small animals. A common approach
used to solve the related inverse problem in highly diffusive media is to assume
homogeneous optical properties in the specimen, and use the analytical
solutions to the diffusion equation (DE) as the photon transport model (the
“homogeneous model”). However, it is a well known fact that small animals
are strongly heterogeneous, both in absorption and scattering. Furthermore,
it was experimentally demonstrated that the presence of absorption
heterogeneities adversely affects the quality of FDOT solutions based on the
homogeneous model, and that the so-called “Born-Normalization” approach
mitigates such adverse effects [A. Soubret et al, IEEE Trans. on Med. Imag.
24, Oct. 2005]. In this context, our work first extends the homogeneous model
to heterogeneous media by introducing linear perturbations to the DE’s
analytical solutions (the “heterogeneous model”). Then, using data generated
with the heterogeneous model, we characterize the effect of the presence of
both absorption and scattering heterogeneities (and the mitigation of that
effect by Born Normalization) in two contexts: FDOT based on: a) the
homogeneous model (common practice); b) the heterogeneous model
(assuming optical property estimates are made available through such means
as multimodal imaging). Experimental results are also shown.
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Conference 6629: Diffuse Optical Imaging in Tissue
6629-39, Session 6
Multi-channel time-domain fluorescence
mammograph
A. J. Hagen, O. Steinkellner, D. Grosenick, Physikalisch-Technische
Bundesanstalt (Germany); M. Möller, Hochschule fur Technik und
Wirtschaft des Saarlandes (Germany); R. Ziegler, T. Nielsen, Philips
Research Labs. (Germany); K. Lauritsen, PicoQuant GmbH (Germany);
R. Macdonald, H. H. Rinneberg, Physikalisch-Technische Bundesanstalt
(Germany)
We report on the ongoing improvement of an eight-channel scanning timedomain fluorescence mammograph as part of the FLUOROMAMM research
project. It is capable of imaging the distribution of a non-specific fluorescent
contrast agent in the female breast, besides imaging intrinsic absorption
and scattering properties of healthy breast tissue and tumors. The apparatus
is based on the PTB multi-channel laser pulse mammograph, originally
designed for measurements of absorption and scattering coefficients at four
selected wavelengths (lambda = 652 nm, 684 nm, 797nm, and 830 nm). It
was upgraded for time-resolved detection of fluorescence, excited at 735
nm by a ps diode laser with 10 mW output power or alternatively by an
amplified ps diode laser with 36 mW output and detected at wavelengths
greater than 780 nm. Cooled PMTs with GaAs photocathodes are used to
detect laser and fluorescence photons at five positions in transmission and
three positions in reflection. Measurements are performed with the breast
being slightly compressed between two parallel glass plates. The transmitting
and receiving fiber bundles are scanned synchronously over the breast in
steps of typically 2.5 mm. At each scan position, distributions of times of
flight of laser photons are measured by time-correlated single photon counting
at eight detector positions, followed by measurements of distributions of
times of arrival of fluorescence photons. The performance of the fluorescence
mammograph was investigated by using breast-like phantoms with a
fluorescent inhomogeneity with dye enrichment varying between 2:1 and
10:1 over background values.
6629-40, Session 6
Time-resolved imaging of fluorescence
inclusions in optically turbid medium
M. Kacprzak, P. L. Sawosz, A. Liebert, R. Maniewski, Institute of
Biocybernetics and Biomedical Engineering (Poland)
We present results of application of a time-resolved optical system for imaging
of fluorescence excited in an inclusion located in optically turbid medium
and filled with ICG (Indocyanine Green). The developed imaging system
enabled simultaneous acquisition of fluorescence and diffuse reflectance.
Eight independent time-resolved measurement channels based on timecorrelated single photon counting technique were applied. In four of these
channels used for the fluorescence detection sets of filters were applied in
order to block the excitation light. 1x9 optical fibres switches allowed to
illuminate sequentially different spots on the surface of the phantom and
finally 4x4 pixels maps at excitation and emission wavelengths were obtained.
A fish tank was filed with a solution of milk and water with black ink added to
obtain optical properties in the range of the optical properties typical of the
living tissue. A gel ball of diameter of 5mm with precisely controlled
concentration of ICG was immersed in the liquid. The measurements were
performed for inclusion located at different depths and for different ICG
concentrations in the gel ball and in the surrounding liquid. The recorded
distributions of times of arrival of fluorescence photons and diffusely reflected
photons were analyzed by calculation of their statistical moments. This
analysis enables for depth resolved reconstruction of gel ball position inside
the physical phantom. We observed that the contrast in the maps of moments
depends on concentration of the dye in the gel ball as well as in the
surrounding liquid.
fluorescence distribution in presence of high attenuating objects has been
validated on phantoms presenting a fluorescent absorpbent inclusion and
compared to a Born normalized correction approach.
A study was conducted on mice in order to follow up by fDOT lungs at different
stages of tumour development. The mice were imaged after intravenous
injection to the animal of a cancer specific fluorescent marker i.e. 10 nmoles
RAFT-(cRGD)4-Alexa700, successively at 10, 12 and 14 days after the primary
tumour implantation. As expected, the reconstructed fluorescence increases
with the tumour age. A control experiment was conducted in parallel on
healthy mice to ensure that the multiple injections of fluorophore did not
induce parasite fluorescence distribution, and that the system does not detect
fluorescence when no marker injection is done.
These results validate our system performances for studying small animal
lungs tumour evolution. Detection and localization of the fluorophore fixations
follows the tumour development.
6629-42, Session 7
MRI-guided NIR spectroscopy of breast cancer
tumors: pilot studies
B. W. Pogue, Dartmouth College (USA)
MR Imaging can be enhanced through inclusion of NIR spectroscopy data,
when the MR is used to guide the mesh creation and provide the spatial
template upon which NIR spectral data is recovered. Recovery of Hemoglobin,
Oxgyen Saturation, Water, and exogenous agents is feasible, as well as
ultrastructural features related to NIR light scattering. The quality and accuracy
of the recovered data is somewhat dependent upon the type of regularization
used in the image reconstruction, and how the spatial prior information is
applied. Representative images in female breast cancer are presented,
showing that fibrogladular tissue can be used as the guide to recover
chromorphore values within this region. The potential use of this system is in
analyzing potential false positive or potential false-negative MRI exams. In
the case of a potential false positive image from MRI, where multiple regions
enhance with Gd-DTPA injection, then NIR can be used to quantify the
chromophore values in those regions. In the case of potential false negative
images, where a tumor does not enhance, the tumor region appears similar
to fibrogladular tissue and using soft-prior implementation the recovery of
heterogeneities within otherwise homogeneous volumes can be obtained.
These procedures are outlined in simulation and case studies with a functional
NIR spectroscopy system coupled into a Philips 3T MRI breast coil.
6629-43, Session 7
The twente photoacoustic mammoscope (PAM):
first clinical results
S. Manohar, S. Vaartjes, J. v. Hespen, J. Klaase, F. v. d. Engh, W.
Steenbergen, T. G. van Leeuwen, Univ. Twente (Netherlands)
6629-41, Session 7
The Twente Photoacoustic Mammoscope (PAM) is based on generating laserinduced ultrasound from absorbing structures in the breast. Excitation uses
pulsed laser light of wavelength 1064 nm and pulse width of 5 ns. Ultrasound
detection is performed using a flat detector matrix. Image reconstruction is
based on a phased-array algorithm. We report on the first results of a pilot
study aimed at studying the feasibility of using this instrument in detecting
tumours in the breasts of human patients. Patients with suspect breasts were
examined using PAM after conventional x-ray mammography and breast
sonography but before biopsy. Measurements were performed in regionsof-interest of the breast in craniocaudal views. Reconstructed images are
compared with the conventional radiological images and the results analyzed
in the light of pathology of resected tumours. Of the 5 technically acceptable
patient measurements, regions of higher optical absorption are clearly
identified in 4 cases. These correlate well with conventional radiological
images and with pathological results. The first results strongly support the
hypothesis of using photoacoustics to detect angiogenesis-driven optical
absorption contrast associated with malignancies in the human breast.
fDOT for in vivo follow-up of tumor development
in mice lungs
6629-44, Session 7
A. Koenig, L. Hervé, A. Da Silva, J. Dinten, J. Boutet, M. Berger, Lab.
d’Electronique de Technologie de l’Information (France); V. Josserand,
ANIMAGE (France); J. Coll, Institut Albert Bonniot (France); P. Peltié, P.
Rizo, Lab. d’Electronique de Technologie de l’Information (France)
This paper presents in vivo experiments conducted on cancerous mice
bearing mammary murine tumours.
In order to reconstruct the fluorescence yield even in highly attenuating and
heterogeneous regions like lungs, we developed a fDOT reconstruction
method which at first corrects the light propagation model from optical
heterogeneities by using the transmitted excitation light measurements. The
same approach is also designed to enable working without immersing the
mouse in adaptation liquid.
The 3D fluorescence map is then reconstructed from the emitted signal of
fluorescence and from the corrected propagation model by an ART (Algebraic
Reconstruction Technique) algorithm. The system ability to reconstruct
European Conferences on Biomedical Optics 2007 •
Breast cancer detection, characterization, and
therapy monitoring using diffuse optical
methods
R. Choe, S. D. Konecky, A. Corlu, K. Lee, C. Zhou, T. Durduran, M. A.
Rosen, M. D. Schnall, B. J. Czerniecki, J. C. Tchou, B. Chance, A. G.
Yodh, Univ. of Pennsylvania (USA)
Our group at the University of Pennsylvania has developed (1) a parallel plane
CCD-based DOT system with an emphasis on high number of source and
detector positions for reliable three-dimensional image reconstruction and
(2) a portable hand-held DCS system for blood flow measurements. These
instruments were used for characterizing optical contrast due to breast cancer
and for monitoring neoadjuvant chemotherapy.
From DOT data (N ~=50), total hemoglobin concentration, blood oxygen
saturation, reduced scattering coefficient distribution was reconstructed in
3-dimension. Then tumor region was selected based on radiology report
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Conference 6629: Diffuse Optical Imaging in Tissue
and/or MRI. The tumor-to-normal contrast was characterized by relative
parameters. These parameters were combined to define the Optical Index
(OI). There is a clear distinction between benign and malignant breast tumor.
For neoadjuvant chemotherapy patients with good response to therapy
showed decrease of optical contrast with the tumor volume shrinkage which
agreed with MRI results.
Increased blood flow was observed in tumor regions relative to healthy tissue,
and control subjects did not exhibit significant blood flow heterogeneity. For
both long-term and short-term monitoring studies, relative blood flow between
tumor and normal generally decreased with the progress of the therapy.
We are actively exploring the optical contrast available both by intrinsic
mechanism and extrinsic contrast agent (e.g. Indocyanine Green) in terms of
one-time detection to neoadjuvant chemotherapy monitoring. The most recent
data from the University of Pennsylvania regarding the breast cancer imaging
and spectroscopy will be presented in this meeting.
6629-45, Session 7
Diffuse correlation/wave spectroscopy for
measurement of cerebral blood flow at the
intensive care unit
T. Durduran, C. Zhou, B. Eldow, R. Choe, G. Yu, S. Kasner, B.
Cucchiara, J. H. Greenberg, J. A. Detre, A. G. Yodh, Univ. of Pennsylvania (USA)
Diffuse correlation spectroscopy (DCS) was pioneered in our laboratory and
has recently been translated to the neuro-intensive care unit.
Patients with diseases such as acute ischemic strokes are closely monitored
and managed via a complex mixture of drugs, surgical and non-surgical
interventions which aim to maximize the blood flow to the infarcted regions.
DCS is a very good candidate to monitor microvascular cerebral blood flow
because it is non-invasive, safe and can be operated at the bed-side providing
microvascular CBF at the cortex.
In this preliminary study, we have investigated DCS’ ability to measure the
impairment and recovery of cerebral autoregulation by inducing an intervention
- change of head of bed (HOB) position - which alters the cerebral perfusion
pressure (CPP) on acute stroke patients.
It is expected that if the cerebral autoregulation is intact, then the changes
between two hemispheres (healthy vs infarcted) and between different angles
should be minimal.
However, any impairment of autoregulation would imply that CBF is simply
driven by the CPP.
Optical measurements of relative CBF during HOB manipulations were
obtained from patients with acute hemispheric ischemic stroke (n=17) with
probes placed on the forehead measuring CBF near the frontal poles and
cohort of 12 subjects with no reported disease.
Overall, statistically analyzed results indicate that DCS has a potential for
critical clinical use in the intensive care units and is able to robustly monitor
local, microvascular cerebral blood flow of the cortical regions.
6629-46, Session 7
Rapid intraoperative diagnosis of sentinel node
metastases in breast cancer using elastic
scattering spectroscopy scanning
M. R. Austwick, W. D. Chicken, S. Somasundaram, B. R. Clark, A.
Mosse, M. Falzon, G. Kocjan, Univ. College London (United Kingdom); I.
J. Bigio, Boston Univ. (USA); S. G. Bown, M. Keshtgar, Univ. College
London (United Kingdom)
Lymphatic spread (the presence of metastatic cancer in the lymph nodes)
remains the strongest determination of prognosis in breast carcinoma, but
only a small proportion of newly diagnosed breast cancer patients actually
have lymphatic metastases at presentation. Sentinel node biopsy is able to
accurately determine whether or not patients have lymph node metastases,
and hence enables node-negative patients to avoid the unpleasant side effects
of axillary dissection and recover quicker from surgery. Using intraoperative
detection avoids a second operative procedure for node-positive patients.
Metastases may be detected intraoperatively using pathological techniques,
but all of these techniques are time consuming, resource intensive and yield
imperfect results.
Elastic scattering spectroscopy (ESS) is an optical technique which is sensitive
to the sizes, indices of refraction and structures of the sub cellular components
that change with malignant transformation. Multivariate statistical analysis
can be used to discriminate between spectra from malignant and benign
tissue.
The key attractions of ESS are that no tissue processing is required and that
the result does not require an expert cytologist or pathologist for interpretation.
Once suitable diagnostic algorithms have been developed, the result can be
generated by computer almost instantaneously. By utilising a fibre-optic plate,
the cut surface of an excised node can be interrogated, the spectra analysed,
and a false colour image of the node’s metastatic and normal regions built
up in real time, allowing the rapid intra-operative diagnosis of metastatic
42
European Conferences on Biomedical Optics 2007 •
nodes. The accuracy compares well with touch-imprint cytology, one of the
histopathological methods currently used for intra-operative diagnosis.
6629-47, Session 7
Monitoring hemodynamic responses to
antivascular therapy and ionizing radiation
assessed by diffuse optical spectroscopies
U. Sunar, Univ. of California/San Diego (USA); S. Makonnen, C. Zhou, H.
Wang, G. Yu, T. Durduran, W. M. F. Lee, A. G. Yodh, Univ. of Pennsylvania (USA)
In this study diffuse optical methods were used to monitor two different
therapies in K1735 malignant mouse melanoma tumor models: antivascular
therapy and radiation therapy. Antivascular therapy induced acute changes
(within an hour) and radiation therapy induced longitudinal changes (within 2
weeks) in hemodynamic parameters. An antivascular drug, Combretastatin
A-4 3-O-Phosphate (CA4P, 2.5 mg/200 µ l PBS/mouse), significantly
decreased tissue blood flow (64%) and oxygenation (67%) within one hour
post injection. In the longitudinal study, single-fraction ionizing radiation (12Gy
x 1) induced significant reduction in tissue blood flow (36%) and oxygenation
(52%) 14 days after radiation. The results also correlated well with contrast
enhanced ultrasound, tumor histology, and nitroimidazole hypoxia marker
(EF5). We conclude that noninvasive diffuse optical spectroscopies are
potential tools in monitoring acute and longitudinal effects of therapies with
quantification of both tissue blood flow and oxygenation.
6629-48, Session 7
Radiotherapy dosimetry assessment with optical
projection tomography
G. Zacharakis, Foundation for Research and Technology-Hellas
(Greece); A. Papadakis, Univ. Hospital of Heraklion (Greece); F.
Zacharopoulou, Univ. General Hospital of Herakleion (Greece); A.
Garofalakis, Foundation for Research and Technology-Hellas (Greece);
T. Maris, Univ. General Hospital of Herakleion (Greece); J. Ripoll,
Foundation for Research and Technology-Hellas (Greece)
Recent advances in radiotherapy have created the need to develop novel
methods for the accurate, three-dimensional assessment of the applied
radiation dose during specific radiotherapy plans. Here we present a study
based on the use of polymer gel dosimeters in combination with a novel
Optical Projection Tomography system, which allows the association of optical
properties, namely the attenuation coefficient, to the irradiation dose. Polymer
gel dosimeters are polymerized after X-ray irradiation via free radical
production during water radiolysis resulting to increased optical opacity as
well as change of the nuclear magnetic resonance relaxation times, thus
making it possible to study them with both optical and MRI techniques. The
optical tomographic system employs a sensitive CCD camera, a rotation
stage allowing full 360 degrees rotation and a homogeneous white light source
transilluminating the samples. This setup allows the calculation of the optical
attenuation coefficient which can then be directly related to the applied
radiotherapy dose, as well as the definition of the surface of the sample in
space. The experimental procedure involves the recording of transillumination
images of the polymer samples in steps of 1 degree to get the desired
resolution. Data analysis is performed by back propagating the photons using
an inverse Radon transform resulting to the reconstruction of three
dimensional images of the attenuation coefficient or equivalently the dose
distribution. The sensitivity and dynamic range offered by the technique covers
the range of radiotherapy doses in modern clinical practice and are compared
with the corresponding achieved with MRI.
6629-49, Session 7
Early prediction of treatment response of head
and neck cancers with diffuse optical
spectroscopies
U. Sunar, Univ. of California/San Diego (USA); S. Kim, R. Choe, H.
Poptani, H. Quon, T. Durduran, C. Zhou, G. Yu, S. Nioka, B. Chance, A.
G. Yodh, Univ. of Pennsylvania (USA)
Radiation therapy efficacy is known to be dependent on oxygen status. The
oxygen-sensitive micro-electrode needle method provides a reference
standard for measurement of tumor oxygenation, however it is invasive and
inconvenient for clinical use. Thus, there is a need for reliable non-invasive
techniques that measure tumor response. Tumor blood flow (BF)
measurements are particularly attractive for this application, since blood flow
has been correlated with tumor oxygenation; however, most of the perfusion
imaging techniques needs contrast agent injection. The diffuse optical
methods (Diffuse correlation spectroscopy (DCS) and diffuse reflectance
spectroscopy (DRS)) offer a noninvasive, rapid, portable and low-cost
alternative for repetitive, bedside monitoring of therapies with both blood
flow and oxygenation quantification. Here we present therapeutic response
of 10 patients, 8 of which responded and 7 of which did not responded to
chemo-radiation therapy. Among these patients, 3 of them were also
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Conference 6629: Diffuse Optical Imaging in Tissue
measured by Gd-MRI independently at the same day. Our results suggest
early clinical tumor response to radiation therapy can be picked up by diffuse
optical spectroscopies. The data clearly show early changes even within two
weeks of therapy and correlation with treatment response and Gd-MRI
measurements. Since a main aim of therapy diagnostics is to predict the
response earlier, early blood flow and oxygenation changes suggest the
potential of daily measurements during the first two weeks. Due to low
accessibility of the other methods, optical methods have advantages for daily
based therapy monitoring.
European Conferences on Biomedical Optics 2007 •
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Conf. 6630: Confocal, Multiphoton, and Nonlinear Microscopic Imaging
Room 4A
Sunday-Monday 17-18 June 2007
Part of Proceedings of SPIE Vol. 6630 Confocal, Multiphoton, and Nonlinear Microscopic Imaging III
6630-01, Session 1
Two-color intranuclear distance measurements
of gene regions in human lymphocytes
S. Fenz, Forschungszentrum Jülich GmbH (Germany); H. Mathée, G.
Kreth, D. Baddeley, Ruprecht-Karls-Univ. Heidelberg (Germany); Y.
Weiland, Ruprecht-Karls-Univ. Heidelberg (Georgia); J. SchwarzFinsterle, C. G. Cremer, U. J. Birk, Ruprecht-Karls-Univ. Heidelberg
(Germany)
The method of Spectral Precision Distance Microscopy (SPDM) has been
applied to determine distances between two FISH (Fluorescence-in situHybridisation)-labeled gene regions on chromosome 9 after calibration of
the chromatic aberrations. To this end we applied methods to correct for
chromatic aberrations of the microscope optics alone and also of the sample
induced spherical aberrations due to mismatch of the refractive indices. Using
a confocal microscope and a threshold based position determination
algorithm, positions could be measured with an accuracy of about 60nm
inside of fixed cell nuclei. Results have been compared to those obtained
from a model based position determination algorithm. Distances obtained
from the measurements have been verified using a 3D computer model of
the cell nucleus. In principle, this SPDM approach could be combined with
novel fluorescence microscopes to increase the optical resolution. However,
precision of the distance measurements is limited by variations of the refractive
index throughout the specimens.
6630-02, Session 1
In vivo imaging of structures in Caenorhabditis
elegans using non-linear (TPEF-SHG-THG)
microscopy
E. J. Gualda, G. Filippidis, M. Mari, C. Fotakis, G. Voglis, N.
Tavernarakis, Foundation for Research and Technology-Hellas (Greece)
In the last few years, the non-linear optical measurements, used in conjunction
with microscopy observations, have created new opportunities of research
and technological achievements in the fields of biology and medicine. These
imaging technologies represent the forefront pool of research in cell biology.
Due to their inherent advantages in comparison with the conventional
microscopy (increased resolution, ability to section deep within tissues,
minimization of photodamage and photobleaching effects), the non-linear
microscopy techniques comprise a unique and extremely powerful tool for
the extraction of valuable and unique information from biological samples. In
this study, we present the detailed imaging and mapping of the nematode
Caenorhabditis elegans (C. elegans) at microscopic level by performing TwoPhoton Excitation Fluorescence (TPEF), Second-Harmonic Generation (SHG)
and Third Harmonic Generation (THG) measurements. A compact, reliable,
inexpensive non-linear imaging system has been developed. Femtosecond
laser pulses (1028nm) were utilized for the excitation of the biological samples.
The use of 1028nm wavelength as excitation source minimizes the
photodamage effects and the unwanted heating (due to the water absorption)
of the biological specimens. The emitted THG signal lies in the near UV part
of the spectrum (343nm). Detailed and specific structural and anatomical
features from the worm were collected by recording THG signals.
Consummative, unique information concerning the morphology and the
functions of the nematode were obtained by implementing the combination
of THG, SHG and TPEF image contrast modalities to the same microscope
6630-03, Session 1
Functional imaging of skeletal muscle fiber in
different physiological states by second
harmonic generation
V. Nucciotti, C. Stringari, L. Sacconi, F. Vanzi, C. Tesi, N. Piroddi, C.
Poggesi, Univ. degli Studi di Firenze (Italy); C. Castiglioni, A. Milani,
Politecnico di Milano (Italy); M. Linari, G. Piazzesi, V. Lombardi, F. S.
Pavone, Univ. degli Studi di Firenze (Italy)
The intrinsically ordered arrays of proteins in skeletal muscle allows imaging
of this tissue by Second Harmonic Generation (SHG). Biochemical and
colocalization studies have gathered an increasing wealth of clues for the
attribution of the molecular origin of the muscle SHG signal to the motor
protein myosin. Thus, SHG represents a potentially very
powerful tool in the investigation of structural dynamics occurring in muscle
during active production of force. A full characterization of the polarizationdependence of the SHG signal represents a very selective information on
the orientation of the emitting proteins and their dynamics during contraction,
provided that different physiological states of muscle (relaxed, rigor and active)
exhibit distinct patterns of SHG polarization dependence. Here polarization
44
European Conferences on Biomedical Optics 2007 •
data are obtained from single frog muscle fibers at rest and during isometric
contraction and interpreted, by means of a model, in terms of an average
orientation of the SHG emitters which are structured with a cylindrical
symmetry about the fiber axis. Optimizing the setup for accurate polarization
measurements with SHG, we developed a line scan imaging method allowing
measurement of SHG polarization curves in different physiological states.
We demonstrate that muscle fiber displays a measurable variation of the
orientation of SHG emitters with the transition from rest to isometric
contraction.
6630-04, Session 1
Surgical visualization of rabbit cornea after
photorefractive keratectomy by multiphoton
microscopy
C. Hsueh, W. Lo, National Taiwan Univ. (Taiwan); T. Wang, F. Hu,
National Taiwan Univ. Hospital (Taiwan); C. Dong, National Taiwan Univ.
(Taiwan)
Photorefractive keratectomy (PRK) is a surgery to correct mild to moderate
presbyopia, hyperopia, and astigmatism. By removing the epithelial layer of
the cornea with a scrubber or blade first and using the excimer laser to alter
the shape of the cornea achieves clearer focus of vision. The excimer laser
doesn’t burn tissue and evaporates molecules of the cornea precisely attaining
to sculpt the surface of the cornea.
In this work we use multiphoton microscopy to observe the post surgery
structure variation at both submicron resolution and over a large region within
the tissue. Since collagen can be induced to generate strong second harmonic
generation (SHG) signal, multiphoton excitation provide direct visualization
of collagen orientation within corneal stroma. In addition, since the SHG
intensity of collagen tissue deteriorates with reducing the thickness of the
cornea, our methodology can be used to characterize the extent of corneal
stroma damage from the PRK procedure. Finally, the influence of PRK on the
morphology and distribution of keratocytes can also be investigated by
detecting multiphoton excited autofluorescence from the cells.
6630-05, Session 1
Post surgical visualization of rabbit cornea after
conductive keratoplasty by multiphoton
microscopy
W. Lo, National Taiwan Univ. (Taiwan); T. Wang, F. Hu, National Taiwan
Univ. Hospital (Taiwan) and National Taiwan Univ. Medical Collage
(Taiwan); C. Dong, National Taiwan Univ. (Taiwan)
Conductive keratoplasty (CK) is a new refractive surgery for presbyopia and
hyperopia patients. By applying radio frequency current at the peripheral
regions of cornea, collagen, the most abundant composition of corneal
stroma, shrinks due to the heat absorbed. The shrinkage at the periphery
alters the corneal architecture and achieves clearer focus for near vision.
In this work we use multiphoton microscopy to observe the post surgery
structure variation at both submicron resolution and over a large region within
the tissue. Since collagen can be induced to generate strong second harmonic
generation (SHG) signal, multiphoton excitation provide direct visualization
of collagen orientation within corneal stroma. In addition, since the SHG
intensity of collagen tissue deteriorates with increasing thermal damage [13], our methodology can be used to characterize the extent of corneal stroma
damage from the CK procedure. Finally, the influence of CK on the morphology
and distribution of keratocytes can also be investigated by detecting
multiphoton excited autofluorescence from the cells.
This study can lead to the improved understanding of refractive surgical
procedures resulting in the development of more effective procedures.
REFERENCES
[1] S. J. Lin, W. Lo, H. Y. Tan, J. Y. Chan, W. L. Chen, S. H. Wang, Y. Sun, W.
C. Lin, J. S. Chen, C. J. Hsu, J. W. Tjiu, H. S. Yu, S. H. Jee, and C. Y. Dong,
“Prediction of heat-induced collagen shrinkage by use of second harmonic
generation microscopy,” Journal of Biomedical Optics, vol. 11, pp. -, 2006.
[2] Y. Sun, W. L. Chen, S. J. Lin, S. H. Jee, Y. F. Chen, L. C. Lin, P. T. C. So,
and C. Y. Dong, “Investigating mechanisms of collagen thermal denaturation
by high resolution second-harmonic generation imaging,” Biophysical Journal,
vol. 91, pp. 2620-2625, 2006.
[3] S. J. Lin, C. Y. Hsiao, Y. Sun, W. Lo, W. C. Lin, G. J. Jan, S. H. Jee, and C.
Y. Dong, “Monitoring the thermally induced structural transitions of collagen
by use of second-harmonic generation microscopy,” Optics Letters, vol. 30,
pp. 622-624, 2005.
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Conf. 6630: Confocal, Multiphoton, and Nonlinear Microscopic Imaging
6630-06, Session 1
Enhanced fluorescence cell imaging with metalcoated slides
E. Le Moal, E. Fort, École Supérieure de Physique et de Chimie
Industrielles (France); S. Lévêque-Fort, Univ. Paris-Sud II (France); A.
Janin, H. Murata, Univ. Paris VII (France); F. P. Cordelières, M. FontaineAupart, Univ. Paris-Sud II (France); C. Ricolleau, École Supérieure de
Physique et de Chimie Industrielles (France)
Fluorescence microscopy has become the method of choice in the majority
of life-science applications. We will show how the use of mirror slides can
significantly enhance the fluorescence signal using standard air microscope
objectives. This technique offers sufficient gain to achieve high-sensitivity
imaging with wide field of observation and large depth of focus, two major
breakthroughs for routine analysis and high-throughput screening applications
on cells and tissue samples. Mirror slides enhance the fluorescence signal
over the entire visible spectrum and over micrometer thicknesses allowing
multicolor labeling and thick sample imaging like cells and tissues.
We will present two applications on cell cultures and tissues that exemplify
the need for such active substrates in thick sample imaging. The first one
concerns the study of epithelium degeneration, in the context of research on
cancer (see Figure 1). In the second one, we applied mirror slides to tissue
imaging for medical diagnosis, in the follow-up of the survival and stability of
sex-mismatched human grafts.
6630-07, Session 2
Multi-wavelength multiphoton FLIM with direct
detection
W. Becker, A. Bergmann, Becker & Hickl GmbH (Germany)
We present a fluorescence lifetime imaging technique with simultaneous
spectral and temporal resolution. The recording process is based on a multidimensional TCSPC (time-correlated single photon counting) technique. The
optical part is fully compatible with the commonly used multi-photon
microscopes and direct (non-descanned) detection.. A fibre bundle is used
to transfer the fluorescence light into a spectrograph. A lens projects an
image of the back aperture of the microscope lens on the input of the fibre
bundle. The input of the bundle has a circular shape; the output has the
shape of the input slit of the spectrograph. Thus, even scattered light from
deep sample layers is efficiently transferred into the detection system. The
spectrum at the output of the spectrograph is projected on the cathode of a
16-channel multi-anode PMT. For every detected photon, the electronics
determines the time of the photon in the pulse period of the excitation laser,
the PMT channel number, and the position of the laser beam in the scanning
area. The recording process builds up a four-dimensional photon distribution
over these parameters. The method delivers a near-ideal counting efficiency
and a time-resolution only limited by the transit time-spread in the PMT.
6630-08, Session 2
Full photon information data structure applied to
laser scanning microscopes enabling FLIM,
FRET, and FCS data analysis
U. Ortmann, B. Krämer, F. Koberling, PicoQuant GmbH (Germany)
Confocal Laser Scanning Microscopes (LSMs) are widely used tools in
biochemistry, cell biology and other related sciences. The capabilities of these
instruments can be greatly enhanced by adding the time as another dimension
of information. In that way new measurement modes like FLIM, lifetime-FRET
and FCS become feasible.
The presented upgrade is based on picosecond pulsed diode lasers together
with a Time-Tagged Time-Resolved (TTTR) data acquisition. This advanced
form of time-correlated single photon counting (TCSPC) is the key to the
new capabilities, because it acquires and immediately stores the full photon
information (temporal on two different time scales, spatial, color, etc.) in a
generalized format. Virtually all algorithms and methods for the analysis of
photon dynamics and lifetime imaging can be applied to the resulting
information-rich TTTR data.
The pulsed diode lasers are integrated in a special laser coupling unit that
allows easily to switch from CW laser excitation (typical for LSM experiments)
to pulsed lasers used for FLIM. The light detection is accomplished by single
photon sensitive detectors, which itselfs greatly increases the sensitivity. Onthe-fly processing of the TTTR data stream allows a real time visualization of
the acquired fluorescence lifetime image. Multiple color detection is a very
straightforward extension. In connection with the time-resolved measurement
mode it makes possible to routinely perform lifetime-FRET imaging. We will
demonstrate these capabilities by mapping the relative hydrophobicity inside
of hepatocytes (HepG2) using the NBD (nitrobenzoxadiazol) dye as a probe
(sample courtesy of Prof. Andreas Herrmann, Humboldt University, Berlin)
and by lifetime based FRET imaging of transcription factor proteins in the
nucleus of living cells, confirming a dimerization process (data acquired at
the FRET workshop of Prof. Ammasi Periasamy, WM Keck Center for Cellular
Imaging, University of Virginia, Charlottesville).
European Conferences on Biomedical Optics 2007 •
However, the confocal optics of LSMs can be utilized not only for imaging,
but also for single point measurements. For example, fluorescence correlation
spectroscopy (FCS) can be performed after parking the beam into a selected
location. FCS is possible because the necessary timing information is available
now for every detected photon. The ultimate power of the TTTR mode in
connection with the versatility of the upgrade package becomes even more
obvious when the photon records are processed off-line by the dedicated
software. Making use of the timing information available on two different
time scales (relative to the excitation pulse and relative to the start of the
acquisition) advanced FCS techniques can be implemented on the same
(imaging) microscope. We will demonstrate the impact of nanosecond timegating on FCS results obtained by measurement of very diluted samples,
where the elastic and Raman scattering are the main signal contributions.
6630-09, Session 2
Microscopic fluorescence lifetime and
hyperspectral imaging with digital micromirror
illuminator
A. Bednarkiewicz, M. Bouhifd, M. P. Whelan, Joint Research Ctr. (Italy)
An experimental setup for Fluorescence Lifetime Imaging (FLIM) and Hyper
Spectral Imaging (HIS) is described which is based on the combination of a
digital micro-mirror device illuminator (DMDI) with an optical microscope.
Spatially selective illumination is obtained by tilting the relevant group of
micro-mirrors thus reflecting the excitation light from a UV picosecond laser
diode towards chosen points on the sample. The resulting fluorescence signal
is collected by a single detector (i.e. photomultiplier in photon counting mode
for FLIM, CCD spectrophotometer for HSI) from the whole field of view. Image
reconstruction is facilitated by either raster scanning over the sample or by
directly accessing specific regions of interest. The unique features of the
DMD illuminator allow a Global Analysis (GA) to be first executed to
significantly speed up the on-line experimental data analysis associated with
subsequent raster-scanning. The GA thus supplies good initial values for
fitting parameters, which in turn decreases the computation time needed to
obtain a satisfactory quality-of-fit. Experimental results will be presented that
clearly show the possibilities for temporal and spectral unmixing within images
acquired on phantoms and biological samples. The advantages of our
approach in comparison to typical electro-mechanical or electro-optical raster
scanning approaches include more efficient on-line data analysis, adjustable
spatial resolution, and random rapid access to single or multiple regions of
interest on the sample.
6630-10, Session 2
Development of a TIRF-FLIM microscope for
biomedical applications
P. Blandin, S. Lévêque-Fort, F. P. H. Druon, M. Hanna, P. M. Georges,
Univ. Paris-Sud II (France); R. Briandet, Institut National de la Recherche Agronomique (France); Z. Lenkei, École Supérieure de Physique et
de Chimie Industrielles (France); M. Fontaine-Aupart, Univ. Paris-Sud II
(France)
Total internal reflection fluorescence microscopy (TIRFM) is a powerful optical
technique to observe single molecule fluorescence at surfaces. Associated
with fluorescence lifetime imaging, TIRFM enables to measure contrasts
independent of fluorophores concentration and reveal probe environment
(pH imaging, ion mapping,...) with subwavelength axial resolution.
We develop a home made device based on an Olympus IX 71 in the through
the objective TIRF configuration. The entrance of the laser beam on the back
aperture of the high numerical aperture objective (Olympus TIRFM 1,45) is
finely controlled to switch easily from epifluorescence configuration to TIRF.
For the excitation, we use a commercial tunable pulsed laser with a low
repetition rate (few tens MHz), which permits to observe lifetime until several
nanoseconds. A high rate imager (HRI Kentech) read by a cooled CCD camera
(Orca EG Hamamatsu) allows to record time gated fluorescence images to
obtain FLIM map for large field of view (typically 100µm x 100µm).
After a complete characterization of the setup, we will demonstrate its
capability to tackle biological problems near an interface (adhesion
mechanism, membrane traffic ...). . TIRF can give access to the very first
layers of bacterial biofilm involved in significant problems in medical, industrial
and environmental areas, and so help to understand their adhesion
mechanisms. Neuropharmacology is another wide domain of application, as
the events of the plasma membrane can be separated from endosomal events.
Previous studies show that sub-neuronal localization profoundly modifies
activation- induced trafficking of the type 1 cannabinoid receptor (CB1R)
receptors. Preliminary TIRF-FLIM images of CB1R in a physiologically
relevant cellular context will also be presented.
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Conf. 6630: Confocal, Multiphoton, and Nonlinear Microscopic Imaging
6630-11, Session 3
3) Preza, C., “Rotational diversity phase estimation from differential
interference contrast microscopy images,” Journal of the Optical Society of
America A, 17(3), 415 424, 2000.
Refractive index determination by indexmismatch-induced spherical aberration
P. Su, T. Fwu, H. Vladimir, C. Dong, National Taiwan Univ. (Taiwan)
6630-14, Session 3
Two-photon microscopy has been successfully applied in biological and
material sciences since 1990. However, it is known that the resolution of
two-photon imaging is adversely affected from the index mismatch induced
spherical aberrations. Spherical aberration increases the focal volume and
causes degradation of image resolution as one image deeper into the
specimen. In this work, we propose to use this intrinsic artifact to measure
the refractive index in specimens of uniform refractive indices. Using the
intensity profiles of standard refractive liquids as our database, we can
compare the intensity profile of the unknown specimen with them and measure
the refractive index if the specimen has uniform refractive index.
Two- and one-photon color confocal screening
microscope
6630-12, Session 3
Determination of the confocal volume for
quantitative fluorescence correlation
spectroscopy
S. Rüttinger, Physikalisch-Technische Bundesanstalt (Germany); V.
Buschmann, B. Krämer, F. Koberling, PicoQuant GmbH (Germany); R.
Macdonald, Physikalisch-Technische Bundesanstalt (Germany)
Single molecule detection methods, in particular those based on fluorescent
labels offer the possibility to gain not only qualitative but also quantitative
insight into the function of complex biological systems. Fluorescence
Correlation Spectroscopy (FCS) is one of the favourite techniques to determine
concentrations and diffusion constants as well as molecular brightness in
the pico- to nano-Molar concentration range, with broad applications in
Biology and Chemistry. Although FCS in principle has the potential to measure
absolute concentrations and diffusion coefficients, the necessity to know
the exact size and shape of the confocal volume very often hampers the
possibility to obtain quantitative results and restricted FCS to relative
measurements mainly. The determination of the confocal volume in situ is
difficult because it is sensitive to optical alignment and aberrations, optical
saturation and variations of the index of refraction as observed in biological
specimen.
In the present contribution, we compare different techniques to characterize
the confocal volume and to obtain the confocal parameters by FCS-curve
fitting, a FCS dilution series and confocal bead scanning. The results are
compared in the view of quantitative FCS measurement and analysis. Artifacts
like aberration, cover-slide thickness effects and saturation are compared to
theoretical calculations. It will be shown that the dye ATTO 655 is well-suited
for the mentioned characterization and determination of parameters, because
it has rather low probability for intersystem crossing to triplet states, good
hydrophybility and known diffusion coefficient in water.
6630-13, Session 3
C. Preza, The Univ. of Memphis (USA); J. A. O’Sullivan, Washington
Univ. in St. Louis (USA)
In a recent calibration study (King et al., 2007) we have shown that although
phase -shifting can remove amplitude (absorption) and yield approximate
linear phase gradients, a linear integration method based on a geometric
optics model is fundamentally limited in its ability to compute the specimen’s
true phase from the phase-shifted differential-interference-contrast (DIC)
images. In this paper we present the development of a new iterative method
based on a diffraction imaging model (Preza et al., 1999) for the computation
of a specimen’s complex transmittance function (amplitude and phase) from
DIC images. This new method extends our initial work (RD method presented
in Preza, 2000) which was based on the assumption that the specimen does
not absorb light and thus only the specimen’s phase function or optical path
length (OPL) distribution was computed from rotationally-diverse DIC images.
The new method requires two images acquired with traditional DIC at two
orthogonal shear directions (or specimen orientations) and it is based on an
alternating minimization algorithm (AMA). Simulation results compare
specimen amplitude (absorption) and phase estimated with the AMA method
to the true specimen parameters and to phase estimated with the RD method.
References: 1) King, S. V., Libertun, A. R., Preza, C., Cogswell, C. J.,
“Calibration of a phase-shifting DIC microscope for quantitative phase
imaging”, in Three Dimensional and Multidimensional Microscopy: Image
Acquisition and Processing XIV, C. J. Cogswell, J. A. Conchello, T. Wilson,
eds., Proc. SPIE Vol. 6443, 2007.
2) Preza, C., Snyder, D. L., and Conchello, J. A., “Theoretical development
and experimental evaluation of imaging models for differential interference
contrast microscopy,” Journal of the Optical Society of America A, 16(9):2185
2199, 1999.
European Conferences on Biomedical Optics 2007 •
The goal was to develop an upright microscope platform for the screening of
slides employing one- and two-photon laser scanning techniques.
A highly compact unit was created which combines novel concepts for moving
a slide in three dimensions, keeping it focussed while doing so, scanning a
laser-focus over the sample using novel galvanometer-control concepts,
combining and separating excitation and emission beam und spectrally
dispersing the emitted light by a linearized prism-spectrograp. Spectral
detection is achieved by turning a 128 x 128 back-thinned EMCCD detector
in a continuously reading spectral point-detector.
To make the unit even ore versatile it can be turned into a conventional widefield fluorescence microscope, thus allowing to carry out routine experiments
and to select regions of the sample for a subsequent, more detailed confocal
analysis.
6630-15, Session 4
Advances in lasers for multi photon microscopy
D. P. Armstrong, Coherent Scotland Ltd. (United Kingdom)
Multiphoton Excitation (MPE) microscopy has become an important
bioimaging technique, enabling the study of dynamic processes in living cells
and tissues without causing significant damage. MPE produces highresolution, three-dimensional images, and primarily relies on the use a tunable,
ultrafast laser to excite highly specific fluorophores in order to follow specific
biochemical processes. Key to future advances in MPE is the ability to work
with a wider range of fluorophores, to increase data acquisition speed, and
to improve data signal-to-noise ratio. In terms of laser characteristics, these
requirements translate into wider tuning range, faster tuning speeds, and
higher peak power delivered to the sample. This paper explores the advances
in tunable ultrafast laser and amplifier technology currently under development
to meet these goals and thus power the next generation of MPE
instrumentation.
6630-16, Session 4
Substantial improvement in penetration depths
and photo damage reduction: multiphoton
microscopy beyond one micron
E. Büttner, APE GmbH (Germany); V. Andresen, LaVision BioTec GmbH
(Germany); I. Rimke, APE GmbH (Germany); P. Friedl, Univ. Würzburg
(Germany)
Quantitative determination of specimen
properties using computational differentialinterference contrast (DIC) microscopy
46
J. Walter, TILL I.D. GmbH (Germany); C. Seebacher, Ludwig Maximilians
Univ. Munich (Germany); R. Uhl, Ludwig Maximilians Univ. Munchen
(Germany)
Here we present a multiphoton excitation microscopy setup extending the
excitation wavelengths far beyond one micron. A synchronously pumped fsOPO (OPO PP-Automatic, APE) pumped by a fs-Ti:Sapphire oscillator is used
as the light source. The biological relevant wavelength range from <1050 to
\>1350 nm can be covered with a fixed pump frequency and a single optics
set through hands free, automated tuning. Together with the Ti:Sapphire pump
laser (Coherent Chameleon) excitation wavelengths from 700 to 1600nm are
achieved.
Two separate scanners (LaVision BioTec) are optimized for Ti:Sapphire and
OPO wavelength ranges respectively including dispersion compensation for
maintaining the short pulses at the sample site. An overall transmission of
30-38% up to 1400 nm was achieved.
Measurements on human dermis with excitation above 1 micron, compared
to lower wavelengths, showed doubling of the penetration depths, strongly
reduced photo damage, a 30fold increased excitation efficiency and 10fold
reduced photobleaching of red fluorescent dyes, including RFP and Cy5.5.
Excitation at 1100 nm further leads to a 4fold decrease in autofluorescence,
resulting in a significantly improved signal-to-noise ratio. The resolution is
slightly reduced in comparison to Ti:Sapphire excitation, which corresponds
well to the longer excitation wavelength used.
An OPO pump wavelength around 800nm opens up the possibility to use the
Ti:Sapphire laser to pump the OPO and to excite the sample simultaneously
giving the opportunity to excite dyes such as GFP with the pump laser and
red shifted fluorophores (for instance RFP) with the OPO at the same time.
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Conf. 6630: Confocal, Multiphoton, and Nonlinear Microscopic Imaging
6630-17, Session 4
Coherent light microscopy with a multi-spot
source
R. Riesenberg, M. Kanka, Institut für Physikalische Hochtechnologie
e.V. (Germany)
The coherent illumination of a sample by a pinhole and the detection of
resulting interference patterns by a CCD is known as digital inline holography
/1 ... 3/. Positioning the sample near the source can accomplish a holographic
microscope setup.
We present a coherent light microscopy using a multi-spot source /4 ... 6/.
Advantages are an essential increase of illumination intensity leading to
increased sensitivity /5/ and an increase of numerical detection aperture
resulting in better 3D resolution /6, 7/.
The achieved advantages depend on the dimensions of the pinhole array,
the size of the pinholes and the used areas. The array-device consists of a
Si-chip with a membrane, which is a multi-layer system. Because the diameter
of the pinholes should be < 1 µm, the optically active thickness of the device
membrane should be 0.5 µm or less.
The light damping (caused by reflection and absorption) of the state-of-theart pinhole array device reaches more than 4 orders of magnitude. As a result
for full laser illumination of the membrane an additional damped plane wave
with low intensity passes the area of the membrane. The plane wave acts as
an offset in the interferogram and reduces the contrast. The contrast limits
the advantages of the coherent microscopy. Dimensions of the multi-spot
device are discussed in relation to the coherent light microscopy.
Furthermore we work on a coherent multi-spot source with diffractive elements
in combination with the pinhole device for an additional contrast enhancement
by more than two orders.
An experimental set up and results of imaging of bacteria as well as of test
standards in µm scale are given.
[1] D. Gabor: “A new microscopic principle” in Nature 161, 777-778 (1948)
[2] D. Gabor: “Microscopy by Reconstructed Wavefronts” in Proc. Roy. Soc.
A 197, 457- 484 (1949)
[3] H.J.Kreuzer, R.A.Pawlitzek, „Digital in-line holography” in Europhysics
News 34, 62-65 (2003)
[4] R. Riesenberg, “Pinhole-arrays and lenseless holographic micro-imaging”,
DGaO-Proceedings A 26, 106th Conference of the DgaO (2005) http://
www.dgao.de/
[5] R. Riesenberg, M. Kanka, J. Bergmann „Unconventional Imaging by
Synthetic Aperture” DGaO-Proceedings A 25, 107th Conference of the DgaO
(2006), ) http://www.dgao.de/
[6] M. Kanka, R. Riesenberg, “Wide field holographic microscopy with pinhole
arrays”, OPTO 2006, Sensor+Test 2006 Proceedings, pp. 69-72
[7] M. Kanka, R. Riesenberg, “Advanced coherent 3D micro-imaging”,
submitted to the same conference
6630-18, Session 4
Phase reconstruction by multiple plane
detection for holographic microscopy
A. Grjasnow, R. Riesenberg, A. Wuttig, Institut für Physikalische
Hochtechnologie e.V. (Germany)
Phase contrast microscopy is a tool for characterizing micro-optical devices
as well as biological samples like cells. As a technique for absolute phase
retrieval inline holographic microscopy is known. In the latter case the
interference patterns between sample and reference wave are detected by a
CCD. Any distortion of the reference wave, e.g. from the surfaces of a test
slide, causes reconstruction errors. The reference usually is disturbed by the
sample, therefore it is necessary to limit the size of the sample.
We present a measurement technique which generates the same information
as a hologram but does not use any reference wave. Self-interference patterns
of the object wave are measured in a set of planes of different distances
from the sample. The obtained set of interference images is used for
reconstruction of the full 3D image.
The technique was demonstrated for microscopic resolution in case of
amplitude objects [7], [8]. The paper focuses on imaging and reconstruction
of phase objects and of the estimation of the absolute phase.
The technique is experimentally applied to phase test standards. The
dependencies of lateral resolution and phase noise on the number of
measured planes (typically 3 ... 6 ) and on the distances of these planes from
one to each other and from the sample are discussed. Phase differences of
p/3 with a lateral resolution of 2 µm are well resolved. Furthermore
experimental results of imaging bacteria E. Coli and Pleurosigma are
presented.
References:
[1] D. Gabor: “Microscopy by reconstructed wavefronts” in Proc. Roy. Soc.
A 197: 454 - 487 (1949)
[2] R.W. Gerchberg and W.O. Saxton: “A practical algorithm for the
determination of phase from image and diffraction plane pictures” in Optik
(Stuttgart), Vol.35, No.2, pp.237-246 (1972)
European Conferences on Biomedical Optics 2007 •
[3] G. Yang: “Gerchberg-Saxton and Yang-Gu algorithms for phase retrieval
in a nonunitary transform system: a comparison” in Appl. Optics 33(2): 209218 (1994)
[4] G. Pedrini, W. Osten, Y. Zhang: “Wave-front reconstruction from a sequence
of interferograms recorded at different planes “ in Opt. Letters 30(8): 833835 (2005)
[5] Y. Zhang, G. Pedrini, W. Osten, H.J. Tiziani: “Whole optical wavefields
reconstruction from double or multi in-line holograms by phase retrieval
algorithm “ in Opt. Express 11(24): 3234-3241 (2003)
[6] R. Riesenberg: “Pinhole array and lenseless microscopic micro-imaging”,
DGaO-Proceedings(2005), 106th Conference of the DGaO, http://
www.dgao.de/
[7] A. Grjasnow, R. Riesenberg, A. Wuttig: “Lenseless coherent imaging by
multi-plane interference detection”, DGaO-Proceedings(2005), 106th
Conference of the DGaO, http://www.dgao.de/
[8] A. Grjasnow, R. Riesenberg, A. Wuttig: “Microscopy by multi-plane
interference detection” in SENSOR+TEST 2006 Proceedings, OPTO & IRS(c)˜
2006 Conference
[9] P. Almoro, G. Pedrini, W. Osten: “Complete wavefront reconstruction using
sequential intensity measurements of a volume speckle field” in Appl. Optics
45(34): 8596 - 8605 (2006)
6630-19, Session 4
STED microscopy far beyond the diffraction limit
employing beam scanning in a regular
microscope
V. Westphal, S. W. Hell, Max-Planck-Institut für biophysikalische
Chemie (Germany)
Stimulated emission depletion (STED) microcopy utilizes the strong
nonlinearity generated at a reversible saturable optical linear fluorescent
transition to overcome the diffraction barrier [1]. The basic idea of STED:
Directly after the excitation pulse molecules in the diffraction-limited focal
volume are in the excited and ready to fluoresce. Before they can do so,
molecules in the focal periphery will be quenched while leaving the ones in
the center untouched. We can now only detect the fluorescence from a
reduced volume, effectively increasing the resolution. This depletion is
accomplished through stimulated emission using a second red-shifted and
delayed laser pulse, with a (diffraction limited) zero intensity in the center of
the excited volume [2]. The resolution enhancement so far has been
demonstrated to about an order of magnitude [3].
So far all the STED microscope setups have been based on stage-scanning
the sample, with only very limited wide-field inspection capabilities. This new
setup employs a regular microscopy body as a user front-end, featuring
normal eye-pieces and a cooled CCD camera for sample selection.
Furthermore the setup was optimized for improved stability and easy
alignment, simplifying every-day use. Previous stage scanning setups needed
minutes to acquire a single image. Therefore we incorporated beam-scanning
into the system, which allows for rapid image acquisition up to 1 Hz.
On the conference we will show our first results of this new STED microscope.
We believe it has high potential for a wide range of applications, utilizing a
simplified user front-end.
REFERENCES
[1] S. W. Hell,”Toward fluorescence nanoscopy,” Nature Biotechnol. 21, 13471355 (2003)
[2] V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup and S. W. Hell,”Laserdiode-stimulated emission depletion microscopy,” Appl. Phys. Lett. 82, 31253127 (2003)
[3] V. Westphal, S.W. Hell, “Nanoscale Resolution in the Focal Plane of an
Optical Microscope” Physical Review Letters 94, 143903 (2005)
6630-20, Session 4
Advanced fluorescence microscopy using light
emitting diodes
G. T. Kennedy, V. Poher, I. H. Munro, D. S. Elson, P. M. W. French, M. A.
A. Neil, Imperial College London (United Kingdom)
Light emitting diodes are a rapidly evolving technology with the brightness
of available devices developing according to their own version of Moore’s
law and doubling approximately every 18-24 months. The available devices
today cover a wide spectral range and are now bright enough to compete
with conventional lamp technologies as suitable sources for fluorescence
microscopy. In addition, they offer the significant advantage that the light
output can be electrically controlled on nanosecond timescales and they
can be patterned using semiconductor processing techniques to produce
programmable structured light sources.
We demonstrate how these advantages can be used in the realisation of a
range advanced optical fluorescence microscopy techniques. We show how
cheap of the shelf devices can be driven sinusoidally or in pulsed mode as
sources in both frequency and time domain fluorescence lifetime imaging
systems - replacing the acousto-optic modulators or ultrafast laser systems
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Conf. 6630: Confocal, Multiphoton, and Nonlinear Microscopic Imaging
conventionally used. We also show how a structured LED consisting of an
array of lines can be used programmable to realise optical sectioned imaging
by structured illumination and line scanning confocal techniques - all in a
single compact solid-state device with no moving parts.
6630-24, Session 5
Aberration-free refocusing in high numerical
aperture microscopy
T. Wilson, Univ. of Oxford (United Kingdom)
6630-21, Session 4
Programmable optics for confocal and multiphoton microscopy
M. A. A. Neil, B. R. Boruah, Imperial College London (United Kingdom)
We describe a beam scanning optical microscope system that uses a
programmable holographic optical element to control the optical field in the
illumination pupil of the microscope objective. The system uses a computer
controlled, fast, ferroelectric liquid crystal spatial light modulator as the
programmable optical element to display arbitrary computer generated
holograms. In conjunction with simple polarisation optics these produce
arbitrary optical wave fields that can be projected into the pupil plane of the
scanning microscope by a galvo-mirror pair arranged to minimises the
movement of the field across the pupil as the beam is scanned. We
demonstrate how the programmable optical element allows the control of
aberrations in the illumination path and present a novel method for sensing
those aberrations based on helical doughnut beams. The control over
polarisation afforded by the system also allows us to arbitrary polarisation
distributions in the illumination pupil such as radially polarisation, which when
focused in a high NA objective results in a dominant axially polarisation
component in the focus. We investigate the use of this microscope to probe
the orientation and reorientation of fluorescent markers on the molecular
level.
A common requirement in many kinds of microscopy is to obtain a series of
high- resolution, through-focus images. An approach often adopted is to
move the specimen axially so that different planes are brought into focus. A
practical drawback to this approach is that mechanical movement is inevitably
slow and, in some applications, may be impractical, for example when the
scanning movements affect the specimen. An optical method of refocusing
is clearly preferable as this permits imaging to be carried out without disturbing
the specimen. Furthermore, such an approach could enable higher axial scan
rates than were previously possible. In the simplest case, optical refocusing
can be performed by repositioning the detector. However, when used in
conjunction with high numerical aperture (NA) objectives, this method
inevitably produces spherically aberrated images. In this paper we present
the theory and results of a new system, where the camera remains fixed and
refocusing is performed by a different means using a second high NA lens
and mirror. We will show that if this is designed correctly then perfect diffraction
limited images of all planes in the specimen can be acquired by scanning the
reference mirror. In addition to this, we will also present results from a number
of different confocal systems that have been built to exploit this principle of
operation.
6630-25, Session 5
6630-22, Session 4
Two-photon luminescence imaging of cancerous
tissue using gold nanorods as bright contrast
agents
Spherical aberration cancellation in polarized
photon-pairs confocal laser scanning
microscopy
N. J. Durr, T. Larson, D. K. Smith, The Univ. of Texas/Austin (USA); B. A.
Korgel, The Univ. of Texas/Austin (USA) and Texas Materials Institute
(USA); K. V. Sokolov, The Univ. of Texas M.D. Anderson Cancer Ctr.
(USA); A. Ben-Yakar, The Univ. of Texas/Austin (USA)
C. Chang, National Central Univ. (Taiwan); C. Chou, National Yang Ming
Univ. (Taiwan) and National Central Univ. (Taiwan); H. Huang, National
Yang Ming Univ. (Taiwan); H. Chang, National Central Univ. (Taiwan); W.
Kuo, National Taiwan Normal Univ. (Taiwan); H. Yau, National Central
Univ. (Taiwan)
Refractive index mismatch in specimen induces spherical aberration in
conventional confocal microscopy with high numerical aperture objective.
This results in the axial resolution degradation apparently. In this study, we
developed a polarized photon-pairs confocal laser scanning microscopy
(PCLSM) which is able to reduce the spherical aberration effectively. The
theory of image formation of PCLSM is derived of which the spherical
aberration is cancelled theoretically based on the properties of propagation
of polarized photon-pair in PCLSM. In the experiment, two different
thicknesses (1.3 mm and 0.46 mm) of glass plates are introduced into PCLSM
in order to produce spherical aberration. Then, the experimental results verify
the ability of PCLSM on spherical aberration reduction. The scattering effect
of specimen on the depolarization and decorrelation of polarized photonpairs decreases the ability of spherical aberration cancellation in PCLSM is
discussed.
6630-23, Session 5
High throughput, high content microscopic
imaging
P. T. C. So, Massachusetts Institute of Technology (USA)
High throughput and high content imaging tools are vital in biological and
medical studies. In this presentation, we will describe a new tool: two-photon
tissue cytometry. This new method allows high throughput and high content
biological imaging of whole small animal organs with sub-micron resolution.
Two-photon tissue cytometry is developed to address the problem that cellular
states are strongly influenced by cell-cell and cell-matrix interactions.
Traditional cytometry has limited ability to quantify structure and biochemistry
of intact tissues in three dimensions (3D). We extend the technique of image
cytometry to quantify 3D tissue physiology and pathology states with
subcellular resolution. Based on a high-speed multiphoton microscope for
3D imaging and an automated computer-controlled specimen stage, we can
quantify cellular and tissue morphology and biochemistry in a high throughput
manner. With the excellent penetration depth of multiphoton microscopy, it
can assay tissue structures with subcellular resolution down to a few hundred
micrometers in most tissues. By incorporating an automated microtome
system, we can further advance this technique for the quantification of tissue
morphology throughout whole small animal organs with volume on the order
of a cubic centimeter spanning five orders of magnitude in length scale.
Applications in cardiology, cancer metastasis, carcinogenesis, and muscle
physiology will be presented.
48
European Conferences on Biomedical Optics 2007 •
We demonstrate the use of gold nanorods as molecularly targeted contrast
agents for two-photon luminescence (TPL) imaging of cancerous cells deep
inside a tissue phantom. Gold nanorods are a desirable alternative to
traditional two-photon contrast agents because of their brightness, ease of
synthesis, tunable optical properties, biocompatibility, and resistance to
photobleaching. We synthesized gold nanorods of 50 nm x 15 nm size with a
longitudinal surface plasmon resonance of 760 nm. The nanorods were
conjugated to anti-epidermal growth factor and labeled to A431 human
epithelial skin cancer cells in a collagen matrix tissue phantom. Using a
custom-built two-photon microscope, we found that excitation power needed
for similar emission intensity in TPL imaging of labeled cells was 64 times
less than that needed for autofluorescence imaging of unlabeled cells, which
would correspond to a more than 4,000 times increase in emission intensity
under equal excitation energy. TPL images show two-photon luminescence
signal comes primarily from the membrane of the cells, indicating successful
labeling of epidermal growth factor receptor. We also measured strong twophoton emission from gold nanorods in the tissue phantoms from a depth
up to 75 microns, which is of major significance from the point of view of in
vivo bioimaging. Furthermore, the increase in excitation energy required for
consistent emission signal collection as imaging depth was increased was
the same in both labeled and unlabeled phantom, suggesting that, at the
concentrations used, the addition of gold nanorods did not appreciably
increase the bulk scattering coefficient of the tissue. The remarkable TPL
brightness of gold nanorods in comparison to two-photon autofluorescence
(TPAF) signal makes them an attractive contrast agent for early detection of
epithelium cancers.
6630-26, Session 5
A new, easy to use light source for CARS
microscopy based on an optical parametric
oscillator
I. Rimke, APE GmbH (Germany); C. L. Evans, Harvard Univ. (USA); E.
Büttner, APE GmbH (Germany); S. Xie, Harvard Univ. (USA)
A new, broadly tuneable synchronously pumped picosecond optical
parametric oscillator (OPO) for Coherent anti-Stokes Raman Scattering
(CARS) microscopy is presented. It is based on a non-critically phase-matched
LBO crystal, pumped by the second harmonic (532 nm) of a mode-locked
Nd:Vanadate laser.
The tuning range covers 680 nm to 990 nm (Signal beam) and 1150 nm to
2450 nm (Idler beam), thus completely substituting picosecond - Ti:Sapphire
lasers. By using the Signal and Idler as pump and Stokes beams for CARS
microscopy, this translates into a vibrational frequency range of ?1350 \>10.000 cm-1.
Both beams are extracted from the same cavity mirror and therefore propagate
collinearly. Due to the mechanism of their generation, Signal and Idler are
optically synchronized, and thus, perfectly overlap in space and in time with
no jitter.
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The 5 ps pulses generated are close to transform limited and of excellent
beam quality (M(c)˜ < 1,1) and show a high pointing stability. The output
power for Signal and Idler is about 2 W \@ 4 W pump power leading to 50%
overall conversion efficiency.
The perfect spatial and temporal overlap, stable operation, and broad
tuneability makes the described OPO an ideal and nearly hands-free laser
source for CARS microscopy. The longer operational wavelength range results
in higher penetration depths and lower sample photodamage than previously
reported systems. Thus, our CARS source is optimized to image highly
heterogeneous tissue samples, as will be shown in several applications.
The latest methods for further sensitivity improvements will be presented.
6630-27, Session 5
Application of multiplex CARS spectroscopy and
microscopy in biomedical sciences
H. A. Rinia, Univ. van Amsterdam (Netherlands); M. Bonn, FOM Institute
for Atomic and Molecular Physics (Netherlands); E. M. Vartiainen,
Lappeenrannan Teknillinen Yliopisto (Finland); C. B. Schaffer, Cornell
Univ. (USA); M. Müller, Univ. van Amsterdam (Netherlands)
Coherent Anti-Stokes Raman scattering (CARS) is the non-linear analogue
of spontaneous Raman scattering. Due to the coherent and non-linear nature
of the process, the CARS signal is about 4 to 5 orders of magnitude larger
than the linear Raman signal and the signal is easily discriminated from
background luminescence. Also, CARS offers the possibility of 3-D imaging
thanks to its inherent optical sectioning capabilities. These virtues make
CARS microscopy a promising tool in biomedical imaging. Multiplex CARS
microscopy and spectroscopy provide chemical, physical and quantitative
information on the studied sample. Due to the coherent addition of both
resonant and non-resonant contributions, the lineshape of CARS spectra is
complicated. This is especially the case for biological samples which give
rise to highly congested spectra. In order to extract information from our
measured spectra, we apply a direct phase retrieval algorithm based on the
maximum entropy method. We have used multiplex-CARS spectroscopy to
quantitatively measure the oxygen saturation level of hemoglobin. Unlike other
optically based techniques developed to determine blood oxygenation, CARS
has, in principle, the capability to resolve the signal from individual vessels
located in (highly scattering) tissue. Because the oxygenation state of the
blood is a key determinate of physiological state, direct determination of the
oxygenation of blood in vessels in live tissue would provide elusive information
in studies concerning vascular dysfunction. The experimental results are
discussed and future potential of the technique in biomedical applications is
indicated.
6630-28, Session 5
Confocal Raman microscopy for investigation of
differentiating living tumor cells
C. Scalfi-Happ, S. Fulda, Univ. Ulm (Germany); A. Jauss, WITec GMBH
(Germany); O. Hollricher, WITec GmbH (Germany); C. Hauser, R. W.
Steiner, A. C. Rueck, Univ. Ulm (Germany)
The investigation of living cells at physiological conditions requires sensitive,
sophisticated, non invasive methods. In this study, Raman spectroscopy, a
chemically sensitive measuring technique, is combined with high resolution
confocal microscopy to obtain a complete Raman spectrum at every confocal
image point.
Neuroblastoma is the most common solid extra-cranial tumor in children,
having the unique feature to spontaneously differentiate, eventually leading
to complete remission. Since differentiating agents are used for neuroblastoma
therapy, there is a special need to develop non-invasive and sensitive new
methods to monitor neuroblastoma cell differentiation.
Neuroblastoma cells at different degrees of differentiation were analysed with
the confocal Raman microscope alpha300 R (WITec GmbH, Germany), using
a frequency doubled Nd:YAG laser at 532 nm and 10 mW for excitation.
Integration time per spectrum was 80-100 ms. A 60x water immersion lens
with a numerical aperture of 1,0 allows to reach a lateral resolution in
submicrometer range.
Raman images of cells at different degrees of differentiation were generated
from these data sets by either integrating over specific Raman bands or by
basis analysis using reference spectra. The automated evaluation of all spectra
as linear combinations of basic spectra results in spectral unmixed images
providing insight into the chemical composition of the sample. With this
procedure, different cell organelles, cytosol, membranes could be
distinguished.
The results of this work may have applications in monitoring of molecular
changes and distribution of biomolecules and in particular of low molecular
weight markers as they occur during the differentiation of neuroblastoma
cells.
European Conferences on Biomedical Optics 2007 •
6630-36, Poster Session
Simultaneous imaging of confocal fluorescence
and Raman spectrum
M. Ahn, Korea Advanced Institute of Science and Technology (South
Korea)
The confocal fluorescence microscopy provides higher spatial resolution than
the widefield fluorescence microscope. The advantage of confocal
fluorescence microscopy is the ability to acquire high resolution images of
fluorescent specimens non-invasively. And the advantage of confocal Raman
microscopy is the ability to provide the chemical characteristics of specimens
from spectroscopy. To obtain simultaneously high resolution images and
chemical characteristics of specimens, the fluorescence signals of cell and
the Raman spectrum of cell itself should be separated. By separating two
kinds of signals, the fluorescence image and the Raman spectrum are
acquired simultaneously at the same position.
In general case, the Raman spectrum acquired from the dyed cell contains
the fluorescence signal. Because the fluorescence intensity is much larger
than the Raman spectrum intensity, the Raman spectrum cannot be isolated
from the fluorescence signal. Even though the Raman spectrum from the
dyed cell can be measured from the fluorescence signal, it contains both the
spectrum of fluorophore itself and the spectrum of cell itself. Thus the Raman
spectrum of the cell itself is hard to be obtained.
In this paper, we demonstrate a confocal fluorescence microscopy combined
with the Raman microscopy. And we propose a method that eliminates the
Raman spectrum of dye itself from the Raman spectrum of the dyed cell and
that obtains simultaneously the fluorescence image from the dyed cell and
the Raman spectrum of cell itself without replacing the cell. By using the
proposed method, we compare the fluorescence image with the Raman image
and analyze them.
6630-37, Poster Session
Improvement of axial resolution in confocal
microscopy using heterodyne illumination
S. Lee, Korea Advanced Institute of Science and Technology (South
Korea)
In this thesis, a new technique for improving the axial resolution of confocal
microscope is proposed. This approach is based on the generation of a
frequency domain field confined focal spot, which is made by overlapping
two different frequency beams axially. In the vicinity of overlap region, the
interference between two beams is occurred. The signal due to the
interference has a beating with a frequency of . If the only information
generated at the overlap region is extracted, the higher axial resolution will
be obtained. This is why the effective region made by the interference is
smaller in axial direction than an ordinary single beam.
The analytic expression of the 3D intensity point spread function (IPSF)and
optical transfer function (OTF) are derived and calculated numerically.
The numerical results show that the full width half maximum (FWHM) of the
IPSF is improved by factor of 1.74 maintaining maximum side lobes below
0.5.
6630-38, Poster Session
Design of high efficiency and simple multichannel spectral detector for confocal scanning
microscopy
I. Song, S. Lee, D. Gweon, Korea Advanced Institute of Science and
Technology (South Korea)
High Content Screening (HCS) system has been useful as an early drugdiscovery platform for improving the quality of targets, hits, and leads and
shorten time and cost of drug development. HCS is defined as the automation
of high-content cell biological investigation of arrayed cells including the key
operation of experimental design, sample preparation, image acquisition,
archiving, processing and analysis, and cellular knowledge mining. Confocal
microscopy is commonly used as image acquisition tool for HCS system.
HCS system commonly needs to acquire many information simultaneously,
confocal microscopy in a HCS system is faced with screening a various
fluorescent probes in a same time. In this article, we propose a new spectral
detector scheme which measures three fluorescences simultaneously with
high efficiency and flexibility. The proposed spectral detector utilizes acoustooptic tunable tilter (AOTF) and equilateral dispersion prism. Excitation laser
is incident to AOTF at an angle of first order and go to scanning optics and
objective lens. Fluorescent lights come back to AOTF and transmit it at zero
order angle. The excitation light reflected from specimen and slide glass is
diffracted at AOTF so that spatially separated with fluorescent light. In zero
order angle, AOTF acts like a dispersing prism so fluorescent light is dispersing
chromatically. The dispersion angle is amplified by using dispersing prism
and fluorescent light, spatially decoded by wavelength, is focused on multichannel PMT. Placing a slit would control detectable wavelength region at
each detect area.
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49
Conf. 6630: Confocal, Multiphoton, and Nonlinear Microscopic Imaging
6630-39, Poster Session
Intravital multiphoton microscopy for imaging
hepatobiliary function
F. Li, T. Sun, C. Dong, National Taiwan Univ. (Taiwan)
Liver is the chemical factory in body responsible for important functions such
as metabolism and detoxification. When liver can not be regenerated in time
to amend damages that has occurred, failure of hepatic functions such as
liver failure and metabolic disease can result. Traditionally, the study of liver
pathology has depended on histological techniques, but such methods are
limited to ex-vivo observation. In order to study hepatic metabolism in vivo,
we have designed a hepatic imaging chamber made of biocompatible titanium
alloy (6V4Al-Ti, ELI grade). In combination with multiphoton and second
harmonic generation microscopy, our approach allows the intravital
observation of hepatic intravital activities to be achieved. Processes such as
hepatic metabolism and disease progression can be studied using this
methodology.
fluorescent light from dye molecules, because of good signal-to-noise ratio
and time resolution. Using the APD, however, measurement is performed in
a single point at once, so lacking spatial resolution. Then we develop a FCS
system, which uses an electron-multiplying charge-coupled device (EM-CCD)
for spatially resolved FCS measurement.
In our FCS system, an EM-CCD camera (C9100-2, Hamamatsu Photonics),
which is operated in the fastest mode (16 x 16 pixel, time interval of 3 ms), is
used for detection and imaging of fluorescence. Excitation light from a Nd:
YAG laser of wavelength of 532 nm is incident to an observing area on a
sample. The sample, which consists of fluorescent beads (diameter is 100
nm) dispersed in water, is filled in between two cover glass slips (spacing is
3um). Observation volume of FCS is estimated to 26.5 fL under our
experimental condition of a cell size of 2.1 x 2.1 x 3 micron in x, y, and z
direction, respectively. We tested 3 samples, which have different
concentrations of fluorescent beads, and successfully investigated the
difference of correlation coefficients of FCS signal. As a consequence EMCCD can be used as a detector of spatially resolved FCS. This method can
be applied to a measurement of local diffusion coefficient of molecules in
living cells.
6630-40, Poster Session
Multifocal multispectral descanned detection in
2-PLSM
T. Bergmann, M. Tiemann, J. Martini, K. Tönsing, D. Anselmetti,
Bielefeld Univ. (Germany)
We present a new detection method for a 2PLSM (2-photon laser scanning
microscope) that allows a fast and easy access to spectrally resolved, threedimensional images without the use of filters. In our setup eight fluorescent
foci are directed through a straight vision prism in the descanned detection
path. This prism spectrally splits up the fluorescence beamlets, resulting in
eight parallel spectral fluorescence lines. These lines are imaged onto a slit
blocking array in front of a 8x8 multi anode PMT. Each PMT row detects an
8-fold spectral characteristic of its corresponding focus, while each column
detects one of the 8 foci. The region of interest in the sample is scanned by
the two scanning mirrors in x- and y-direction. In order to achieve a threedimensional image, the exciting foci are scanned in z-direction (depth) inside
the sample by varying the objective lense’s position. As a result of the eight
foci eight spectrally resolved images of slightly shifted sample regions are
generated and added up after the measurement, maintaining the spectral
information.
We present spectrally resolved 3D-data of various biological samples like
pollen grains, Hela-cells and algae.
6630-41, Poster Session
Fiber laser-based light source for CARS
microspectroscopy
E. R. Andresen, C. K. Nielsen, J. Thøgersen, S. R. Keiding, Åarhus Univ.
(Denmark)
Coherent anti-Stokes Raman scattering (CARS) microspectroscopy employs
two pulses (pump and Stokes), which simultaneously probe a few selected
vibrations.
This selective probing makes CARS faster than conventional Raman
spectroscopy. We demonstrate an alternative light source for CARS
microspectroscopy based on a home-built fiber laser and a photonic-crystal
fiber. The light source simultaneously delivers a near-transform-limited
picosecond pump pulse at 1033 nm and a frequency-shifted, near-transformlimited femtosecond Stokes pulse, tunable from 1033 nm to 1400 nm. This
corresponds to a range 0 - 2500 cm-1, so that Raman-active vibrations in
this frequency range can be probed.
The spectral resolution is 5 cm-1, given by the spectral width of the pump
pulse. The frequency range that can be probed simultaneously is 200 cm-1wide, given by the spectral width of the Stokes pulse. The achievable pulse
powers are 50 mW for the pump and 2 mW for the Stokes pulse. The repetition
rate is 35 MHz.
We demonstrate the capability of this light source by performing CARS
microspectroscopy and comparing CARS spectra with Raman spectra. This
light source represents a reduction in the expenses associated with the light
source for CARS microspectroscopy compared to traditional approaches
based on bulk lasers.
6630-42, Poster Session
Spatially resolved fluorescence correlation
spectroscopy based on electron multiplying
CCD
M. Matsumoto, T. Sugiura, K. Minato, Nara Institute of Science and
Technology (Japan)
Fluorescence correlation spectroscopy (FCS) is widely used for investigation
of concentration, diffusion coefficients, and dynamics of single molecules. In
present FCS system an avalanche photo diode (APD) is widely used to detect
50
European Conferences on Biomedical Optics 2007 •
6630-43, Poster Session
Evaluation of a new method for the
determination of experimental PSF of a widefield microscope using white-light and a linear
sensor
M. P. Macedo, Instituto Superior de Engenharia de Coimbra (Portugal);
A. J. Barata, A. G. Fernandes, C. M. B. A. Correia, Univ. de Coimbra
(Portugal)
In the biology field there is a high demand for three-dimensional (3D)
microscopy techniques owing to the need for obtaining depth information.
Confocal microscopy is the reference method but it has the drawback of
slow image acquisition time. Different configurations were developed to
obviate this such as spinning-disk that basically consists in a set of parallel
confocal microscopes and line scanning that uses line illumination so the
scanning in the fast axis is not required. Amongst other 3D techniques there
is the digital microscopy that normally uses a wide-field microscope to acquire
2D images (optical sections) of the specimen containing substantial
contributions from out-of-focus portions. To remove this, a computational
method is used that is based on the definition of a model for the process of
image formation and recording. A compromise should be taken between a
more accurate model with best results or a simplified one with a lower
computational processing time. This work is based on a laboratory prototype
of a wide-field microscope with white-light illumination using a CMOS linear
image sensor. It aims at the application of one simplified model that takes
advantage of a new method for the determination of experimental point spread
function (PSF) that uses a specimen consisting of a set of lines in twoperpendicular directions due to the anisotropy that results from detector
geometry. The 2D and 3D PSF were built. A simulation of the application of
the model was developed using Matlab ((Mathworks (c)) using different
simulated specimens. It will be presented the application of this model to the
imaging of a real specimen and its preliminary results.
6630-44, Poster Session
A time-gated hyperspectral fluorescence
lifetime imaging microscope
H. B. Manning, D. M. Owen, E. Auksorius, P. de Beule, C. B. Talbot, C.
W. Dunsby, I. H. Munro, A. I. Magee, M. A. A. Neil, P. M. W. French,
Imperial College London (United Kingdom)
Spectrally resolved fluorescence lifetime imaging provides an enormous
amount of information that may be used to analyse complex fluorescence
signals in many situations, including imaging autofluorescence of unstained
tissues and multiplexed fluorescence experiments. Here we present a
microscope that is capable of rapidly acquiring optically sectioned, spectrally
resolved, fluorescence lifetime images at continuously tunable excitation
wavelengths in the visible and u.v. spectral region.
Pulsed wavelength-tunable light is used for line illumination of the sample;
the resulting fluorescence is then collected through the input slit of a compact
imaging spectrograph. The resulting x-? image is incident on the input
photocathode of a gated optical intensifier to provide time-gating, and the
output intensified image is recorded by a cooled electron multiplying CCD
camera. Scanning of the microscope stage then allows a full x, y, ?, t data
stack to be acquired as quickly as a few 10’s of seconds.
The tunable excitation source is based on a 3m tapered microstructured
optical fibre pumped by a 7W 1.06µm fibre laser generating a continuum of
light extending from 350nm to 2000 nm. This novel light source, with its
extended u.v. capability, is widely applicable to the study of tissue
autofluorescence.
The hyperspectral FLIM microscope is demonstrated in its application to
tissue autofluorescence and also to the characterisation of the novel phasesensitive membrane dye, di-4-ANEPPDHQ in model membranes and live
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Conf. 6630: Confocal, Multiphoton, and Nonlinear Microscopic Imaging
cells, for which a significant variation of lifetime with emission wavelength
was observed. The unmixing of the di-4-ANEPPDHQ signal from a CFP
construct indicates the potential for multiplexed fluorescence experiments.
6630-45, Poster Session
Fast three-dimensional random access multiphoton microscopy for functional recording of
neuronal activity
P. Saggau, Baylor College of Medicine (USA); D. Reddy, Rice Univ. (USA)
The dendritic processes of neurons have been shown to possess active and
dynamic properties that give them the ability to modulate synaptic integration
and shape individual synaptic responses. Effectively studying these properties
at multiple locations on a live neuron in highly light scattering brain tissue
requires an imaging/recording mechanism with high spatio-temporal resolution
as well as optical sectioning and random access site selection capabilities.
Our lab has made significant steps in developing such a system by combining
the spatial resolution and optical sectioning ability of advanced imaging
techniques such as confocal and multi-photon microscopy with the temporal
resolution and random access capability provided by acousto-optic laser
scanning. However, all systems that have been developed to date restrict
fast imaging to two-dimensional (2D) scan patterns. This severely limits the
extent to which many neurons can be studied since they represent complex
three-dimensional (3D) structures. We have previously demonstrated a scheme
for fast 3D scanning which utilizes a unique arrangement of acousto-optic
deflectors and does not require axial movements of the objective lens. We
have also shown how, when used with the ultra-fast laser pulses needed in
multi-photon microscopy, this scheme inherently compensates for the spatial
dispersion which would otherwise significantly reduce the resolution of
acousto-optic based multi-photon microscopy. We have now coupled this
scanning scheme to a modified commercial research microscope and use the
combined system to effectively image user-defined sites of interest on
fluorescent 3D structures with positioning times that are in the low microsecond
(µs) range. The resulting random-access scanning mechanism allows for
functional imaging of complex 3D structures such as neuronal dendrites at
several thousand volumes per second.
6630-46, Poster Session
Biological applications of microscope profiler
S. Han, Veeco Tucson Inc. (USA); E. L. Novak, Veeco Instruments Inc.
(USA); J. Reed, M. A. Teitell, J. K. Gimzewski, Univ. of California/Los
Angeles (USA)
Interferometric profilers have been widely employed in semiconductor, data
storage, MEMS and research fields for more than two decades. These systems
are known to produce rapid, accurate, and repeatable characterization of
surface roughness, form, film thickness, and more recently dynamic behavior
of MEMS and other moving devices. Recently, however, optical profiling has
been playing a more and more important role in biosensor, living biological
system and biomedical applications.
The measurement of living cells presents technical challenges for standard
interferometric profilers. The system must be able to measure objects in fluid,
where the index of the cell and fluid is sometimes very similar. Also, the
timescales of change for different experiments involving living cells can vary
greatly, requiring a variety of measurement methodologies to maximize data,
including strobed interferometry and video-rate deformation calculations. In
addition, the responses of individual cells can differ significantly from one
another in a given experiment, making measurement of large numbers of cells
in parallel very important to achieve proper statistical sampling of the
population. This presents challenges both in how to best trade field-of-view
for lateral resolution and for how each surface image is analyzed, as the
software intelligently track and log information from each of many different
cells simultaneously.
Measurement of inorganic parts, such as cantilever arrays, removes some of
the challenges associated with cells. However, the need for varying temporal
range, parallelism of measurement across parts, and intelligent analyses that
allow the researcher to easily connect theory with the implemented part are
still present. This paper describes how multiple interferometric techniques,
implemented on a single system, can be combined to provide viable
measurements of both biosensors and cells, enabling collection of data in
environments and with timescales not previously achievable.
6630-29, Session 6
Two-photon microscopy of non-melanoma skin
cancer: initial experience and diagnostic criteria
ex vivo
M. B. Ericson, Göteborg Univ. (Sweden) and Consultant (Sweden); J.
Paoli, Göteborg Univ. (Sweden); A. Odu, Linköping Univ. (Sweden); M.
Smedh, A. K. Wennberg, Göteborg Univ. (Sweden)
Multiphoton microscopy is an interesting optical technique, which allows for
non-invasive imaging of highly light scattering media such as human skin.
European Conferences on Biomedical Optics 2007 •
Recent reports have showed the potential of applying this technique for 3D
visualisation of cell structures of biological tissue without previous sectioning
of the tissue samples. In this study, we have applied two-photon microscopy
on excised lesions of human non-melanoma skin cancer ex vivo in order to
find diagnostic criteria using this technique. The skin samples have been
investigated by a multiphoton microscopy system based on a fs-pulsed
Ti:sapphire laser connected to confocal microscope. The autofluorescence
of the skin was detected using excitation at 780 nm. The cell nuclei distribution
turned out to be one important parameter which can be used for characterising
between tumour and normal tissue. We are now developing a technique for
automatic detection and characterisation of tissue, based on an image
analysis algorithm. The detection of cell nuclei has been found crucial for
this purpose. The goal is to develop a fast characterisation algorithm that
can be used on line in connection to in vivo investigations. This would allow
for a true non-invasive biopsy technique in the future.
6630-30, Session 6
Multiphoton tomograph DermaInspect(r): non
invasive powerful tool for in vivo evaluation of
the human skin compounds
R. Le Harzic, Fraunhofer-Institut für Biomedizinische Technik (Germany);
R. Bückle, JenLab GmbH (Germany); A. Ehlers, Fraunhofer-Institut für
Biomedizinische Technik (Germany); A. Colonna, L’Oreal (Germany); C.
Hadjur, F. Leroy, F. Flament, R. Bazin, B. Piot, L’Oreal (France); I.
Riemann, K. König, Fraunhofer-Institut für Biomedizinische Technik
(Germany)
In vivo simultaneous collagen and elastin measurements using the multiphoton
tomograph dermaInspect have been performed in the dermis of the skin. We
have demonstrated the ability of simultaneous measurements of
autofluorescence (AF) and Second Harmonic Generation (SHG) with a new
developed device using 2 PMTs for time-correlated single photon counting.
Collagen structures are able to generate second harmonics (SHG) in contrast
to elastin (AF). The comparison of the images and ratios of SHG / AF recorded
in the depth at the outer and inner side of the forearm of two European
female volunteers (31 and 60 years) shows differences in collagen and elastic
fibres density. It decreases with depth for the 60 years old volunteer compared
to the 31 years old one. More collagen is observable for a young skin
compared to an older one.
Furthermore, a comparison between an Asian (29 years old) and an European
(31 years old) skin has been studied. In that work AF and SHG images in the
epidermis and dermis have been performed separately. Measurements and
comparisons of images, ratios of SHG / AF and SAAID show some differences
between both skins and that more collagen is present in the dermis than
elastin for both type skin which allow to control the effects of photoaging.
The DermaInspect device demonstrates the ability to perform in vivo
multiphoton images in various type of skin
6630-31, Session 6
Adjustable mirror arm for in-vivo two-photon
microscopy
N. Koop, M. Ehrke, G. Hüttmann, Univ. zu Lübeck (Germany)
The Derma-Inspect two photon imaging system (JenLab) allows in-vivo
autofluorescence imaging of human skin with subcellular resolution. Main
drawback of the system are difficulties to access certain skin areas due to
the fixed position of the scan head. A mirror arm, which can be attached to
the DermaInspect with minimal modifications, is presented, which allows to
extend the two-photon diagnosis to difficult locations (e.g. in the face).
Femtosecond excitation and resulting tissue fluorescence are relayed by the
combination of dielectric mirrors and lenses in a 4-f configuration. The joints
are easy to move, but can be locked in any desired position in order to prevent
motion artifacts during imaging. Excellent fluorescence and FLIM images
were obtained with the mirror arm.
6630-32, Session 6
Spectrally encoded confocal imaging in vivo
through a handheld probe
C. Boudoux, Massachusetts Institute of Technology (USA); D. Yelin, W.
Y. Oh, M. S. Shishkov, B. E. Bouma, G. J. Tearney, Harvard Medical
School (USA)
Spectrally encoded confocal microscopy (SECM) enables reflectance confocal
microscopy to be conducted within the confines of a small-diameter, flexible
probe, thus extending the number of potential medical applications of this
technique. In this work, we present a novel design for a handheld confocal
microscope capable of cellular and subcellular imaging in vivo. For our system,
scanning the fast axis of the image was accomplished by illuminating a high
density holographic transmission grating with a rapidly tuned, wavelength
swept source (center 1310 nm, bandwidth 70 nm). The slow axis of the image
was scanned by mounting the grating on a galvanometer. The scanned grating
was imaged onto the pupil of a microscope objective (LOMO, 0.75NA, 40x,
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Conf. 6630: Confocal, Multiphoton, and Nonlinear Microscopic Imaging
water immersion) by a telecentric telescope, housed in a stainless steel tubing
(outer diameter: 15 mm). The laser, detection, and acquisition electronics
were packaged into a portable console. The SECM system yielded images
with a 350x350 micrometer field of view, a transverse resolution of 1.4 µm,
and an axial resolution (optical sectioning thickness) of 6 µm. With the probe,
we obtained reflectance confocal microscopy images of human skin in vivo
at 10 frames per second. Images showed microstructural features of the
epidermis and dermis including cell membranes, nuclei, and blood cells
flowing through capillaries within the dermal papillae. These results support
the use of this tool for visualizing histopathologic features in situ to facilitate
intraoperative tissue identification . Clinical pilot studies to investigate this
device for evaluating laryngeal superficial lamina propria microstructure are
also underway.
6630-33, Session 6
Utilizing nonlinear optical microscopy to
investigate the development of early cancer in
nude mice in vivo
C. Wang, F. Li, S. Lin, W. Lo, C. Dong, National Taiwan Univ. (Taiwan)
In this investigation, we used nonlinear optical microscopy to image and
analyze normal and carcinogen DMBA treated skin tissues of nude mice in
vivo. Using the results obtained from two-photon autofluroescence and
second harmonic generation (SHG) images and the application of ASI
(Autofluorescence versus SHG Index), we can visualize the interaction
between mouse skin cells and connective tissue.
We found that as the imaging depth increases, ASI has different distribution
in normal and treated skin tissues. Since the DMBA treated skin eventually
became squamous cell carcinoma (SCC), our results show that the
physiological changes to mouse skin en route to become cancer can be
effectively tracked by multiphoton microscopy. We envision this approach
can be effective in studying intravital tumor biology, leading to improved
tumor treatment procedures.
6630-34, Session 6
Investigation of depilatory mechanism by use of
multiphoton fluorescent microscopy
C. Lin, J. Lee, S. Lin, S. Jee, C. Dong, National Taiwan Univ. (Taiwan)
Transdermal drug delivery provides a non-invasive route of drug
administration, and can be a alternative method to oral delivery and injection.
The stratum corneum (SC) of skin acts as the main barrier to transdermal
drug delivery. Studies suggest that depilatory enhances permeability of drug
through the epidermis. However, the detailed pathway and mechanism of
transdermal delivery are not completely understood. Previous studies have
found that depilatory changes the keratinocytes of epidermis, and cause the
protein and the lipid extraction of the SC to become disordered.
In this study, we use multi-photon microscopy to visualize and quantify
fluorescence probes delivery. The aim of this study is to use multi-photon
fluorescent microscopy to study the delivery pathway due to depilatory
enhanced delivery of fluorescence probes.
6630-35, Session 6
Multiphoton Microscopy for the Investigation of
trans-cutaneous drug delivery
F. Stracke, Fraunhofer-Institut für Biomedizinische Technik (Germany);
M. Schneider, B. Weiss, C. Lehr, U. F. Schäfer, Univ. des Saarlandes
(Germany); K. König, Fraunhofer-Institut für Biomedizinische Technik
(Germany)
The trans-cutaneous pathway for drug delivery is of particular interest since
it allows a simple and non-invasive administration of pharmaceutically relevant
compounds. As the skin is an effective barrier for many of these compounds,
various strategies have been developed to enable and control the transcutaneous transport. Here we discuss, how multiphoton microscopy and
spectral imaging can be valuable tools for the analysis of the penetration
pathways of topically applied drugs. A time dependent study of the cutaneous
penetration of a fluorescent drug model released from a nano-particular carrier
is presented. The localization of single nano-particles in human skin (ex vivo)
and the discrimination of different fluorescent compounds, as the drug model,
the particle’s label and the cutaneous endo-fluorescence by spectral imaging
and selective excitation is shown.
Multiphoton imaging techniques were found to be excellent methods for the
non-invasive evaluation of cutaneous drug delivery strategies and analysis
of dermal penetration pathways down to the sub-cellular level.
52
European Conferences on Biomedical Optics 2007 •
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Conference 6631: Novel Optical Instrumentation for Biomedical Applications
Room 11 • Sunday-Tuesday 17-19 June 2007
Part of Proceedings of SPIE Vol. 6631 Novel Optical Instrumentation for Biomedical Applications III
6631-01, Session 1
Improvement of depth resolution on
photoacoustic imaging using multiphoton
absorption
Y. Yamaoka, T. Takamatsu, Kyoto Prefectural Univ. of Medicine (Japan)
Commercial imaging systems, such as CT and MRI, are frequently used as
powerful tools for observing the deep part of human body. However, they
cannot precisely observe several-tens micrometer-sized structure for lack of
spatial resolution. In this paper, we propose a photoacoustic imaging using
multiphoton absorption technique to generate ultrasonic waves for improving
depth resolution. Since the multiphoton absorption occurs at only the focus
point and employed infrared pulses deeply penetrate in living tissues, it
enables us to extract characteristic features embedded in the living tissue.
When nanosecond pulses from 1064nm Nd:YAG laser were focused on
Rhodamine B/chloroform solution (absorption peak: 540 nm), the peak
intensity of generated photoacoustic signal was in proportional to the square
of the input pulse energy. This result means that the photoacoustic signals
can be induced by the two-photon absorption of infrared nanosecond pulse
laser and also can be detected by a commercial MHz transducer. Furthermore,
in order to evaluate the depth resolution of multiphoton-photoacoustic
imaging, we investigated the dependence of photoacoustic signal on depth
position using 1mm-thick phantom in water bath. As a result, we found that
the depth resolution of two-photon photoacoustic imaging (1064nm) is
improved compared with that of one-photon photoacoustic imaging (532nm).
It is concluded that the evolution of the multiphoton-photoacoustic imaging
renders the investigation of biomedical phenomena at the deep layer in the
living tissue.
6631-02, Session 1
Photoacoustic image reconstruction methods: a
quantitative analysis
J. I. Sperl, General Electric Co. (Germany); K. Zell, Technische Univ.
München (Germany); P. Menzenbach, Innolas GmbH (Germany); C.
Haisch, Technische Univ. München (Germany); S. Ketzer, M. Marquart,
H. Koenig, M. W. Vogel, General Electric Co. (Germany)
Photoacoustic imaging is a promising new way to generate unprecedented
contrast in ultrasound diagnostic imaging. It differs from other medical imaging
approaches, in that it provides spatially resolved information about optical
absorption of targeted tissue structures. Because the data acquisition process
deviates from standard clinical ultrasound, choice of the proper image
reconstruction method is crucial for successful application of the technique.
In the literature, multiple approaches have been advocated, and the purpose
of this paper is to compare four reconstruction techniques. Thereby, we
focused on lateral resolution, reconstruction speed and SNR.
We generated experimental data, acquired using 7ns 532nm laser pulses
and a PVDF-needle hydrophone, as well as simulated data. We reconstructed
images of the pressure distribution using four different methods: delay-andsum (DS), circular backprojection (CB), Fourier transform (FT), and generalized
2D Hough transform (HT).
All methods were able to depict the point sources properly. DS and CB
produce blurred images containing typical superposition artifacts. HT shows
an excellent SNR but suffers from the longer computation time. The FT is the
fastest and the best resolving algorithm.
In our study, we found the FT and the HT to outperform the superposition
methods. Regarding the limitations of the HT with respect to complex source
shapes and its numerical inefficiency, the FT has the best overall performance.
It allows a theoretically exact reconstruction in real-time. Thus Fourier
transform based image reconstruction methods should be implemented to
full utilize the new contrast mechanisms in full resolution and fidelity.
6631-03, Session 1
Two-dimensional image reconstruction for
photoacoustic tomography with line detectors
G. Paltauf, R. Nuster, Karl-Franzens-Univ. Graz (Austria); M. Haltmeier,
Leopold-Franzens-Univ. Innsbruck (Austria); P. Burgholzer, Upper
Austrian Research GmbH (Austria)
It has been recently shown that three-dimensional photoacoustic tomography
(PAT) is possible if the ultrasound detectors receiving acoustic waves from
the photoacoustic source are in at least one dimension much larger than the
size of the object to be imaged. An example is PAT with integrating line
detectors, which requires a two-step image reconstruction procedure: In the
first step signals from a scan of the line along the object are used to reconstruct
a projection of the initial photoacoustic pressure in the object, which involves
an inversion of the two-dimensional acoustic wave propagation. In the second
step, projections taken at different angles are combined to give a set of linear
European Conferences on Biomedical Optics 2007 •
Radon transforms of the initial pressure in the object, which can be inverted
using standard methods. In many practical applications the line detector
cannot scan along a curve that totally encloses the object, resulting in a
limited detection view problem for the first step. Strategies are presented
and analyzed how to solve this problem of two-dimensional photoacoustic
image reconstruction with different shapes of the scanning curve, such as a
combination of lines or a spherical arc. Time and frequency domain
reconstruction algorithms are compared and tested with experimental signals.
6631-04, Session 1
OPUS: optoacoustic imaging combined with
conventional ultrasound for breast cancer
detection
C. Haisch, K. Zell, Technische Univ. München (Germany); J. I. Sperl,
General Electric Co. (Germany); M. W. Vogel, General Electric Co.
(USA); P. Menzenbach, InnoLas GmbH (Germany); R. Niessner,
Technische Univ. München (Germany)
Besides x-ray imaging, ultrasound imaging is the most common method for
breast cancer screening. The intention of our work is to develop optoacoustical
imaging as an ad-on to a conventional system. While ultrasound imaging
measures acoustical properties of tissue, optoacoustics generates an imaging
of the distribution of optical absorption in tissue. Hence, it can be a valuable
tool, because acoustical properties of different tissues show only a slight
variation whereas the optical properties can strongly differ. Additionally,
optoacoustics can reveal physiological parameters like oxygen saturation of
blood.
For preliminary studies, we employed a 10 Hz laser at 532 nm with 7 ns
pulse duration (Spitlight 200, InnoLas, Krailling, Germany) to induce
optoacoustic effect, while an ultrasound device (General Electric - Global
Research, Garching, Germany) is responsible for the detection. The laser
pulse is delivered fiber-optically to the ultrasound transducer and coupled
into the acoustical field of view. Homogeneous illumination is vital in order to
achieve unblurred images, furthermore, maximum pulse intensities have to
be adjusted in accordance with standards for medical equipment. The
ultrasound instrument generates the trigger signal which controls the laser
pulsing in order to apply ultrasound instrument’s imaging procedures without
major modifications to generate an optoacoustic image. First experiments
were performed on tissue phantoms. These phantoms have been specially
designed regarding their acoustical as well as their optical properties.
In the next step, the laser is replaced by a 100 Hz laser system (InnoLas) to
achieve a higher frame rate and so allows moving the sensor head more
rapidly. Furthermore, the laser is coupled to an optic-parametric oscillator
(OPO) to be able to tune the wavelength of the laser pulses in a range from
680 nm to 2500 nm, which allows to select wavelengths compromising high
spectral information content with high skin transmission. Different wavelengths
will be compared.
6631-05, Session 1
Development of waveguide sensors for the
application in photoacoustic tomography
R. Nuster, G. Paltauf, H. Ditlbacher, Karl-Franzens-Univ. Graz (Austria);
P. Burgholzer, Upper Austrian Research GmbH (Austria)
Photoacoustic tomography (PAT) is based on the recording of the acoustic
signals excited by illumination of a sample with short laser pulses. PAT with
integrating line detectors is a promising alternative to the current methods
using arrays of small ultrasound transducers or single detectors scanning
around the object. The use of an optical waveguide as an integrating line
detector is obvious. An arriving acoustic pulse modifies the dimensions of
the waveguide, as well as the refractive index of the waveguide material and
of the surrounding liquid. This results in a change of amplitude and phase of
the transmitted light. We chose Polystyrol (PS) as slab waveguide material
because it is acoustically well matched to water. The sensor development
and its application to imaging of small biological samples is subject of this
work.
6631-06, Session 2
Photoacoustic tomography using a fiber based
Fabry-Perot interferometer as an integrating line
detector and image reconstruction by modelbased time reversal method
H. Grün, Upper Austrian Research GmbH (Austria); M. Haltmeier,
Leopold-Franzens-Univ. Innsbruck (Austria); G. Paltauf, Karl-FranzensUniv. Graz (Austria); P. Burgholzer, Upper Austrian Research GmbH
(Austria)
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Conference 6631: Novel Optical Instrumentation for Biomedical Applications
Photoacoustic imaging is based on the generation of acoustic waves in a
semitransparent sample (e.g. soft tissue) after illumination with short pulses
of light or radio waves. The goal is to recover the spatial distribution of
absorbed energy density inside the sample from acoustic pressure signals
measured outside the sample (photoacoustic inverses problem).
If the acoustic pressure outside the illuminated sample is measured with a
large-aperture detector, the signal at a certain time is given by an integral of
the generated acoustic pressure distribution over an area that is determined
by the shape of the detector. For example a planar detector measures the
projections of the initial pressure distribution over planes parallel to the
detector plane, which is the Radon transform of the initial pressure distribution.
Stable and exact three-dimensional imaging with a planar integrating detector
requires measurements in all directions of space and so the receiver plane
has to be rotated to cover the entire detection surface.
We have recently presented a simpler set up for exact imaging which requires
only a single rotation axis and therefore the fragmentation of the area detector
into line detectors perpendicular to the rotation axis. Using a two-dimensional
reconstruction method and applying the inverse two-dimensional radon
transform afterwards gives an exact reconstruction of the three-dimensional
sample with this set up.
In order to achieve high resolution, a fiber-based Fabry-Perot (FP)
interferometer is used. It is a single mode fiber with two fiber bragg gratings
on both ends of the line detector. Thermal shifts and vibrations are
compensated by frequency locking of the laser. The high resolution and good
performance of this integrating line detector has been demonstrated by
photoacoustic measurements with line grid samples and phantoms using a
model-based time reversal method for image reconstruction. The time
reversed pressure field can be calculated directly by retransmitting the
measured pressure on the detector positions in reversed temporal order.
The work is supported by the Austrian Science Fund (FWF), project P18172N2. Additionally, the work of M.H. has been supported by the FWF, project
Y123-INF.
6631-09, Session 2
Concomitant acoustic property measurements
in a photoacoustic imager
S. Manohar, R. Willemink, F. v. d. Heijden, K. Slump, T. G. van Leeuwen,
Univ. Twente (Netherlands)
Photoacoustics is a hybrid imaging technique that combines the contrast
available to optical imaging with the resolution that is possessed by ultrasound
imaging. The technique is based on generating ultrasound from absorbing
structures in tissue using pulsed light. In computerized tomography (CT)
photoacoustic imaging, reconstruction of the optical absorption image in a
subject, is performed by filtered acoustic backprojection. Here the
backprojection is performed along circular paths into image space instead
of along straight lines as in x-ray CT imaging. For this the speed-of-sound
through the subject is usually assumed. An unsuitable speed-of-sound can
compromise resolution and contrast. We discuss here a method of actually
measuring speed-of-sound tomograms using ultrasound transmission
through the subject under photoacoustic investigation. This is achieved in a
simple approach that does not require any additional ultrasound transmitter.
The method uses a carbon-fibre that is placed in the imager in the path of
the illumination which generates ultrasound by the photoacoustic effect and
behaves as an ultrasound source. Measuring the time-of-flight of this
ultrasound transient by the same detector used for conventional
photoacoustics, allows a speed-of-sound image to be reconstructed. This
concept is validated on phantoms.
6631-10, Session 3
Simultaneous acquisition of time-domain fNIRS
and fMRI during motor activity
6631-07, Session 2
A. Torricelli, D. Contini, A. Pifferi, L. Spinelli, R. Cubeddu, Politecnico di
Milano (Italy); L. Nocetti, A. A. Manginelli, P. Baraldi, Univ. degli Studi di
Modena e Reggio Emilia (Italy)
Development of a small animal photoacoustic
imager: system performance with phantom
studies
A time-domain fNIRS system was developed for simultaneous acquisition
with fMRI. Preliminary results during motor activity indicate good sensitivity
and temporal resolution of the system. To our knowledge this is the first
time-domain fNIRS and fMRI study on human brain.
C. Panneman, S. Manohar, R. Willemink, J. G. C. van Hespen, T. G. van
Leeuwen, Univ. Twente (Netherlands)
6631-11, Session 3
Photoacoustic imaging is a non-invasive technique that is capable of
interrogating optical absorption contrast in tissue while retaining high
resolution. It is based on illuminating the subject under investigation with
nanosecond pulses of near-infrared light, and detecting the ultrasound that
is generated using ultrasound detectors. We describe here a miniaturized
imager intended for imaging mice. We discuss various instrumental aspects
and present performance studies in terms of sensitivity, frequency response
and resolution. Studies on well-characterized phantoms carrying tumour
simulating inserts are shown. Imaging results of rod-shaped gold
nanoparticles embedded in phantoms are also shown. These nanorods having
absorption peaks at 800 nm are potential contrast enhancers for
photoacoustic imaging. Finally imaging results on sacrificed mice are
presented.
6631-08, Session 2
Gold nanorods: contrast agents for
photoacoustic imaging?
C. Ungureanu, R. G. Rayavarapu, S. Manohar, T. G. van Leeuwen, Univ.
Twente (Netherlands)
Photoacoustic imaging is a new noninvasive imaging technique which can
be used to obtain images of living tissue with high resolution. This technique
uses pulsed laser light to induce thermoelastic expansion in an absorbing
structure inside the tissue. This expansion generates an ultrasound wave
which is detected by a proper ultrasound detector. By analyzing the detected
wave, information regarding size, structure and position of the absorbing
structure can be determined. Photoacoustic imaging has potential in the
detection of cancer in the human breast. In order to improve the sensitivity
and specificity of the technique, the use of the contrast agents may be
required. These agents can help to increase the contrast between normal
and suspicious tissue. The high optical absorption of gold nanorods at nearinfrared wavelengths makes them suitable as contrast agents in photoacoustic
imaging. By conjugating the nanorods with an appropriate protein it could
be possible to accumulate these particles at a tumor site. The results of
modeling and phantom experiments presented in this article attempt to
answer questions related to photoacoustic contrast enhancement such as:
What are the optimal sizes and aspect ratios of gold nanorods to achieve
maximum enhancement? What is the number density of particles required to
accumulate at a cancer site to increase local contrast? Could non-linear
phenomena contribute to photoacoustic signals and under which conditions?
Could changes occur to various stealth and targeting coatings employed in
these particles during and after laser irradiation?
54
European Conferences on Biomedical Optics 2007 •
Time-resolved diffuse reflectance at small
source-detector separation using a time-gated
single-photon avalanche diode
A. Pifferi, A. Torricelli, L. Spinelli, D. Contini, R. Cubeddu, Politecnico di
Milano (Italy); F. Martelli, G. Zaccanti, Univ. degli Studi di Firenze (Italy);
A. Tosi, A. Dalla Mora, F. Zappa, S. Cova, Politecnico di Milano (Italy)
A key issue in photon migration applications is to increase the penetration
depth of the measurement. A common assumption is that the larger the
source-detector (interfiber) distance, the deeper the probed regions. On the
other hand - for a time-resolved reflectance measurement - the mean
penetration depth is independent from the interfiber distance, while it
increases for longer arrival times of photons. Following this concept we have
demonstrated theoretically that time-resolved reflectance at null interfiber
distance provides higher number of photons at any arrival time, higher
contrast, and better spatial resolution as compared to longer interfiber
distances. In this paper, we demonstrate the feasibility of time-resolved diffuse
reflectance at a small source-detector separation using a single-photon
avalanche diode (SPAD) operated in time-gated mode. A key advantage of
SPAD is the possibility to switch it on and off at a fast pace. Thus, by gating
off the early photons that would otherwise saturate the time-correlated single
photon counting electronics, it is possible to detect long lived photons carrying information on deeper structures. Shifting the enabling gate in steps
of 500 ps, and adjusting the injected laser power, photon time distributions
at an interfiber distance of 0.2 cm were obtained on a tissue phantom with a
reduced scattering coefficient of 10 cm-1, and an absorption coefficient of
0.1 cm-1, with a dynamic range of 6 decades and collecting photons at
arrival times up to 4 ns. The reconstructed photon-time distribution - when
fitted with the Diffusion model - yields the same optical properties as for a
measurement at a large interfiber distance, demonstrating that the proposed
technique is capable to accurately detect the whole photon distribution even
at late times.
6631-12, Session 3
Estimation of biomedical optical properties by
simultaneous use of diffuse reflectometry and
photothermal radiometry: investigation of light
propagation models
E. S. R. Fonseca, Univ. da Beira Interior (Portugal); M. E. P. de Jesus,
Univ. da Beira Interior (Portugal) and Unidade de Detecção Remota
(Portugal)
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Conference 6631: Novel Optical Instrumentation for Biomedical Applications
The estimation of optical properties of highly turbid and opaque biological
tissue is a difficult task since conventional purely optical methods rapidly
loose sensitivity as the mean photon path length decreases. Photothermal
methods, such as pulsed or frequency domain photothermal radiometry (FDPTR), on the other hand, show remarkable sensitivity in experimental
conditions that produce very feeble optical signals. Photothermal Radiometry
is primarily sensitive to absorption coefficient yielding considerably higher
estimation errors on scattering coefficients. Conversely, purely optical
methods such as Local Diffuse Reflectance (LDR) depend mainly on the
scattering coefficient and yield much better estimates of this parameter.
Therefore, at moderate transport albedos, the combination of photothermal
and reflectance methods can improve considerably the sensitivity of detection
of tissue optical properties.
Recently, we have proposed a novel method that combines FD-PTR with
LDR, aimed at improving sensitivity on the determination of both optical
properties. Signal analysis was performed by global fitting the experimental
data to forward models based on Monte-Carlo simulations. Although this
approach is accurate, the associated computational burden often limits its
use as a forward model. Therefore, the application of analytical models based
on the diffusion approximation offers a faster alternative. In this work, we
propose the calculation of the diffuse reflectance and the fluence rate profiles
under the delta-P1 approximation. This approach is known to approximate
fluence rate expressions better close to collimated sources and boundaries
than the standard diffusion approximation (SDA). We extend this study to the
calculation of the diffuse reflectance profiles. The ability of the combined
delta-P1 based model to provide good estimates of the absorption, scattering
and anisotropy coefficients is tested against Monte-Carlo simulations over a
wide range of scattering to absorption ratios. A comparative study with the
SDA and the delta-P0 models is also presented.
Experimental validation of the proposed method is accomplished by a set of
measurements on solid absorbing and scattering phantoms.
6631-13, Session 3
New approaches in laser speckle biomedical
imaging: nonergodictiy correction and active
speckle sampling
P. V. Zakharov, A. Völker, Univ. de Fribourg (Switzerland); A. Buck, B.
Weber, Univ. Hospital Zürich (Switzerland); F. Scheffold, Univ. de
Fribourg (Switzerland)
We discuss new approaches to laser speckle biomedical imaging with the
goal to establish a quantitative link between the measured signal and the
local dynamic properties, such as Brownian motion or blood flow. We further
report on technical improvements with respect to the statistical accuracy of
the laser speckle image and the recording time of the technique [1-3].<br\>
We demonstrate that the presence of a static component in laser speckle
imaging (LSI) signal can significantly complicate the quantitative interpretation
of the imaging data. Based on Monte-Carlo simulations and model
experiments we show that the error in the mean particles velocity extracted
using traditional approaches can reach a several orders of magnitude while
using a proper theoretical treatment the error can be substantially reduced
[3].<br\>
We further discuss improved dynamic light imaging techniques based on
scrambling the incident laser beam with a ground glass. As a consequence
we can substantially reduce the statistical noise and/or reduce the data
acquisition time thus allowing accurate quantitative data interpretation
[1,3].<br\><br\>
[1] P. Zakharov and F. Scheffold, SPIE Newsroom 11/2006, DOI: 10.1117/
2.1200609.0397<br\>
[2] P. Zakharov et. al. Phys. Rev. E 73, 011413, 2006<br\>
[3] P. Zakharov et. al., Quantitative modeling of laser speckle imaging, Optics
Letters, Vol. 31, Issue 23, pp. 3465-3467, 2006; and Laser speckle imaging
with an active noise reduction scheme, Optics Express 13, No. 24, p. 9782,
2005
6631-14, Session 3
Heterodyne interference microscopy for noninvasive cell morphometry
M. P. Whelan, F. Lakestani, D. Rembges, M. G. Sacco, Joint Research
Ctr. (Italy)
Abstract contained in supplemental file.
6631-15, Session 3
Dynamics measurement of both the integral
refractive index and cell morphometry with
digital holography microscopy
P. P. Marquet, Ctr. Hospitalier Univ. Vaudois (Switzerland); Y. Emery,
LyncéeTec SA (Switzerland); T. Colomb, F. Charriere, J. G. Köhn, C. D.
Depeursinge, B. Rappaz, P. Jourdain, P. J. Magistretti, École
Polytechnique Fédérale de Lausanne (Switzerland)
European Conferences on Biomedical Optics 2007 •
Recently, new emerging quantitative phase microscopy techniques (QPM)
have demonstrated their capability to provide non-invasive accurate 3D
imaging of transparent living cells (Carl et al. 2004; Marquet et al. 2005;
Popescu et al. 2005; Curl et al. 2006). Specifically, phase shifts, induced by
living cells on the transmitted wave front, provide information concerning 3D
cell morphology as well as intracellular refractive.
However, the information concerning cell morphology and refractive index is
intrinsically mixed, making relevant analyses of cellular processes in terms
of the phase signal difficult. To overcome this drawback, we have recently
developed (Rappaz et al.) a decoupling method, based on a transmission
Digital holographic microcopy technique, which allows to separately measure,
the cellular thickness and the integral intracellular refractive index from the
numerically reconstructed quantitative phase images of living cells. Practically,
it consists in perfusing cells consecutively with two perfusion solutions having
different refractive indices.
Currently, we are devising, a real time decoupling method based on the
numerical reconstruction of two holograms recorded for two different
wavelengths, in the off-axis configuration, on a CCD camera and requiring
the utilization of extracellular dyes in the perfusion solution. It results in the
possibility to study cell-shape dynamics, with a sub wavelength axial
sensitivity, as well as the transient local variations of the integral intracellular
refractive index, related to the intracellular protein concentration and water
fluxes, in physiological and pathophysiological processes.
6631-42, Poster Session
Monte Carlo simulation of photon
transillumination time of flight
P. Vacas-Jacques, M. Strojnik, Ctr. de Investigaciones en Óptica, A.C.
(Mexico); G. Paez, Ctr. de Investigaciones en Optica, A.C. (Mexico)
We have proposed an interferometric setup for performing tomographic
diagnosis. In the present study we extend the interferometric analysis to
partially coherent (temporal) radiation. Temporal coherence limits the amount
of delay introduced by the scattered photons, because incoherent photons
do not produce interference. Thus, short coherence time differentiates
between wanted pass-through and scattered radiation. The proposed as
metric of discrimination is therefore coherence, tested in an interferometric
setup.
We develop a stochastic Monte Carlo (MC) simulation to determine the
average photon migration time in turbid media, in general, and a tissue, in
particular. More specifically, we evaluate the time of flight necessary for the
radiation to traverse the tissue under test, with one or most two scattering
events. This distribution function relating time of arrival of photons on the
detector versus tissue characteristics determines the signal-to-noise ratio
for the interferometric measurements. The density of scattering centers within
tissue is represented here as a material coherence function.
In this talk, we will present the results of the MC simulation studies and their
dependence on the coherence annihilation attributes of the tissue scattering
centers. Further, a discriminate function will be introduced to differentiate
between the signal generated by a healthy and a deteriorating tissue, for
several types of altered tissues. The results will determine the type of source
and its coherence characteristics. Our initial expectation is that super
luminescent diodes (SLED) will be adequate radiation sources, a super
continuum source may be required for optimal performance.
6631-43, Poster Session
Characterization and optimization of an
integrating sphere based detector for the
estimation of tissue optical properties
D. F. Moscu, J. E. Hayward, T. J. Farrell, M. S. Patterson, McMaster
Univ. (Canada) and Juravinski Cancer Ctr. (Canada)
An integrating sphere system has been developed to non-invasively study
the optical properties of biological tissues over a broad spectral range. Using
the integrating sphere as both a diffuse illumination source and a detector
provides a technically simple measurement apparatus with numerous
advantages. A primary advantage is the reduction of the effect of spatial
inhomogeneities on the determination of optical properties, afforded by the
increased area of detection through the port-opening of the sphere, which
challenges many fibre-based, spatially-resolved measurements. Through a
single measurement of total diffuse reflectance, an estimation of the albedo
of homogeneous, liquid phantoms can be made for those cases where
scattering is greater than a determined threshold: 1 mm^(-1) for the present
probe design. Further estimations can be made to describe the absorption
environment. The effects of the sphere geometry, particularly port-opening
size, on the accuracy of the estimated albedo, the relevant scattering
threshold, and the accuracy of estimated absorption properties will be
discussed. These results will be used to modify the design of the integrating
sphere as an efficient illuminator and light collector, in order to optimize its
use in determining the optical properties of biological tissues.
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Conference 6631: Novel Optical Instrumentation for Biomedical Applications
6631-44, Poster Session
6631-47, Poster Session
Laser-Doppler spectrum decomposition method:
experimental validation
All-reflective digital microscope system for rapid
histological and immunofluourescent imaging of
tissue
N. S. Zolek, Physik-Tech Bundesanstalt (Poland); A. Liebert, Institute of
Biocybernetics and Biomedical Engineering (Poland); R. Maniewski,
Physik-Tech Bundesanstalt (Poland)
Aim of this study is to validate usefulness of Laser-Doppler spectrum
decomposition method in estimation of speed distribution of particles.
Decomposition method is based on assumption
that measured laser-Doppler spectrum can be approximated by linear
combination of probability distributions of single Doppler scattering calculated
for different speeds of particles and anisotropy of light scattering in the
medium. The Doppler shift probability distributions were calculated using
Monte-Carlo for Henyey-Greenstein scattering phase
function. This decompostion method allows to obtain distribution of speeds
of moving particles in the medium, not only average speed as it was possible
in laser-Doppler perfusion monitors.
Recently we reported that the method was positively verified on spectra
generated for different speed distributions using Monte Carlo simulations. In
this study we present results of application of the decomposition procedure
in analysis of laser-Doppler spectra obtained in physical phantom
experiments. A diluted solution of milk was pumped through a tube capillary
(diameter 1mm) with different speeds. The dependence of the obtained
distributions of speed of moving particles on the speed of flow was observed.
Laser-Doppler spectra obtained during in-vivo experiment were also
successfully decomposed. A healthy volunteer was investigated and the
spectra of laser-Doppler signal during postocclusive hyperemia test were
recorded and analyzed.
We conclude that the spectrum decomposition procedure can be successfully
applied in analysis of the measured laser-Doppler spectra and the amount of
information provided by laser-Doppler technique can be significantly
increased.
6631-45, Poster Session
Image transmission by multimode optical fiber
for microendoscopy
T. Rozzi, A. Lucesoli, Univ. Politecnica delle Marche (Italy)
The aim of this work is the utilization of individual multimode fibers for the
purposes of microendoscopy. In the present contribution we discuss the
question of image aberration induced by intermodal dispersion along the
fiber and by scattering at the truncated fiber end, and we propose a restoration
algorithm. Under the LP hypothesis, we firstly derive analytically the scattering
matrix of the “fiber-to-air” interface and we quantify the extent of intermodal
coupling. Results show that intermodal coupling is weak and it can be
neglected for not too large core diameters. On the other hand, intermodal
dispersion induces serious aberration but in this work we demonstrate that it
may be computed and corrected. We implemented a restoration algorithm
based on the separation and equalization of the contribution of each mode,
for both step index and graded index fibers. Simulations show that fibers
with a diameter of few tens of microns can transmit even quite detailed images,
and the proposed algorithm is effective for both the above types of fibers, for
different fiber lengths and for a variety of images. Experimental tests were
performed by transmitting a Gaussian beam through a graded index silica
fiber (diameter 62.25 µm, NA=0.27). After applying the proposed postprocessing to the aberrated image exiting from the fiber, we obtained an
error of 0.25 µm on the FWHM of the original Gaussian beam. In conclusion,
it appears possible to “capture” an external image and transmit the same
through the fiber towards an observer at the other fiber end, after appropriate
phase correction.
6631-46, Poster Session
Time-gated real-time pump-probe imaging
spectroscopy
R. Ferrari, C. D’Andrea, A. L. Bassi, G. Valentini, R. Cubeddu,
Politecnico di Milano (Italy)
An experimental technique which allows one to perform pump-probe transient
absorption spectroscopy in real-time is an important tool to study irreversible
processes. This is particularly interesting in the case of biological samples
which easily deteriorate upon exposure to light pulses, with the formation of
permanent photoproducts and structural changes. In particular pump-probe
spectroscopy can provide fundamental information for the design of optical
chromophores. In this work a real-time pump-probe imaging spectroscopy
system has been realized and we have explored the possibility to further
reduce the number of laser pulses by using a time-gated camera. We have
demonstrated an improvement of the signal to noise ratio which allows a
reduction of the acquisition time. We believe that the use of a time-gated
camera can provide an important step towards the final goal of pump-probe
single shot spectroscopy.
56
European Conferences on Biomedical Optics 2007 •
R. J. Filkins, S. Yazdanfar, K. Tasimi, K. Kenny, E. Dixon, G. Abramovich,
M. Meyers, M. Montalto, GE Global Research (USA)
A major concern in the trend towards digital imaging in the anatomical pathology
lab is the time required to scan individual sections and the size of the resulting
images. This is driven primarily by the resolution required to interpret subtle
changes in tissue architecture. In this paper we demonstrate a digital
microscope that is capable of scanning tissue sections much faster than typical
imaging microscopes. We’ll review the design and fabrication of our digital
microscope system which comprises: a novel all-reflective objective lens, a
field flattening tube lens, a dual camera, image-based auto-focusing
subsystem, an ultra-bright solid state Abbe-type illuminator, and a custom
image processing engine for stitching and compression. As we’ll demonstrate,
the all-reflective objective lens simultaneously provides a larger than typical
field of view, numerical aperture, working distance and range of imaging
wavelengths. The large N.A. objective lens is coupled to a solid state white
light source with a custom beam shaping design. In order to maintain sharp
focus on tissue sections an image-based technique is used, rather traditional
laser-based methods. A means of maintaining focus while acquiring images
rapidly is discussed. Our results include images of hematoxylin and eosin
stained and immunohistochemical stained tissue sections with large amounts
of architectural detail.
6631-48, Poster Session
Flexible hollow polycarbonate fiber for
endoscopic infrared laser treatment
M. Nakazawa, Shimadzu Corp. (Japan); Y. Shi, Fudan Univ. (China); K.
Iwai, Sendai National College of Technology (Japan); Y. Matsuura, Tohoku
Univ. (Japan); X. Zhu, Fudan Univ. (China); M. Miyagi, Sendai National
College of Technology (Japan)
Flexible and uniform polycarbonate (PC) capillary was fabricated from
commercially available PC tube by using optimized glass-drawing technique.
We report on hollow optical fiber based on the PC capillary with silver and
cyclic olefin polymer (COP) inner coatings to enhance the reflection rate at
designed wavelengths. The PC capillary based hollow fiber showed
transmission properties equal to that of glass capillary based ones. Owing to
the smooth inner surface of the PC capillary, low loss coaxial delivery of infrared
and visible pilot beams was possible by selecting proper COP film thickness.
Straight losses for the Er:YAG laser light and a green pilot beam were 0.4 dB/
m and 3 dB/m for hollow PC fiber with 700 micrometer inner diameter. Hollow
PC fibers are safer and more flexible, which makes it possible to deliver infrared
laser power in endoscopic application. Both Er:YAG laser light and green pilot
beam were delivered through an endoscope with low loss even when it was
sharply bent with a bending radius as small as 1 centimeter. We also made
flexible hollow fiber bundle with 40 cm length and 50 cm2 cross-section.
Transmission properties of infrared thermal image by using the bundle were
experimentally discussed.
6631-49, Poster Session
Determination of agar tissue phantoms depth
profiles with pulsed photothermal radiometry
M. Milanic, B. B. Majaron, Jozef Stefan Institut (Slovenia); S. J. Nelson,
Beckman Laser Institute (USA)
Pulsed photothermal radiometry (PPTR) could be used for non-invasive depth
profiling of skin vascular lesions (e.g., port wine stain birthmarks), aimed towards
optimizing laser therapy on an individual patient basis.
Optimal configuration of the experimental setup must be found and its
performance characterized on samples with well defined structure, before
introducing the technique into clinical practice. The aim of our study was to
determine how sample structure and width of spectral acquisition band affect
the accuracy of measured depth profiles.
We constructed tissue phantoms composed of multiple layers of agar and of
thin layers of absorbers between the agar layers. Three phantoms had a single
absorber layer at various depths between 100 and 500 µm, and one phantom
had two absorber layers. In each sample we induced a non-homogeneous
temperature profile with a 585 nm pulsed laser and acquired the resulting
radiometric signal with a fast InSb infrared camera. We tested two
configurations of the acquisition system, one using the customary 3-5 um
spectral band and one with a custom 4.5 µm cut-on filter. The laser-induced
temperature depth profiles were reconstructed from measured PPTR signals
using a custom algorithm and compared with sample structure as determined
by histology and optical coherent tomography (OCT).
PPTR determined temperature profiles correlate well with sample structure in
all samples. Determination of the absorbing layer depth shows good
repeatability with spatial resolution decreasing with depth. Spectral filtering
improved the accuracy of reconstructed profiles for shallow absorption layers
(100-200 µm).
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Conference 6631: Novel Optical Instrumentation for Biomedical Applications
PPTR technique enables reliable determination of structure in tissue phantoms
with thin absorbing layers. Narrowing of the spectral acquisition band (to 4.5
- 5.3 µm) improves reconstruction of objects near the surface.
6631-50, Poster Session
Design and implementation of detection
schemes for spectral photoplethysmography
and photo-acoustics
I. S. Abdulhalim II, G. Tsvilikhovski, B. Epstein, Ben-Gurion Univ. of the
Negev (Israel)
Schemes for simultaneous detection of multi-wavelength
photoplethysmography (PPG) and photoacoustic (PA) signals are designed
and implemented to improve the information content of such measurements.
Spectral PA and PPG techniques are believed to give comprehensive clinical
information by measuring small changes in blood content in arteries and
capillaries. The signal measured in the PA and PPG has however large dc
content which is usually removed by filtering of low frequency components.
When simultaneous multi-wavelength or spectral detection is required the
detector is not a single pixel, rather a large array of small detectors such as
a CCD or PIN diode array. There are several issues involved in the design of
multi-wavelength PA or PPG such as the light source, filtering and
amplification, in order to obtain clean informative signal. Possibilities of using
existing spectrometers as well as new designs for such AC coupled
spectrometers are being considered. We shall present the design and some
preliminary results of measurements using transmitted signals through the
human finger.
6631-51, Poster Session
Automated slide-screening platform for histo/
pathology
R. Daum, TILL Photonics GmbH (Germany); R. Biandu, LudwigMaximilians-Univ. München (Germany); R. Uhl, TILL Photonics GmbH
(Germany)
The goal was to develop a fully automated screening platform capable of
scanning 300 slides per day. To achieve this we have developed a highly
compact. higly rigid microscope frame made from mineral cast, which has
vibration damping characteristics 14x better than cast aluminum or steel.
Slides are scanned by continuously moving a compact x-y-z-stage with
digitally controlled voice coild focus drive and by employing strobeed RGBillumination from an LED-based light source. This allows to employ a highly
sensitive CCD-camera, which can also be used in the also integrated
epifluorescence mode.
6631-52, Poster Session
Single point and imaging measurements of the
optical clearing process
J. G. Enfield, J. W. O’Doherty, M. J. Leaahy, Univ. of Limerick (Ireland)
Biomedical optics and photomedicine applications are challenged by the
turbid nature of most biological tissue systems. This limits the depth which
light penetrates into the skin and is mainly due to the refractive index mismatch
between the tissue structural components and interstitial matter. The depth
of light penetration into tissues can be improved by administration of nonreactive, biocompatible optical clearing agents with higher refractive index
than the interstitial matter. This leads to equalization of refractive indices
between different tissue components and causes a decrease in overall tissue
scattering, and thus an increase in optical transmittance. In this paper we
examine the effects of optical clearing agents on ex vivo porcine skin using
the immersion method. We examine the change in the reflected light spectrum
over time as the clearing agent enters the skin. This is examined via point
probe measurements and also a wide field imaging technique with a
consumer-end digital camera. Results from the wide field imaging technique
are validated by comparing results with the point probe measurements. Theory
has been developed modeling changes in the reflection spectrum over time
as clearing occurs. Image processing algorithms are under evaluation to
assess the level of optical clearing Psuedo color maps are generated related
to the level of clearing of the tissue, which in turn can be related to the
penetration depth of photons. Optical clearing applications include the ability
of improving optical biopsy, greater visualization of skin erythema and
microvascular angiogenesis as well as improving laser depth in surgery.
6631-53, Poster Session
High-resolution image acquisition using a
compact microlens-coupled detector
D. Unholtz, R. B. Schulz, W. Semmler, J. Peter, Deutsches
Krebsforschungszentrum (Germany)
Recently, a thin optical detector assembly consisting of a microlens array
(MLA) coupled to a large area CMOS sensor through a septum mask was
European Conferences on Biomedical Optics 2007 •
developed. The sensor is placed in the physical focal plane of the MLA. Each
lens of the MLA forms a small image on the sensor surface, with individual
images being separated from each other through the septum mask. The
resulting raw image of the sensor thus shows a multitude of small sub-images.
A low-resolution image can be attained by extracting those pixels located on
the optical axis of each microlens, as reported previously. Herein we describe
an improved post-processing method to extract higher resolution images
(which can be focused to an arbitrary plane) from a single raw sensor image:
Each lens of the MLA results in a mapping from points in object space to
corresponding sensor pixels. By tracing back the light paths from sensor
pixels through the lenses onto an arbitrary focal plane in object space, this
mapping can be inverted. Intensities captured on individual sensor pixels
can be attributed to virtual pixels on that focal plane using the computed
inverse mapping.
As a result, from a single acquisition by the detector, images focused to any
plane in object space can be calculated. In contrary to the approach of
extracting focal point intensities, the spatial resolution is not limited by
microlens pitch. We present experimental examples of extracted images at
various object plane distances and studies determining the spatial resolution.
6631-54, Poster Session
Optical biosensor to monitor sugar level
changes for diabetes patients in real time
A. Rahman, Polytechnic Univ. (USA)
A novel micromachined optical biosensor has been presented to monitor the
sugar/glucose level changes for diabetes patients in real time. Over the years
several optical techniques have been realized to monitor the sugar/glucose
level, however, none of those are capable to monitor the sugar/glucose level
directly from diabetes patient’s body in real time. The major objective of this
paper is to demonstrate a novel micromachined optical biosensor with
theoretical and numerical model. The detailed fabrication processes have
been outlined in a multimode optical fiber which is based on MEMS
fabrication. The sensor is designed by following the basic principle of FabryPerot interferometer. The optical biosensor presented here could be able to
detect and monitor the sugar/glucose level for diabetes patient in real time
and it could also potentially be used in the area of biomedical applications,
nano research, drug delivery, microfluidics, etc.
6631-55, Poster Session
The application of a long period grating sensors
to human respiratory plethysmography
T. D. P. Allsop, K. Carroll, D. J. Webb, I. Bennion, Aston Univ. (United
Kingdom); M. Miller, Univ. Hospital Birmingham NHS Trust (United
Kingdom)
A series of nine in-line curvature sensors on a garment are used to monitor
the thoracic and abdominal movements of a human during respiration for
application to Human Respiratory Plethysmography. These results are used
to obtain volumetric tidal changes of the human torso which showed
agreement with data from a spirometer used simultaneously to recorded the
inspired and expired volume at the mouth during both rhythmic and transient
breathing patterns. The curvature sensors are based upon long period gratings
which are written in a progressive three layered fibre to render them insensitive
to refractive index changes. The sensor consists of the long period grating
laid upon a carbon fibre ribbon, with this then encapsulated in a low
temperature curing silicone rubber. The sensing array is multiplexed and
interrogated using a derivative spectroscopy based technique to monitor
the response of the LPGs’ attenuation bands to curvature. The versatility of
this scheme is demonstrated by applying the same garment and sensors to
various human body types and sizes. It was also found from statistical analysis
of the sensing array data in conjunction with the measurements taken with
the spirometer that 11 to 12 sensors should be required to obtain an absolute
volumetric error of 5%.
6631-56, Poster Session
Imaging correlography applied to high resolution
retinal imaging
B. Thurin, L. Diaz-Santana, City Univ. (United Kingdom)
The resolution of the images obtained from the eye fundus are limited by the
ocular aberrations. As most of the aberration are due to the eye optics, the
light intensity measured in the eye iris plane is only slightly affected by the
ocular aberrations. By illuminating the retina with a coherent laser source
and collecting the light in a pupil plane conjugate, it is possible to apply the
imaging correlography technique proposed by Fienup and Idell. From
processing series of pupil plane images, this technique gives information
about the retina in the form of the squared modulus of the Fourier transform
or, equivalently, the autocorrelation of the diffraction-limited image intensity.
Two factors make this technique suitable for retinal imaging:
1. For this technique to work, changes of phase distribution in the retinal
plane are necessary between each frame. Small eye movements naturally
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Conference 6631: Novel Optical Instrumentation for Biomedical Applications
provide these changes.
2. This method does not provide directly the phase of the Fourier transform.
Therefore it is of most use for centro-symmetric objects like the retinal’s
photoreceptor mosaic, for which the squared modulus of the Fourier transform
is sufficient to estimate the photoreceptors density.
An ocular correlographer has been developed. Preliminary data have been
obtained in vivo showing the feasibility of applying such a technique in the
eye. The main interest of this technique is the simplicity and low cost of its
optical setup. Data collection is very fast and requires very little subject’s
cooperation. Experimental results are compared against simulation based
on the retinal scattering model proposed by Vohnsen et al.
6631-57, Poster Session
Light scattering application for bacterial cell
monitoring during cultivation process
I. Y. Kotsyumbas, I. M. Kushnir, State Scientific-Research Control
Institute of Veterinary Preparations and Fodder Additives (Ukraine); R.
O. Bilyy, Institute of Cell Biology (Ukraine); V. B. Getman, A. I. Bilyi, Ivan
Franko National Univ. of L’viv (Ukraine)
Monitoring of bacterial cell numbers is of great importance not only in
microbiological industry but also for control of liquids contamination in the
food and pharmaceutical industries. Here we describe a novel low-cost and
highly efficient technology for bacterial cell monitoring during cultivation
process. The technology incorporates previously developed monitoring device
and algorithm of its action. The devise analyses light scattered by suspended
bacterial cells. Current stage utilizes monochromatic coherent light and
detects amplitudes and durations of scattered light impulses, it does not
require any labeling of bacterial cell. The system is calibrated using highly
purificated bacteria-free water as standard. Liquid medial are diluted and
analyzed by the proposed technology to determine presence of bacteria.
Detection is done for a range of particle size from 0.1 to 10 um, and thus
particle’s size distribution is determined. We analyzed a set of different
bacterial suspensions and also their changes in quantity and size distribution
during cultivation. Based on the obtained results we conclude that proposed
technology can be very effective for bacteria monitoring during cultivation
process, providing benefits of low simplicity and low cost of analysis with
simultaneous high detection precision.
6631-59, Poster Session
A new optical system for 3-dimensional mapping
of the cornea
S. M. B. Franco, J. B. Almeida, Univ. do Minho (Portugal)
Purpose: Precise measurement of corneal thickness and topography is
important in many areas such as refractive surgery, diagnosis and
management of corneal disease as well as the evaluation of corneal tolerance
to new contact lens materials.
In this work the authors present a new optical corneal tomographer that
uses two Scheimpflug cameras attached to an innovative illumination system
that allows a rotary scanning of the entire cornea.
Method: The measurements are made from corneal optical sections obtained
by illumination with a collimated beam expanded in a fan by a small cylindrical
lens. This lens is provided with motor driven rotation in order to perform
automated rotary scan of the whole cornea. The authors expect to achieve a
scanning speed that will allow producing complete tomography maps without
consideration of eye movements.
Two Scheimpflug cameras are used to capture the images of the optical
sections.
Results: With this system it’s possible to obtain 3-D representation of the
corneal thickness as well as corneal topography. Maps of the corneal
thickness and elevation maps are shown.
Conclusions: Although under development, this new optical system allows
the measurement of the thickness of the whole cornea as well as 3-D mapping
of both corneal surfaces. As Scheimpflug cameras are used, it’s expected to
obtained data from the lens too.
Methods:
An optical setup that conjugated the patient’s pupil with the plane of the
wavefront sensor was constructed. 50 right eyes of 25 mail and 25 female
patients with highly aberrated eyes (mean RMS of High Order Aberration
(HOA) was 3 microns) were measured using both sensors. Both the HS sensor
and the cylinder symmetry sensor had diameters of 10 mm and a lenslet
spacing of 500 microns. A special mechanical adaptor was constructed in
order to interchange both sensors without changing the position of the
conjugated plane. Patients were dilated with tropicamid solution at 1% and
mean pupil size was 7 mm.
Results:
Zernike coefficients up to 10th order (66 terms) were fit to aberration data
and mean deviation among sensors for HOA was computed for each eye
and the root mean square error (RMSE) was computed for all eyes between
both sensors. The RMSE (in microns) between both sensors for all eyes for
LOA was 0.01 microns and for HOA was 0.02 microns.\
Conclusions
Precision of the cylindrical sensor when measuring astigmatic and spherical
surfaces is compatible with the HS Sensor, as has been demonstrated in
previous studies on mechanical eyes [1, 2, 3]. Our goal in the present work
was to verify if similar performance would be obtained on in vivo and highly
aberrated eyes. From the results obtained here, we may affirm that this is
the case and that the lack of tangential slope has little or no effect on wavefront
retrieval. We believe there are some possible benefits of this new sensor
when applied specifically to vision science.
Centration with this new sensor is an absolute process since one always
knows where the optical center is, whereas with the HS sensor it is a relative
process. By “absolute” we are referring only to the procedure of centering
the sensor’s center to the center of the entrance pupil, which is where the
line of site passes. Of course this procedure does not eliminate, as it also
happens for the HA Sensor, the possibility of tilt. One other possible benefit
is fewer loss of data points on the edges of the pupil. These and other aspects
of this new sensor have not been investigated here but are certainly important
topics for future work. We consider the results presented here as statistically
valid data in terms of demonstration that HS and cylindrical sensor render
equivalent results for in vivo eyes.
References
[1] CARVALHO, LA ; Castro ; Chamon ; Schor . Proceedings of the 7th
International Congress of Wavefront Sensing and Optimized Refractive
Corrections: A New Wavefront Sensor With Polar Symmetry: Quantitative
Comparisons With a Shack-Hartmann Wavefront Sensor, J Refract Surg.
2006;22:954-958.
[2] CARVALHO, LA ; Castro ; Schor ; Chamon . Quantitative Comparison of
Different-Shaped Wavefront Sensors and Preliminary results for Defocus
Aberrations on a Mechanical Eye. Arquivos Brasileiros de Oftalmologia, 2006.
[3] CARVALHO, LA ; Castro . The Placido Wavefront Sensor and Preliminary
Measurement on a Mechanical Eye. Optometry and Vision Science, USA, v.
83, n. 2, p. 108-118, 2006.
6631-61, Poster Session
A novel method for 3D wide-angle corneal
topography
L. A. V. Carvalho, Univ. de São Paulo (Brazil)
In this work the instrumentation and software for wide-angle corneal
topography using a Placido based videokeratographer was developed. The
objective is to allow the measurement of a greater area of the cornea using a
simple adaptation to the Placido mire, therefore opening opportunities for
the improvement of other applications which might benefit from this additional
information, such as: contact lens adaptation and design improvement,
algorithms for customized refractive surgery, among others. Results show
that up to 100% more area of the cornea may be mapped using the technique
described here. We present results for a calibration spherical surface and
also for a highly astigmatic cornea and analyze quantitatively the additional
area that is recovered in terms of curvature and true elevation.
6631-62, Poster Session
6631-60, Poster Session
Advanced coherent 3D micro-imaging
A new cylindrical symmetry wavefront sensor for
and preliminary results on in vivo highly
aberrated eyes
M. Kanka, R. Riesenberg, Institut für Physikalische Hochtechnologie
e.V. (Germany)
L. A. V. Carvalho, J. C. Castro, Univ. de São Paulo (Brazil)
Purpose:
The purpose of this work was to present the development of a novel wavefront sensor and preliminary results for highly aberrated in vivo eyes.
Comparisons with the traditional Hartmann-Shack (HS) sensor, which has
Cartesian symmetry and is generally accepted as the “Gold Standard” by
eye-care professionals, were also conducted for comparison.
58
European Conferences on Biomedical Optics 2007 •
The inline holography is considered with a pinhole as a light source and a
CCD for detection of interferences. The use of a divergent reference wave
generated by a pinhole and a placement of small samples near to the pinhole
enables the detection of sub-µm details by CCD pixels in the 10 µm scale.
This is a lensless microscopic imaging /1, 2/.
We discuss the coherent imaging by illumination of a single source as well as
by a multi-spot source /3/. As a single source a pinhole is used with diameter
of 0.8 ... 2 µm. As multi-spot sources serve pinhole-arrays with 9 and 16
pinholes. It was shown that the lateral resolution can be essential increased
in case of using an illumination array /4/.
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Conference 6631: Novel Optical Instrumentation for Biomedical Applications
In the paper the increase of spatial resolution in the normal direction to the
source will be presented. Multi-spot sources enable a sample illumination
from different directions in one measurement. The spatial resolution in normal
direction is discussed in dependence on the position of the samples, the
influence of the pinhole diameter and its distances in the array.
The limits actually are given by the CCD. We use a CCD with 8 Mpixels, 3.5
µm pixel pitch and a dynamic range of 12 bits. The spatial resolution
advantages by using multi-spot sources reach the factor 4.
References
[1]D. Gabor, “Microscopy by reconstructed wavefronts”, Proc. Roy. Soc. A
197, pp. 454-487, 1949.
[2]H. J. Kreuzer, R. A. Pawlitzek, “Digital in-line holography”, Europhysics
News 34, pp. 62-65, 2003.
[3]M. Kanka, R. Riesenberg, “Wide field holographic microscopy with pinhole
arrays”, Sensor+Test 2006, Proceedings, pp. 69-72.
[4]R. Riesenberg, M. Kanka, J. Bergmann, „Unconventional Imaging by
Synthetic Aperture”, DGaO-Proceedings, A 25, 107th Conference of the
DGaO, 2006.
6631-16, Session 4
Lipid particle detection by means digital
holography and lateral shear interferometry
L. Miccio, Istituto Nazionale di Ottica Applicata (Italy); A. Finizio, S. M.
De Nicola, Istituto di Cibernetica Eduardo Caianiello (Italy); P. Ferraro,
Istituto Nazionale Ottica Applicata (Italy)
Digital Holography (DH) in microscope configuration thanks to the numerical
reconstruction procedure is a flexible and useful tool for analysis of biological
material. We present the investigation of a lipid particles using a DHM
employed in combination with Lateral Shear Interferometry (LSI). The optical
setup is based on a Mach-Zehnder interferometer in transmission geometry.
The sample cell is placed in one interferometer arm while the other one is
used as a reference beam. By means of the Rayleigh-Sommerfield integral is
possible to retrieve the complex object field and then to calculate the
amplitude and phase of the laser light transmitted by the sample. Traditional
microscopy allows to obtain amplitude contrast image only, DH, instead,
enables to calculate the phase map of the complex wave that is simply related
to the optical phase difference (OPD) experienced by the light when it is
transmitted through the object. In this way it is possible to obtain phase
contrast image that is very useful for biological materials that often present
low amplitude contrast for quantitative amplitude image. The main difficulty
of this technique is to remove the optical aberrations produced by the optical
setup components. Several methods have been proposed, such as
subtraction of a reference phase map (without sample) [1] or numerical
multiplication of a parametric lens [2]. We propose a fast and effective solution
of this problem based on LSI. We digitally introduce a lateral shear of one
pixel in and directions calculating the phase difference , between the
actual phase map and its sheared replica in both directions. include a linear
term due to defocus aberration and the object phase difference. The linear
term can be easily eliminated by numerical integration. This technique allows
to retrieve the correct phase contrast image removing optical aberration,
avoiding unwrapping problems.
6631-17, Session 4
Erythrocytes analysis with a digital holographic
microscope
B. Rappaz, École Polytechnique Fédérale de Lausanne (Switzerland); A.
Barbul, Tel-Aviv Univ. (Israel); F. Charrière, J. G. Köhn, École
Polytechnique Fédérale de Lausanne (Switzerland); R. Korenstein, TelAviv Univ. (Israel); C. D. Depeursinge, P. J. Magistretti, P. P. Marquet,
École Polytechnique Fédérale de Lausanne (Switzerland)
Digital holographic microscopy (DHM) is a technique that allows obtaining,
from a single recorded hologram, quantitative phase image of living cell with
interferometric accuracy (Marquet, Rappaz et al., Opt. Lett. 30, 468-70, 2005).
Specifically, the optical phase shift induced by the specimen on the
transmitted wave front can be regarded as a powerful endogenous contrast
agent, depending on both the thickness and the refractive index of the sample.
We have recently proposed (Rappaz, Marquet et al., Opt. Exp. 13, 93619373, 2005) a new and efficient decoupling procedure allowing to directly
obtain separate measurements of the thickness and the integral refractive
index of a given living cell. Consequently, it has been possible, for the first
time to our knowledge, to accurately measure (with a precision of 0.0003)
the mean refractive index of living erythrocytes. This value has permitted to
calculate the mean cell hemoglobin concentration (MCHC), (Barer and Joseph,
Quarterly Journal of Microscopical Science 95, 399-423, 1954), a parameter
which is altered in various blood diseases including anemia. On the other
hand, the cellular thickness measurements allow to calculate the volume
and shape of erythrocytes. Interestingly enough, DHM, thanks to its
subwavelength phase shift measurements, was found to yield an efficient
tool to assess erythrocyte cell membrane fluctuations (ECMF).
European Conferences on Biomedical Optics 2007 •
6631-18, Session 4
Single-pulsed digital holographic topometry
S. Hirsch, Ctr. of Advanced European Studies and Research (Germany);
S. Heintz, Ctr. of Advanced European Studies and Research (Germany)
and Furtwangen Univ. (Germany); A. Thelen, N. Gisbert, Ctr. of
Advanced European Studies and Research (Germany); P. Hering, Univ.
Düsseldorf (Germany) and Ctr. of Advanced European Studies and
Research (Germany)
For the planning and documentation of maxillofacial surgery highly resolved
tissue information is needed. In our approach, the surface of an object is
displayed and measured with pulsed holography. With a single laser pulse
(Nd:YAG) of 20 ns the object surface is recorded on a CCD sensor, movement
artefacts are systematically avoided.
With the kowledge of the recording parameters, the original wave field is
synthesized numerically from the holographic interference pattern. The
calculated slices are combined into an image stack, representing the digitized
real image. This wave field represents the object geometrically correct, but
focussed and unfocussed regions overlay. The focussed regions are identified
numerically and combined into a height map, the texture information is
extracted from the real image simultaneously. Both, height and texture are
combined, yielding pixel-precise textured surface models.
With this novel method it is possible to capture the surface of moving objects,
even 3d motion series are possible. Skin can be detected in the real image,
giving the potential application for facial measurements.
Compared our analog holographic topometry, there are still limitations
regarding the extend of the imageable field and the axial resolution. Yet there
is no consumption of material and an optical reconstruction is dispensable.
The quick display of the reconstructed real image allows a direct appraisal of
the object topology. This method is a valueable tool for the surface
visualization of living subjects, offering potential for completely new fields of
application.
6631-19, Session 4
Optical imaging of the surface profiles of
biological cells and tissues with nanometer
resolution
C. Lai, I. Hsu, Chung Yuan Christian Univ. (Taiwan)
We proposed and developed an optical system for imaging of the surface
profiles of biological cells and tissues with nanometer resolution. In
comparison with traditional techniques for profilometry such as atomic force
microscopy (AFM) and scanning electron microscopy (SEM), optical methods
possess many advantages that they are noninvasive, sensitive and fast.
Furthermore, our system is a low-cost system which can perform the imaging
of the surface morphology in a large area without any special preparation of
the sample even for the sample with a surface of large roughness. The system
consists of two interferometers in which one is in the configuration of a
Michelson interferometer and the other is in the configuration of a MachZehnder interferometer. The former is used for scanning of the surface profile
of the sample, and the latter is used to compensate the phase shift due to
the different traveling ranges of the reference mirror in successive scannings.
The phase difference between the interferograms detected in the two
interferometers is proportional to the surface position of the sample at that
point. Therefore, we can obtain the surface profile of the sample when
executing a two dimensional scanning. The system was demonstrated to
possess the axial resolution of about 2 nm and its lateral resolution is at the
diffraction limit. We used the system for the imaging of various biological
cells and tissues. The system was also used to investigate the process of
apoptosis of cells by dynamically imaging the morphological change of their
surfaces.
6631-21, Session 4
High-resolution adaptive holographic
interferometer for biomedical application
G. E. Dovgalenko, ITT Technical Institute (USA); A. Dagdanova, Eastern
Virginia Medical School (USA)
We realized new adaptive holographic sensor and interferometer, which allows
to visualize high-resolution 3D images of diffuse reflected objects in Continue
Hologram Registration Regime- CHRR. The coupled laser wave nonlinear
theory was applied for optimization of hologram recording in crystals
symmetry 23 and optimization of experimental set up. Experimentally
demonstrated 8000-lines/mm dynamical holographic image sensor on doped
23 symmetry photosensitive crystal, which used 633 nm, 15mW HeNe laser.
The results were applied for holographic visualization of Cryogenic and
Ultrasonic near field images of Surgical Medical Instrument. Proposed
holographic CHRR interferometer allows to get image contrast 100:1 for
diffuse reflected objects. We realized 11641 lines/mm dynamical holograms
in CHRR “light by light” processing using 442 nm HeCd laser.
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Conference 6631: Novel Optical Instrumentation for Biomedical Applications
Application of CHRR interferometer for hologram registration of moving
biological object in “vivo” has been demonstrated.
Experimental results show that proposed interferometer gives outstanding
performance for development of high resolution and contrast holographic
images in CHRR and is promising instrument for biomedical application.
6631-22, Session 4
New spectral imaging techniques for blood
oximetry in the retina
G. D. Muyo Nieto, I. Alabboud, Heriot-Watt Univ. (United Kingdom); D.
Mordant, A. I. McNaught, Cheltenham General Hospital (United
Kingdom); A. R. Harvey, Heriot-Watt Univ. (United Kingdom)
Hyperspectral imaging presents a unique opportunity for direct and
quantitative mapping of retinal biochemistry - particularly of the vasculature
where blood oximetry is enabled by the strong change of absorption spectra
with oxygenation. This is particularly pertinent both to research and to clinical
investigation and diagnosis of retinal diseases such as diabetes, glaucoma
and age-related macular degeneration. The optimal exploitation of
hyperspectral imaging however, presents a set of challenging problems,
including; the effects of optical clutter and poorly characterised and controlled
optical environment of the retina; the erratic motion of the eye ball; and the
compounding effects of the optical sensitivity of the retina and the low
numerical aperture of the eye.
We have developed two spectral imaging techniques to address these issues.
The first one, a time sequential technique, makes use of conventional fundus
camera into which a liquid crystal tuneable filter has been integrated so as to
spectrally filter the illumination. Whilst this constitutes a flexible experimental
tool for investigation of retinal spectral characteristics, its time-sequential
nature introduces some artefacts and the duration of the recording process
is an issue for clinical applications The second technique is based on a unique
image replicating imaging spectrometer (IRIS) that employs polarising
interferometry and beam spliiting to form multiple spectral images of the
retina onto a single detector array. This enables the recording of a spectral
image in, typically, eight bands, in a single snapshot. Results of early clinical
trials acquired with these two techniques together with a physical model
which enables oximetry map will be reported. These initial results show that
the snapshot IRIS technique eradicates calibration and misregistration
problems associated with the time-sequential technique (random movement
of eyeball and increased time to record the images) which makes it ideal as
a clinical tool. Applications in other areas of medical and biophotonic imaging
will also be mentioned.
6631-23, Session 4
Real time assessment of RF cardiac tissue
ablation with optical spectroscopy
S. G. Demos, Lawrence Livermore National Lab. (USA) and Univ. of
California/Davis (USA); S. Sharareh, Biosense Webster, Inc. (USA)
A novel approach to characterize critical parameters in real time during RF
ablation of cardiac tissue is demonstrated by incorporating the use of a fiberoptic probe on a typical ablation catheter. RF ablation is used to treat atrial
fibrillation, a heart condition that causes abnormal electrical signals, known
as cardiac arrhythmias, to be generated in the endocardial tissue resulting in
irregular beating of the heart. The RF energy is delivered locally via ablation
electrode catheters that can be inserted percutaneously under local
anesthesia into a femoral, brachial, subclavian, or internal jugular vein and
positioned in the heart. Current methods have limited effectiveness in
measuring lesion formation parameters in real-time or associated adverse
conditions.
The optical spectroscopy approach discussed in this work allows for critical
parameters of the process leading to the formation of the lesion to be
evaluated in real time including such parameters as, catheter- tissue proximity,
lesion formation, depth of penetration of the lesion, cross-sectional area of
the lesion in the tissue, formation of char during the ablation, recognition of
char from non-charred tissue, formation of coagulum around the ablation
site, differentiation of coagulated from non-coagulated blood, differentiation
of ablated from healthy tissue, and recognition of steam formation in the
tissue for prevention of steam pop. These assessments are accomplished
by analyzing the spectral characteristics of the diffusely reflected light from
the tip of the ablation catheter via the incorporation of fibers to deliver the
illumination and collect the backscattered light.
6631-24, Session 5
On a new wavefront sensor for in vivo
measurements
L. A. V. Carvalho, Univ. de São Paulo (Brazil)
Purpose:
The purpose of this work was to conduct comparisons on highly aberrated
in vivo eyes using two different symmetry sensors: the traditional HartmannShack sensor, which has Cartesian symmetry, and a recently proposed
60
European Conferences on Biomedical Optics 2007 •
wavefront sensor, which has cylindrical symmetry.
Methods:
An optical setup which conjugated the patients pupil with the plane of the
wavefront sensor was constructed. 200 eyes of 50 mail and 50 female patients
with highly aberrated eyes (mean RMS of 4 microns) were measured using
both sensors. Both the Hartmann-Shack and the cylindrical sensors had
diameter of 10 mm and a lenslet spacing of 600 microns. A special mechanical
adaptor was constructed in order to interchange both sensors without
changing the position of the conjugated plane. Patients were dilated with
tropicamide solution at 1% and mean pupil size was 7 mm.
Results:
Zernike coefficients up to 10th order (66 terms) were fit to aberration data
and mean deviation among sensors was computed for each eye and the
root mean square error (RMSE) was computed for all eyes between both
sensors. The RMSE (in microns) between both sensors for LOA was 0.01
microns and for HOA was 0.02 microns.
Conclusions
Precision of the new sensor when measuring astigmatic and spherical
surfaces is compatible with the SH Sensor, as has been proven in previous
studies on mechanical eyes. Our goal in the present work was to see if similar
performance would be obtained in vivo and highly aberrated eyes. From the
results obtained here we may affirm that this is the case and that the lack of
tangential slope has little or no effect at all on aberration retrieval. We believe
there there are some possible advantages of this new sensor when applied
specifically to vision science. Centration with this new sensor is an absolute
process since one always knows where the optical center is, whereas with
the Hartmann-Shack sensor it is a relative process. By “absolute” we are
referring only to the procedure of centering the sensor center to the center of
the entrance pupil, which is where the line of site passes. Of course this
procedure does not eliminate, as it also happens for the Hartmann-Shack
Sensor, the possibility of tilt. We consider the results present here and
statistically valid data, but further investigations by other laboratories is
suggested and welcome by the authors.
6631-25, Session 5
Laser Doppler perfusion imaging with a highspeed CMOS-camera
M. Draijer, E. Hondebrink, W. Steenbergen, T. G. van Leeuwen, Univ.
Twente (Netherlands)
Laser Doppler Perfusion Imaging (LDPI) is used for determining e.g. the skin
perfusion in burns and during drug uptake, and cerebral blood flow.
Current LDPI-instruments scan the area under investigation and a single photo
detector captures the photoelectric current to obtain a perfusion map. In
that case the imaging time for a perfusion map of 64 x 64 pixels is around 5
minutes. This long imaging time increases the chance of moving artifacts
and is unpleasant for the patient.
Our goal is to decrease the imaging time drastically by making use of a high
speed CMOS-camera. By illuminating the area under investigation and
simultaneously at high speed taking images with the camera, it is possible to
obtain a perfusion map of the area under investigation much faster than with
the commonly used Laser Doppler Perfusion Imagers.
Our setup consists of a high speed CMOS-camera with a maximum frame
rate of 100,000 fps, a 671 nm laser and beam shaping optics. The required
time for obtaining a 128 x 128 pixel perfusion map is less then 5 seconds.
Only 1% of this time is measurement time, the remaining time is used for
transferring the data to the computer and calculating the perfusion map.
We will show perfusion maps of port wine stains and of semi real-time
applications like reactive hyperemia and use of perfusion increasing creme.
Furthermore we will show a comparison with a commercial available LDPIinstrument.
6631-26, Session 5
Real time diffuse reflectance polarization
spectroscopy imaging to evaluate skin
microcirculation
J. W. O’Doherty, Univ. of Limerick (Ireland); J. Henricson, Univ. Hospital
Linköping (Sweden); G. E. Nilsson, WheelsBridge AB (Sweden); M. J.
Leahy, Univ. of Limerick (Ireland); C. Anderson, Univ. Hospital Linköping
(Sweden)
This paper describes the design of a real time imaging system to investigate
the human microcirculation. The system utilizes polarization spectroscopy,
where the polarizing filters are arranged over the custom built light source
and camera detector with their pass directions perpendicularly oriented so
that the surface reflections from the skin surface are suppressed. Thus the
signal accepted by a consumer-end camera with 3 CCDs (one for each color
plane) is composed only of the light that has penetrated through to the reticular
dermis of skin tissue. Software has been developed that allows the real time
acquisition of 5 frames per second while applying a dedicated image
processing algorithm to the 256x256 pixel frame. The closest technology in
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Conference 6631: Novel Optical Instrumentation for Biomedical Applications
the area, line scanning laser Doppler imaging (LDI), requires 5 seconds for a
50x64 LDI image, and can lead to misinterpretation of temporal variability as
spatial heterogeneity in the tissue RBC concentration. Theory has been
developed, and an image processing algorithm obtained showing the
vasoconstriction or vasodilation of an area of tissue with a theoretical
resolution of 16µm when using the zooming functions. A video flow model
describes a linear relationship between the algorithm output and red blood
cell (RBC) concentration. Applications of this new technology include skin
care product development, investigation of the spatial and temporal areas of
skin blanching and erythema, and skin toxicology assessment.
6631-27, Session 5
Polarimetric surface plasmon resonance
imaging biosensor
A. Duval, F. Bardin, A. Aide, A. Bellemain, J. Moreau, M. T. G. Canva,
Institut d’Optique (France)
We report the realization of a surface plasmon resonance imaging biosensor
capable of dynamically characterizing optical anisotropy by means of
polarimetric measurements.
Our approach relies on a light beam propagating through a high refractive
index glass-prism (Kretschmann-Raether configuration) in order to excite a
surface plasmon wave along a metal-dielectric interface. This evanescent
wave probes the metal-dielectric vicinities with sub-nanometer sensitivity,
thus resolving optical characteristics of adsorbed biomolecular targets. Fixing
wavelength and angle of incidence of the beam enables real-time monitoring
of adsorptions and desorptions of targets onto the whole surface of the chip,
allowing for example characterization of DNA:DNA interaction kinetics with
applications to genetic diagnosis [1, 2].
The polarimetric surface plasmon resonance imaging device is depicted in
figure 1 and uses a pyramid of high index glass and two orthogonal SPR
imaging sensor arms. The interface is probed along two orthogonal directions.
A signal difference in reflection between the two arms allows us to resolve
the optical anisotropy of the dielectric medium, keeping the parallel and realtime capabilities of the system. Additional information can be obtained by
varying the angle of incidence of the light beam or tuning its wavelength. We
believe that this type of sensor will be useful for studying collective
biomolecular assemblies’ conformational changes.
6631-28, Session 6
Rigid and flexible multiphoton fluorescence
endoscopes
S. Schenkl, A. Ehlers, Fraunhofer-Institut für Biomedizinische Technik
(Germany); R. Le Harzic, JenLab GmbH (Germany); I. Riemann, D.
Sauer, Fraunhofer-Institut für Biomedizinische Technik (Germany); B.
Messerschmidt, Grintech GmbH (Germany); M. Kaatz, FriedrichSchiller-Univ. Jena (Germany); K. König, Fraunhofer-Institut für
Biomedizinische Technik (Germany)
Multi-photon autofluorescence imaging offers minimal-invasive examination
of cells without the need of staining and complicated confocal detection
systems. Therefore, it is especially interesting for non-invasive clinical
diagnostics.
To extend this sophisticated technique from superficial regions to deep lying
cell layers, inner body and specimens, difficult of access, the bulky optics
need to be reduced in diameter. This is done by tiny GRIN-optics, based on
a radial gradient in the reflective index. Of especial interest for multi-photon
applications is the newly developed GRIN-lens assembly with increased
numerical aperture. High resolution images of plant tissue, hair and cells
show the enhanced image quality.
The rigid GRIN-endoscopes are applied in wound healing studies. Here, the
GRIN-lens with the diameters smaller than 3 mm enter small skin depressions.
It reproduces the focus of the conventional laser scanning tomograph tens
of mm apart in the specimen under study. We will present clinical
measurements of elastin and SHG of collagen of in vivo human skin of ulser
curis.
In the flexible endoscope, the light is delivered by a fiber to the specimen
and the region of interest controlled using a miniaturized dedicated 2D scanner
device based on piezoceramics bimorph actuators. The well-characterized
photonic crystal fiber supports the high laser power of the femtosecond
excitation impulses without the generation of non-linearities. A sensitive PMT
detector detects the fluorescence. First fluorescence images through a fiberGRIN lens combination will be presented.
6631-29, Session 6
Combined Raman spectroscopy-optical
coherence tomography
C. A. Patil, Vanderbilt Univ. (USA); N. Bosschaart, Univ. Twente
(Netherlands); D. J. Faber, Univ. van Amsterdam (Netherlands); T. G. van
Leeuwen, Univ. Twente (Netherlands); A. Mahadevan-Jansen, Vanderbilt
Univ. (USA)
European Conferences on Biomedical Optics 2007 •
Raman Spectroscopy (RS) and Optical Coherence Tomography (OCT) are
two techniques whose application in characterizing and evaluating
pathological tissue types has been demonstrated in vivo. While Raman
spectra provide information related to the specific biochemical composition
of a tissue type, OCT provides morphological information in the form of high
resolution tomographic images. In this abstract, we present a combined
device capable of sequential OCT imaging and RS acquisition along a
common optical axis. The device is realized by simply modifying the sample
arm of an OCT system to allow for co-alignment of a Raman probe beam
with the OCT beam. We have developed two implementations of the device,
using both time-domain and fourier-domain OCT systems. We will
demonstrate the utility of the device to: 1. guide positioning of the RS probe
beam using OCT for spatially precise spectral acquisition, and 2. characterize
a structural anomaly in an OCT image plane using RS in optical phantoms.
Additionally we will present preliminary in-vitro results demonstrating the RSOCT’s ability to confirm the biochemical content of structural anomalies within
an OCT image as well as guide RS in pathological human skin samples.
6631-30, Session 6
High-resolution imaging using random
fluorescent probes
P. Lecaruyer de Lainsecq, E. Fort, Univ. Paris VII (France); S. Fort, Univ.
Paris-Sud-XI (France); N. Tran Hong, Institute of Physics & Electronics
(Vietnam)
Individual fluorescent nanoparticles can be located with a nanometric
precision. These nanoparticules can thus be used as random probes to scan
the propagating and evanescent electromagnetic field and provide highresolution imaging of various samples.
The spatial resolution of an optical system is limited by the Rayleigh criterion.
Using visible light and a high numerical aperture objective allows achieving
250 nm lateral resolution. Centroid localization, which has been used for
single-particule tracking, permits determination of the position of the particle
to a much better precision than the length scale defined by the Rayleigh
criterion. Recently, this approach has been used to image molecular motor
and cells with a nanometer-resolution [1,2]. The fluorophores are local probes
of the electromagnetic field. When placed in a liquid, their ergodic random
motion permits to scan the entire volume available. We will show the ability
of this method to image lithographic structures with a resolution about one
order of magnitude better than the diffraction limit.
Another interesting aspect of these fluorescent probes is their ability to map
the EM field with a high-resolution. These probes are sensitive to propagating
EM field but also to the evanescent one. Hence, they can be used as local
near field probes. In that context, we will show results of this new imaging
method that can be defined as a multiplexed ergodic scanning near field
optical microscope (SNOM). We will compare this technique with the
conventional SNOM technique.
REFERENCES:
[1] A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha Y. E. Goldman, and P. R.
Selvin “Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with
1.5-nm Localization,” Science 300, 2061 (2003)
[2] N. F. Scherer, “Pointillist microscopy,” Nature Nanotech., 1, 19 (2006).
6631-31, Session 6
Two-photon, two-color in vivo flow cytometry to
noninvasively monitor multiple circulating cell
lines
E. R. Tkaczyk, C. F. Zhong, J. Y. Ye, K. Luker, G. D. Luker, J. R. Baker,
Jr., T. B. Norris, Univ. of Michigan (USA)
Circulating cells of various types, including breast cancer cells, recently have
gained considerable attention as new prognostic markers in cancer and
response to therapy. To detect and quantify multiple distinct populations of
cells circulating simultaneously in the blood of living animals, we developed
a novel optical system for two-channel, two-photon flow cytometry in vivo.
We used this system to investigate the circulation dynamics in mice of human
MCF-7 and MDA-MB-435 breast cancer cells, which have low and high
metastatic potential, respectively. After co-injection of both cell types into
mice, markedly greater numbers of MCF-7 cells were present in the circulation
at early time points. While low metastatic MCF-7 cells were cleared from the
vascular system within 24 hours, net numbers of metastatic MDA-MB-435
cells in the circulation remained constant over time. We also used two-photon
flow cytometry to non-invasively enumerate a population of fluorescentlylabeled red blood cells for more than two weeks, demonstrating that this
technique also can quantify dynamics of abundant cells in the circulation for
prolonged periods of time. When we replace the commercial (80-MHz) NIR
excitation laser with a reduced-repetition-rate (20-MHz) mode-locked
oscillator, the signal is enhanced four-fold, enabling detection in blood of cell
lines expressing the fluorescent protein T4 or GFP variants tdTomato or
mPlum. The technique of two-color, two-photon flow cytometry greatly
enhances the capabilities of in vivo flow cytometry to investigate dynamics
of circulating cells in cancer and other medically important diseases.
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Conference 6631: Novel Optical Instrumentation for Biomedical Applications
6631-32, Session 6
Fluorescence imaging of experimental
rheumatoid arthritis in vivo using a fast flyingspot scanner
J. Berger, J. Voigt, F. Seifert, B. Ebert, R. Macdonald, PhysikalischTechnische Bundesanstalt (Germany); I. Gemeinhardt, O. Gemeinhardt,
J. Schnorr, M. Taupitz, Charité-Univ. Medizin Berlin (Germany); A. Vater,
S. Vollmer, K. Licha, M. Schirner, Bayer Schering Pharma AG (Germany)
We have developed a flying-spot scanner for imaging of rheumatoid arthritis
in the near infrared (NIR) spectral range following intravenous administration
of contrast agents. The scanning imaging system comprises a compact cw
diode laser emitting at a wavelength of 756 nm. The laser beam is coupled
into a scan head using a dichroic mirror in reflection geometry. An area of
240 mm x 240 mm can be scanned with 256 x 256 image points within one
second. Emitted fluorescence light is captured by the scan head and passes
the dichroic mirror. The focused transmitted fluorescence light is detected
by an avalanche photodiode.
Images of rats with experimental arthritis were analyzed by applying
appropriate regions of interest located at the area of ankle joints. After injection
of a contrast agent all ankle joints show a rapid increase in fluorescence
intensity, whereas the inflamed joints show a stronger increase in contrast
compared to healthy ankle joints. Different stages of inflammatory joint
diseases in the experimental rat model in vivo are demarcated.
Comparison of fluorescence imaging with contrast enhanced MR imaging
reveal that NIR cyanine dyes are suitable to monitor early stages of joint
inflammation.
6631-33, Session 6
Spectroscopic imaging in the near field with an
aperture less solid-immersion lens system
T. Merz, R. W. Kessler, Reutlingen Univ. (Germany)
The Raleigh criterium limits the optical characterisation of features smaller
than approximately l/2. The combination of SPM and near field optical
microscopy permits the characterization of the morphology and chemistry
of surfaces and cell structures on a nanometer scale.
To achieve lateral high resolution, we exploit the localised electromagnetic
field of a Solid-Immersion-Lens (SIL). The lens is mounted in a cantilever of
an AFM to support a dynamical scan with constant tip-sample force. This
unit is attached to a Micro-Fluorescence or Raman-spectrometer (Zeiss
UMSP) to allow spectroscopy in the near field.
With our setup three methods can be applied with a lateral resolution less
than 30 nm:
Reflectance-SNOM: The sample is imaged by illuminating the surface through
the SIL and detecting the reflected near-field.
Photon-tunnelling-SNOM: The contrast is generated by the ability of the
photons to tunnel through the energy barrier into the substrate.
Fluorescence- SNOM: The chromophore is excited and the fluorescence is
collected by the SIL. The collection efficiency for the fluorescence is increased
due to the high refractive index of the SIL by a factor of 10.
A further advantage of the SIL system is the high transmission of the SIL
which results in a better S/N ratio. It also provides the ability to illuminate
and collect the light by the same probe. The paper will show the design and
construction principles of this SIL near field microscope spectrometer and
several near-field applications will demonstrate the potential of the system.
6631-34, Session 6
Evaluation of a fiber-optic fluorescence
spectroscopy system to assist neurosurgical
tumor resections
M. A. Ilias, F. Westermark, M. Brantmark, Linköping Univ. (Sweden); S.
Andersson-Engels, Lunds Univ. (Sweden); K. Wårdell, Linköping Univ.
(Sweden)
The highly malignant brain tumor, Glioblastoma Multiform, is difficult to totally
resect without aid due to its similarities to surrounding functioning brain and
infiltrative way of growing. The need for an inexpensive and robust real-time
visualizing system for resection guiding in neurosurgery has been formulated
by research groups all over the world. The main goal is to develop a system
that helps the neurosurgeon to make the decision during the surgical
procedure.
A compact fiber optic system using fluorescence spectroscopy has been
developed for guiding neurosurgical resections. The system is based on a
high power LED at 405 nm and a spectrometer. A spliced optical fiber is
used to guide the excitation light and fluorescence light between the
instrument and the surgical suction tool used in the resection. The tip of the
optical fiber is mounted on a conventional surgical suction tool. The system
is controlled by the surgeon through a computer interface and software
package especially developed for the application. This robust and simple
62
European Conferences on Biomedical Optics 2007 •
instrument has been evaluated in neurosurgical resection procedures. Before
surgery the patients received orally a low dose of 5-amino laevulinic acid,
converted to the fluorescence tumor marker protoporphyrin IX in the malignant
cells.
6631-35, Session 6
Combination of panoramic and fluorescence
endoscopic images to obtain tumor spatial
distribution information useful for bladder
cancer detection
S. Olijnyk, Y. Hernandez-Mier, W. W. Blondel, C. Daul, D. Wolf, École
Nationale Supérieure d’Electricité et de Mécanique - Nancy (France)
Introduction :
Bladder cancer is widely spread. Moreover, carcinoma in situ can be difficult
to diagnose as it may not be visible, and become invasive in 50 % of case .
Non invasive diagnosis methods like Photodynamic or autofluorescence (with
near UV excitation) endoscopy allow to enhance sensitivity and specificity.
Besides, bladder tumors can be multifocal. Multifocality increases the
probability of recurrence and infiltration of bladder muscle. Therefore analysis
of the spatial distribution of tumors could be used to improve diagnosis.
In this work, we explore the feasibility to combine fluorescence and spatial
information on phantoms in order to enhance bladder cancer detection.
Materials & Methods :
We developed a system allowing an acquisition of every other image with
white light or UV excitation alternatively and automatically along the video
sequence.
We also developed an automatic image processing algorithm, based on fast
2D gray level registration and stitching, to build a partial panoramic image
from a cystoscopic sequence of images. Fluorescence information is
extracted from wavelength bandpass filtered images and superimposed over
the cartography. Then, spatial distribution measure of fluorescent spots can
be computed.
This cartography can be positioned on a 3D generic shape of bladder by
selecting some reference points, easily exploited by urologists.
Results & Discussion :
Our first results on phantoms show it is possible to obtain a cartography with
fluorescent spots and extract quantitative information of their spatial
distribution.
Our 3D representation can be a visual support useful to the clinician in
providing a comparison of the localization of eventual lesions from a clinical
exam to another.
Further validation on in vivo cancer model is under progress.
6631-36, Session 7
Laser interference measurement of glucose in
liquids
H. M. El Ghandoor, Ain Shams Univ. (Egypt)
In this study, we are developing a new method, which has been invented by
one of our investigators, for measuring the glucose concentration in liquids.
The method is called the laser sheet method. The proposed method allows
us to perform an accurate measurement of refractive index of liquid samples.
We used this technique to measure glucose concentration in distilled water
(ranging from 10 to 50%). We obtained a good correlation between the glucose
concentration and the refractive index. This method will be further developed
to measure glucose concentration in plasma and in anti-coagulated blood
from human volunteers. If the technique is successful, it has the potential of
becoming the principle of a new blood diagnostic machine based on laser
interference.
6631-37, Session 7
Combination of time-domain optical brain
imaging and DC magnetoencephalography for
studying neurovascular coupling
H. Wabnitz, T. Sander, Physikalisch-Technische Bundesanstalt
(Germany); A. Liebert, Institute of Biocybernetics and Biomedical
Engineering (Poland); M. Möller, Hochschule für Technik und Wirtschaft
des Saarlandes (Germany); S. Leistner, B. Mackert, Charité
Universitätsmedizin Berlin (Germany); R. Macdonald, L. Trahms,
Physikalisch-Technische Bundesanstalt (Germany)
This study aims at measuring slow electrophysiological activity of the brain
simultaneously with its vascular correlate. DC-magnetoencephalography (DCMEG) allows for direct monitoring of slow cortical electric activity. Near-infrared
spectroscopy (NIRS) monitors the vascular brain response by measuring
changes in the concentration of oxy- and deoxyhemoglobin and is compatible
with DC-MEG. Time-domain NIRS allows one to separate between deep
and superficial absorption changes. For the optical measurements we used
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Conference 6631: Novel Optical Instrumentation for Biomedical Applications
our time-domain brain imager with four detection channels. The heads of the
healthy subjects with the NIRS optodes attached were positioned beneath
the MEG SQUID sensor array in a magnetically shielded room. The subject’s
position was periodically modulated at 0.4 Hz relative to the MEG sensor to
achieve sufficient signal-to-noise ratio. Subjects performed finger movements
for 30 s followed by 30 s of rest while the DC-MEG and NIRS signals were
recorded over the contralateral motor cortex. The vascular response as
measured by NIRS turned out to be slower than the corresponding sustained
electrophysiological response as monitored by DC-MEG. The leading slope
of the DC-MEG response was found to be steeper, and the 50% level was
reached about 1s to 3s earlier.
Neurovascular coupling is expected to be altered by neurological diseases,
in particular stroke. Therefore, apart from studies on healthy volunteers, first
exploratory measurements on nine stroke patients were performed. To
overcome the limited time resolution of modulation DC-MEG, first combined
measurements were performed with unmodulated broadband MEG (DC to
several kHz) in an extremely well shielded room.
6631-38, Session 7
A novel optical detector concept for dedicated
and multi-modality in vivo small animal imaging
J. Peter, R. B. Schulz, D. Unholtz, W. Semmler, Deutsches
Krebsforschungszentrum (Germany)
An optical detector suitable for inclusion in tomographic arrangements for
non-contact in vivo bioluminescence and fluorescence imaging applications
is proposed. It consists of a microlens array (MLA) intended for field-of-view
definition, a large-field complementary metal-oxide-semiconductor (CMOS)
chip for light detection, a septum mask for cross-talk suppression, and an
exchangeable filter to block excitation light. Prototype detector units with
sensitive areas of 2.5 cm Å~ 5 cm each were assembled. The CMOS sensor
constitutes a 512 Å~ 1024 photodiode matrix at 48 µm pixel pitch. Refractive
MLAs with plano-convex lenses of 480 µm in diameter and pitch were selected
resulting in a 55 Å~ 105 lens matrix. The CMOS sensor is aligned on the
focal plane of the MLA at 2.2 mm distance. To separate individual microlens
images an opaque multi-bore septum mask of 2.1 mm in thickness and bore
diameters of 400 µm at 480 µm pitch, aligned with the lens pattern, is placed
between MLA and CMOS. Intrinsic spatial detector resolution and sensitivity
was evaluated experimentally as a function of detector-object distance. Four
detector units were mounted on a common rotatable gantry and planar as
well as tomographic imaging experiments were performed. To investigate
the full potential for optical tomography (OT) application a Monte Carlo
simulation study was conducted incorporating up to 20 cylindrically aligned
detectors of optimized geometry. Furthermore, simultaneous OT-PET
experimental data acquisition was performed by placing the prototype
detectors inside the bore of a patient scale positron emission tomography
(PET) scanner, thus gaining complementary information in a single, intrinsically
coregistered experimental study.
6631-39, Session 7
Optical vibrocardiography for non contact
monitoring of the cardiac activity: correlation
with heart sounds from phonocardiography
6631-40, Session 7
Observation of IPL spectra using detector
system incorporating broadband optical filters
D. M. Clarkson, Univ. Hospitals Coventry and Warwickshire NHS Trust
(United Kingdom)
A system is described for time resolution of spectral components of intense
pulsed light system using laptop computer, USB data capture module
interfaced to an array of silicon photodiodes/bandpass optical filters. This
provides information relating to general light spectra output of an IPL device
and also the relative variation of identified spectral components with time.
Use was made of a low cost 8 channel, 16 bit USB analogue I/O module
with maximum throughput rate of 100 kHz. An array of 16 silicon photodiode
detectors
which detected light through bandpass filters of typical
bandwidth 90 nm and 40 nm was interfaced via operational amplifiers to
the I/O module. In addition, with use of analogue multiplexer circuit
controlled by the USB device up to 12 channels could be sampled by
software. An initial set of 11 measurement channels over the wavelength
range 450 nm to 950 nm was investigated with system being modified
to extend from 400 nm to 1100 nm.
The system is used for measurement of a output spectra profiles of a
number of IPL systems. The measurement of spectral output and
associated time frame distribution within identified wavelength bands is
shown to be practical to undertake and to provide useful evaluation of IPL
performance. A summary of initial observations will be presented and
applications in safety estimation and device performance/evaluation
discussed. Initial observations indicate wide variation in pulse waveform
characteristics across range of IPL systems.
6631-41, Session 7
Wavelet-based terahertz local tomography
X. Yin, The Univ. of Adelaide (Australia)
Terahertz Computed Tomography (THz-CT) is a promising approach to
achieving non-invasive solid materials inspection and disease state diagnosis,
with potentially numerous applications in industrial manufacturing and
biomedical engineering. With traditional CT techniques such as X-ray
tomography, full exposure data are needed for inverting the Radon transform
to produce cross section images. For time-domain THz measurements,
requirement of full exposure data is impractical due to the slow measurement
process. In this paper, we apply a wavelet-based algorithm to reconstruct
THz-CT images with a significant reduction in the required measurements.
The algorithm localises the Radon Transform by leveraging the vanishing
moment property inherent in the two-dimensional separable wavelet
transform. Instead of inverting the interior Radon transform, the suggested
approach is obtained via wavelet and scaling ramp filters and the traditional
back projection algorithm for the resultant reconstruction. For comparison,
the traditional filtered back projection algorithm is applied on measured
projection data for a global reconstruction. The current algorithm recovers
an approximation from local terahertz image statistics, and improves the
ability of Terahertz imaging to detect defects and diagnose diseases in solid
materials and clinic application. Feasibility of this method is demonstrated
on two sets of terahertz imaging data.
L. Scalise, M. De Melis, U. Morbiducci, E. P. Tomasini, Univ. Politecnica
delle Marche (Italy)
In the last years optical methods have been introduced and developed for
non-invasive measurement of the main cardiac vital signs. Attention was
focused on methods enabling the characterization of the cardiovascular
dynamics wich then could be used in cinical diagnostics. In particular, laser
Doppler vibrometry (LDV) has been proposed by the authors for the noncontact monitoring of the cardiac activity, using the measurement of the
displacement of the skin surface over the chest wall (optical
Vibrocardiography); it was demonstrated that VCG is able to perform both
heart rate and heart rate variability analysis with high accuracy.
In this work heart sounds by digital Phonocardiography (PCG), the vibration
velocity of the skin on the chest wall by optical VCG, along with
electrocardiogram (lead II), were simultaneously recorded on healty subjects.
We have developed an advanced processing algorithms for signal comparison
(multiresolution analysis together with Hilbert transforms for envelope
calculation) were applied in order to extract temporal and morphological
features from the signals. This allowed us to identify on VCG traces the events
of cardiac mechanics, correlating the heart sounds relative to the closure of
the mitral valve, and the following closure of the aortic and pulmonary valve
with characteristic deflections identifiable on VCG recordings.
Our results confirm the technical feasibility of using optical vibrocardiography
as a non-contact method for the extraction of vital cardiac signs in clinical
practice.
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63
Conf. 6632: Therapeutic Laser Applications and Laser-Tissue Interactions
Room B12 • Monday-Wednesday 18-20 June 2007
Part of Proceedings of SPIE Vol. 6632 Therapeutic Laser Applications and Laser-Tissue Interactions III
6632-01, Session 1
CO2 laser free-form processing of hard tissue
M. Werner, M. M. Ivanenko, Ctr. of Advanced European Studies and
Research (Germany); D. Harbecke, Ctr. of Advanced European Studies
and Research (Germany) and Univ. Düsseldorf (Germany); M. Klasing,
Ctr. of Advanced European Studies and Research (Germany); H.
Steigerwald, Ctr. of Advanced European Studies and Research
(Germany) and Univ. Bonn (Germany); P. Hering, Ctr. of Advanced
European Studies and Research (Germany) and Univ. Düsseldorf
(Germany)
Drilling and surface processing of bone and tooth tissue belong to standard
medical procedures (bores and embeddings for implants, trepanation etc.).
Small circular bores can be generally quickly produced with mechanical drills.
However problems arise at angled drilling, the need to execute drilling
procedures without damaging of sensitive soft tissue structures underneath
the bone or the attempt to mill small non-circular cavities in hard tissue with
high precision. We present investigations on laser hard tissue “milling”, which
can be advantageous for solving these problems.
The processing of bone is done with a CO2 laser (10.6 µm) with pulse durations
of 65 - 80 µs, combined with a PC-controlled fast galvanic laser beam scanner
and a fine water-spray, which helps keeping the ablation process effective
and without thermal side-effects. Damaging underlying soft tissue can be
prevented by monitoring the acoustical ablation signal. The acoustic signals
from the different tissue types exhibit distinct differences in the spectral
composition. Also computer image analysis could be a useful tool to control
the operation progress.
Laser “milling” of non-circular cavities with 1 - 4 mm width and about 10 mm
depth can be especially interesting for dental implantology. In ex-vivo
investigations we found conditions for fast laser processing of these cavities
without thermal damage and with minimized tapering. It included the
exploration of different filling patterns (concentric rings, crosshatch, parallel
lines, etc.), definition of maximal pulse duration, repetition rate and laser
power, and optimal spray position. The optimized results give evidence for
the applicability of pulsed CO2 lasers for biologically tolerable effective
processing of deep cavities in the hard tissue.
6632-02, Session 1
Ultra-short pulse laser processing of hard
tissue, dental restoration materials, and
biocompatibles
E. Wintner, M. Strassl, V. Wieger, A. Yousif, Technische Univ. Wien
(Austria)
6632-03, Session 1
Optimized laser treatment of bone tissue by
means of thermal effect visualization
S. Stopp, D. Guenther, H. Deppe, T. C. Lueth, Technische Univ.
Muenchen (Germany)
In this article a new concept for navigated and model based laser surgery is
presented. To achieve maximal bone ablation effect with only minimal thermal
damage of the bone the expected proportion of the bone ablation volume
and the thermal energy is visualized depending on the position and orientation
of the laser handpiece. For this goal a model of the laser ablation can be
defined. With the relation for each focus distance an energy profile can be
computed. Bone material threshold values are used for the calculation of the
ablation to thermal effects. As a result the areas with pure heating and those
with ablation and heating can be determined. The quotient of these for the
healing relevant data can be build and shown to the surgeon in real time. To
determine the position of the laser focus relative to the bone tissue a CT data
based planning and navigation with an optical measurement system is used.
The presented method allows a more effective laser surgery. The laser can
be applied more effectively, since the surgeon can follow the visualized data.
European Conferences on Biomedical Optics 2007 •
6632-04, Session 1
Partial kidney resection by use of a 1,94µm
thulium fiber laser
D. Theisen-Kunde, Univ. zu Lübeck (Germany); S. Tedsen, Univ zu
Lübeck (Germany); V. Danicke, K. Herrmann, R. Brinkmann, Univ. zu
Lübeck (Germany)
In general surgery as well as in urology there is still a need for fast and precise
dissection devices with reliable haemostasis of dissected blood vessels. A
Laser-Scalpel based on a thulium fiber-laser-system emitting a wavelength
at 1,94 µm and a laser power at 16 W (cw-mode) was used for partial resection
of the kidney in 6 pigs. The high absorption coefficient of water (128 1/cm) at
that wavelength yields to efficient water vaporisation in tissue and therefore
to a precise tissue dissection with defined thermal damage.
After general anaesthesia a median laparotomy was performed to expose
one kidney.
Laser radiation was transmitted via quartz fibre (200 µm core diameter) with
polished distal fibre tip which was held in a stainless steel tube.
Results:
Number of kidneys [n]: 6;
Weight of pigs [kg]:34,8 ± 8,3;
Weight of resected tissue [g]:8,6 ± 7,4;
Resection time [min]:7,3 ± 1,9;
Blood lost [ml]:9,5 ± 9,3;
Ischemic time [min]:0;
Histological evaluation with H&E stained sample showed a carbonised zone
of about 100 - 300µm directly at the dissected edge followed by a thermal
damaged zone of about 500 - 800 µm in width. Thereafter healthy tissue was
found in all histological samples. In conclusion partial kidney resection could
be easily and fast performed by the use of a 1,94 µm Laser-Scalpel system.
Haemostasis was highly sufficient so blood lost was negligible. The first results
indicated that the 1,94 µm Laser-Scalpel system is a very promising dissection
device for urological surgery.
6632-05, Session 1
Ultra-short laser pulses must be scanned for collateral damage-free ablation
of biological tissue and related materials, irrespective of their duration. In
this paper a discussion about different scanning algorithms together with 3
different scanners (x-y, r-phi, and linear) and useful as well as economic (with
respect to laser cost) pulse lengths will be presented. Furthermore, ablation
thresholds and rates on dental and bone tissues (human and bovine) and
also dental restoration materials, with respect to different pulse lenghts, will
be given. Morphological analysis in light and environmental scanning elecron
microscopes will be compared to corresponding Er-laser results. The issue
of laser cleaning of implants employing different wavelengths and pulse
durations will be touched. Additionally, features like the possibility of
accompanying light-induded breakdown spectrocopy will be demonstrated
as a useful feedback technique.
64
The visualization of the position and power depending proportion of bone
ablation volume and thermal energy should offer an effective and gently bone
ablation. The verification should contain information about the feasibility of a
model and navigation based approach of analyze of the thermal bone damage.
Hence, a prognosis of the healing time and the healing success can be given.
In the next steps, the approach will be prototypically realized and evaluated
on clinically feasible phantoms.
Preliminary results on diode-laser assisted
vaporization of prostate tissue
R. Sroka, M. Seitz, O. Reich, A. Bachmann, V. Steinbrecher, A.
Ackermann, C. G. Stief, Ludwig-Maximilians-Univ. München (Germany)
Introduction and objectives: The aim was to identify the capability and the
laser parameter of under water tissue vaporisation by means of a diode laser
(1470 nm). Afterwards the feasibility and postoperative clinical outcome of
vaporization of the prostate was investigated.
Method: After acquiring suitable laser parameters in in-vitro experiments using
a perfused tissue model patients (n=10) suffering from bladder outlet
obstruction due to benign prostatic hyperplasia (BPH) were treated by diode
laser. Their clinical outcome, in terms of acceptance and post-operatively
voiding were evaluated. The diode laser emitted light of the wavelength of
1470 nm at 50 W (Biolitec GmbH) and delivered to the tissue by means of a
side-fire fiber introduced through a 24F continuous-flow cystoscope. Normal
saline was used for irrigation with an additive of 1% ethanol. The prostatic
lobes (volume range 35-80ml) were vaporized within the prostatic capsular
using sweeping and push and pull technique. The mean time of laser
application was 2400 sec (1220-4000 sec) resulting in applied energies of
121 kJ in the mean (range: 61-200kJ).
Results: During laser treatment none of the 10 patients showed any significant
blood loss or any fluid absorption (no ethanol uptake). Foley catheters were
removed between 18 and 168 hours postoperatively (mean: 49.8h±46h). After
removal of the catheter the mean peak urine flow rate increased from 8.9ml/
s ± 2.9ml/s pre-operatively in comparison to 15.7ml/s ± 5 ml/s (p=0.049)
post-operatively. 8/10 patients were satisfied with their voiding outcome.
None of the patients showed appearance of urgency, dysuria, hematuria, or
incontinence but two patients required re-catheterization. After a follow-up
of 1month, 8/10 patients showed evidence of good results and are satisfied
with the outcome. Two patients required consecutive TUR-P. After a followup of 6-month the 8 patients are still satisfied. Conclusions: This very early
and limited experience using a 50W-Diode laser emitting at 1470 nm indicates
a safe and effective treatment modality for quickly relieving bladder outlet
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Conf. 6632: Therapeutic Laser Applications and Laser-Tissue Interactions
obstruction due to BPH. Larger randomized clinical trials to compare this
technique with standard transurethral resection of the prostate and increased
follow-up data are needed to determine its long-term efficacy and durability.
6632-06, Session 1
6632-51, Poster Session
Optical coherence tomography monitoring of
vocal fold femtosecond laser microsurgery
H. Wisweh, Laser Zentrum Hannover e.V. (Germany) and Hannover
Medical School (MHH) (Germany); U. Merkel, A. Hüller, K. Lüerssen,
Medizinische Hochschule Hannover (Germany); H. Lubatschowski,
Laser Zentrum Hannover e.V. (Germany)
The surgery of benign pathological changes of the vocal folds implicate
permanent disphonia of the patient if the bounderies of the vocal fold layers
are disregarded. Precise cutting with a fs-laser combined with simultanous
imaging of the layered structure enables accurate resections with respect to
the layer boundaries.
Earlier works demonstrated the capability of optical coherence tomography
(OCT) for the application on vocal folds. The layered structure can be imaged
with a spatial resolution of 10-20 microns up to a depth of 1.5 mm which
correlates well with the penetration depth of the near-infrared fs-laser beam.
The performance of fs-laser cutting was analyzed on extracted porcine vocal
folds with OCT monitoring. Histopathological sections of the same processed
samples could be well correlated with the OCT images. With adequate laser
parameters thermal effects induced only little damage to the processed tissue.
The dimensions of the thermal necrosis were determined to be smaller than
10 microns.
In conclusion, OCT contolled fs-laser cutting of porcine vocal fold tissue in
the micrometer range with minimal tissue damage is presented.
6632-20, Poster Session
Photodynamic therapy of murine non-malanoma
skin carcinomas with diode laser after topical
application of aluminum phthalocyanine chloride
M. Kyriazi, E. Alexandratou, D. M. Yova, National Technical Univ. of
Athens (Greece); M. Rallis, Univ. of Athens (Greece); T. A. Trebst,
CeramOptec GmbH (Germany)
Topical photodynamic therapy (PDT) with ALA has been extensively used for
skin carcinomas. However, its efficiency is restricted only in superficial
carcinomas due to 630 nm light penetration in tissues. Phthalocyanines, are
second generation photosensitizers presenting improved properties. Among
them is the absorption at higher wavelengths where the light can penetrate
deeper in tissues.
The objectives of this work are to study the pharmacokinetics and
photodynamic efficiency of aluminium phthalocyanine chloride (AlClPc) in
dimethylsulfoxide/Tween 80/water formulation, after topical application in
hairless mice bearing non-melanoma skin carcinomas. The concentration of
photosensitizer in normal skin and tumor biopsies 1-6 hours after application
was assessed by fluorescence spectroscopy of chemical extractions. The
higher concentration of AlClPc in tumor was observed 6 hours after
application. The pharmacokinetic studies also revealed, that the ratio of tumor
to normal skin AlClPc concentration at all the studied drug light intervals was
about 40.
For PDT AlClPc was excited by a diode laser emitting at 670 nm. Seven
different combinations of therapeutic parameters were chosen. The efficiency
was assessed as the percentage of complete tumor remission, the tumor
growth retardation and the cosmetic outcomes. The highest complete
remission 60% was achieved with the combination of 75 mW/cm2 at a final
dose 150 J/cm2. No recurrence rate was observed in any treatment
parameters group and the cosmetic outcome in all completely treated tumors
was excellent. The results show that the effectiveness of PDT is highly
dependent on fluence rate. In addition, they are promising for further
investigation of this PDT scheme in preclinical studies mainly in deeper nonmelanoma skin carcinomas.
6632-50, Poster Session
Characterization of biophysical properties of
rabbit auricle reshaped via diode laser (? =980
nm)
T. A. El Tayeb, The German Univ. in Cairo (Egypt)
Laser cartilage reshaping is a temperature dependent process that results in
stress relaxation with subsequent formation of a new and stable specimen’s
geometry. This temperature dependent process results in mechanical stress
relaxation and is characteristic of a phase transformation. The objective of
this study was to quantitavely measure changes in tensile elongation and
elastic modulus of rabbit auricle cartilage reshaped via diode laser (980 nm)
and irradiated in two different protocols. The results revealed that the laser
irradiation parameter used in cartilage reshaping does not produce significant
European Conferences on Biomedical Optics 2007 •
irreversible changes in mechanical properties of the cartilage tissue. So diode
laser can be listed as suitable tool in cartilage reshaping. Its unique
characteristics (cheap, portable and effective) over the other lasers which
were used in cartilage reshaping encourage us to do this study.
Computerized thermal qualification tool (CTQT)
for in-vitro low-water-content
F. A. Canestri, Agilent Technologies Deutschland GmbH (Germany)
Abstract
This Paper discusses in detail the mathematical identification of the Optical
Absorption Coefficient
? ( ) of the Beer’s law, crucial parameter to study the penetration of laser
beam craters into dry poly(methyl methacrylate) (PMMA) samples exposed
to a steady CO2 laser beams emitting radiation at ??= 10.6 ?m in continuous
mode (CW). In clinical applications, these results are important in order to
precisely quantify and forecast the ablation capabilities of the CO2 laser
beam, to optimize its usage in Operating Room and to particularly address
all the safety issues related to surgical interventions on human tissue.
Currently, the data available on literature do not allow the clear identification
the numerical value of
? ( ) for PMMA at ??= 10.6 ?m with enough, and therefore satisfactory,
accuracy. As seen in other Studies by the same Author, the correct
identification of the optical absorption of PMMA allows isolating with better
accuracy other key time-dependent coefficients, such as the relaxation time,
the surface threshold time and the heat incubation time, all described on
Literature on rather qualitative than quantitative fashion.
Correct bone cement preparation depends on the value of ? ( ) of the PMMA
in order to avoid un-wanted complication to patients during cement removal
via laser techniques.
The laser in use has been configured in different combinations amongst the
following parameters : transverse electromagnetic modes (TEMnm), output
power (I0), exposure times (te) and focal lengths (fk).
Several PMMA blocks (1 cm x 4 cm x 4 cm) have been exposed to the CW
radiation of three commercially available CO2 medical laser devices showing
a TEM11 mode.
Each block was exposed to the beam on a horizontal and well polished surface
of each sample.
A set of 4 focal length (2.5", 5", 7.5" and 15.75" (400 mm)) have been used to
focus the beam on the well polished and dry surface of the PMMA samples.
The resulting craters dimensions have been measured after each exposure
which has been kept at 10 Watt CW beam. The exposure time was ranging
between 0.5 to 2 seconds. Two new equations related to the key
thermodynamic parameters of low-water-content media have been identified.
These allow to isolate the value of ?????????? for the PMMA at 10.6 ?m
which also matches other results reported in Literature for similar compact
media in low-water-content in-vitro conditions, such as the PMMA, compact
bone and dentin.
Thanks to the calculated value of ??and of 3 other time-related parameters,
the Author proposes a Computerized Thermal Qualification Tool (CTQT) which
allows a fast automatic determination of the entire set of relevant
thermodynamic parameters related to other similar low-water-content media
samples in-vitro.
6632-52, Poster Session
The photons propagation into non trivial
geometry biological tissue
I. Krasnikov, A. Seteikin, Amur State Univ. (Russia)
Usually biological tissue has non trivial geometry. Some objects like cancerous
growth consist of many layers which can be of different forms, for example
blood vessels. In this work the authors tried to create a mathematical model
of thermal response of laser irradiated multilayered biological tissue. The
tissue has a number of layers with its own optical-physical characteristics.
We used Monte-Carlo simulation to calculate the propagation of light (laser
beams) in tissue and receive the function of heat source. As we usually have
radial symmetric laser beams we use cylindrical coordinates. The solution of
the 2D heat conduction equation is based on finite-element theory with the
use a predefined number of finite elements. We simulated constant and pulse
laser irradiation and as result there has temperature fields and the dynamics
of heat conduction. The analysis of the results shows that heat is not localized
on the surface, but is collected inside the tissue. By varying the boundary
condition on the surface and type of laser irradiation (constant or pulse) we
can reach high temperature inside the tissue without formation of necrosis at
the same time.
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65
Conf. 6632: Therapeutic Laser Applications and Laser-Tissue Interactions
6632-53, Poster Session
The role of autofluorescence colonoscopy in
diagnosis and management of solitary rectal
ulcer syndrome
A. Z. Kawczyk-Krupka, W. Latos, A. E. Ledwon, A. Kosciarz-Grzesiok,
A. Misiak, S. Kwiatek, A. Sieron, Medical Univ. of Silesia, Katowice
(Poland)
Solitary rectal ulcer syndrome (SRUS) is a chronic disease of the rectum.
Morphology of SRUS varies from ulcerative lesions and polypoid lesions to
edematous, nonulcerated, hyperemic mucosa. Although SRUS is a benign
condition there are studies which suggest that chronic ischaemia which occurs
in the solitary ulcer syndrome may lead to transitional mucosa that is similar
to that adjacent to colorectal carcinomas and adenomas and may lead to
colorectal dysplasia and carcinoma development. The exlusion of primary or
metastatic malignancy is the most important aim in the differential diagnosis
of SRUS. In our study we assess the possibilities of autofluorescence
colonoscopy (AFC) in diagnosis and management of SRUS.
We performed 1946 colonoscopies. After the medical history was taken white
light colonoscopy was performed. The tissue samples were taken for routine
pathological examination. When SRUS was histopathologically confirmed
AFC was performed by means of Xillix OncoLIFE The mean time lapse
between the two colonoscopies was 4 weeks. During AFC numerical colour
value (NCV) of autofluorescence of SRUS lesions was noted.
During 1946 colonoscopies eight persons were diagnosed as having SRUS.
There were three men and five women. In our material the endoscopic
spectrum was: four polypoid lesions, three flat ulcers and one case of isolated,
local erythema with hyperemia. We did not observe autofluorescence increase
in case of polipoid and flat ulcer lesions ( NCV 0,39-0,67; mean 0,525) and
little increase of autofluorescence in case of erythema lesion (NCV- 0,94).
SRUS is a rare disorder of the rectum but it causes differential diagnosis
problems. The most common reason for incorrect diagnosis are inadequate
tissue specimens. AFC allows to reveal subtle areas within the lesions of
more intense autofluorescence and localizes the potential cancertransformating dysplasia. In this way the most representative place, connected
with highest risk of pre- or cancerous changes, for biopsy specimen is
indicated.
6632-54, Poster Session
Regulation of mesenchymal stromal cells
differentiation by a blue laser irradiation
T. Kushibiki, K. Awazu, Osaka Univ. (Japan)
Mesenchymal stromal cells (MSCs) are multipotent cells, which are present
in adult bone marrow, that can replicate as undifferentiated cells and that
have the potential to differentiate to lineages of mesenchymal tissues,
including bone, cartilage, fat, tendon, and muscle. Their rapid and selective
differentiation should provide the potential of new therapeutic approaches
for the restoration of damaged or diseased tissue. However, several
fundamental questions must be answered before it will be feasible to usefully
predict and control MSCs responses to exogenous cytokines or genes. In
particular, a better understanding of how specific factor may alter the fate of
differentiation of MSCs is needed. In recent reports, circadian clock protein
controls osteogenesis in vitro and in vivo. Here we show that a stimulation of
a blue laser irradiation regulates the differentiation of mouse MSCs to
osteoblasts by the structural regulation of a circadian rhythm protein, mouse
Cryptochrome 1 (mCRY1). We found that a blue laser irradiation accelerated
osteogenesis of MSCs. After laser irradiation, mCRY1 protein was
translocated from cytoplasm to nucleus and mCRY1 mRNA level was
downregulated thereafter. Furthermore, knock-down of mCRY1 expression
by an RNA interference technique allowed the cells to inhibit osteogenesis
differentiation by laser irradiation. These results indicate that mCRY1, a bluelight receptor and a master regulator of circadian rhythm, plays important
roles in the regulation of the differentiation of MSCs. Since the differentiation
of MSCs was easily regulated only by a laser irradiation, the potential of new
therapeutic approaches for the restoration of damaged or diseased tissue is
anticipated. Furthermore, our results obtained in this study may prove an
excellent opportunity to gain insights into cross-talk between circadian
rhythms and bone formation.
6632-55, Poster Session
The influence of intravenous laser irradiation of
blood on some metabolic and functional
parameters in intact rabbits and experimental
cerebral ischemia
N. I. Nechipurenko, L. A. Vasilevskaya, Institute of Neurology, Neurosurgery & Physiotherapy (Belarus); J. I. Musienko, Belarusian Medical
Academy for Postgraduate Education (Belarus); G. Maslova, Belarusian
State Univ. (Belarus)
The aim of this study was to evaluate the influence of intravenous laser
irradiation of blood (ILIB) with He-Ne Laser (HNL) (0.63 µm wavelength) on
66
European Conferences on Biomedical Optics 2007 •
the lipid peroxidation (LPO), acid-base status, blood oxygen transport (BOT)
and dermal microhemodynamics (MHD) in intact rabbits and after
experimental local ischemia of brain (LIB). LIB was made by bilateral 3-hours
occlusion of common carotid arteries under thiopental anesthesia. HNL
radiation power of the light guide inserted in otic vein was 2.5 mW. Beginning
with the first day after LIB induction the operated and intact rabbits were
carried out 5-10 in number ten-minutes ILIB procedures. Investigation was
performed after the 1st, 5th and 10th procedure.
Postischemic period was characterized by mixed acidosis with prevalence
of metabolic component, worsening of BOT with hemoglobin-oxygen affinity
lowering, increasing of LPO processes with reduction of glutathione
peroxidase (GP) activity and worsening of dermal MHD. The ILIB course of
intact rabbits makes for increasing of reduced glutathione (GSH) content
and GP activity, stimulation of dermal MHD, rising of buffer base capacity in
blood. The ILIB course after LIB modeling contributes to reactivation of GP
activity, normalization of GSH content and the level of substances reacting
with thiobarbituric acid, hemoglobin-oxygen affinity increasing with
oxyhemoglobin dissociation curve shift leftwards, improving of dermal MHD
indexes.
Thus, ILIB promotes correction of some metabolic and microcirculation
processes in postischemic period. It allows considering that the He-Ne
radiation of the pointed regimen is substantiated pathogenetically in cerebral
ischemia.
6632-56, Poster Session
Near-infrared light propagation in human head:
comparison between finite element code data
and Monte Carlo simulations
C. Mansouri, Groupe ISAIP-ESAIP (France); J. L’Huillier, Ecole Nationale
Supérieure d’Arts et Métiers (France); A. Humeau, Groupe ISAIP-ESAIP
(France)
The low scattering properties of the cerebral spinal fluid (CSF) that surrounds
the brain (gray matter and white matter) has been of particular interest in the
development of the accurate modelling of light propagation in human head
for quantitative near-infrared spectroscopy and diffuse optical imaging.
In this study a model based on multiple layers such as the scalp, skull, CSF
and the brain was designed and investigated to show the effect of the
heterogeneous structure of the head on the light propagation within the brain.
Several simulations based on Monte Carlo and Finite Element (diffusion
equation) codes were performed in time-domain, at source-detector spacing
of 30 mm, in order to show the role played by the optical properties of the
CSF layer on the accurate forward photon migration process.
Results show that the presence of the CSF layer is important to accurately
modelling the light propagation in the head model especially for the low
reduced scattering values varying between 0.001 and 0.1 mm-1, while keeping
the absorption coefficient µa = 0.0033 mm-1.
The Finite Element method fails for early times and for very low reduced
scattering coefficients, but the discrepancy between Monte Carlo and Finite
Element methods is relatively small in detected signal for µs’ = 1 mm-1 .
6632-07, Session 2
Surgical treatment of cerebral ischemia by
means of diode laser: first experimental results
and comparison with theoretical model
T. Lo Feudo, C. Bellecci, P. Gaudio, M. Gelfusa, Univ. degli Studi di
Roma/Tor Vergata (Italy); C. D. Signorelli, G. Iofrida, F. Signorelli, A.
Giaquinta, Univ. degli studi Magna Græcia di Catanzaro (Italy)
In the present paper feasibility and potential advantages of using diode laser
to surgical treatment of cerebral ischemia and intracranial aneurysms will be
evaluated.
A this aim non linear mathematical model was developed and experimentally
validated to investigate the effects of the changes in tissue physical properties,
in terms of anastomotic time, tensile strength and tissue damage. during the
medical laser application.
The numerical simulations have been carried on by a finite-elements based
software package (FEMLAB).
In vitro results of human saphenous veins of inferior limbs (n=32) after 799
nm diode laser soldering, combined with an indocyanine green-enhanced,
will be presented.
The simulations results and their comparison with experimental measurements
will be reported.
6632-08, Session 2
Optical coherence tomography investigations of
endoluminal vein treatment after radiofrequency
and laser light application
R. Sroka, O. Meissner, K. Hunger, G. Barbaryka, C. Burgmeier, R.
Blagova, W. Beyer, T. J. Beck, B. Steckmeier, C. Schmedt, Ludwig-
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Conf. 6632: Therapeutic Laser Applications and Laser-Tissue Interactions
Maximilians-Univ. München (Germany)
Introduction: The purpose of this study was to develop a practicable model
to allow standardized experimental evaluation and comparison of endovenous
thermal occlusion procedures additionally by means of OCT.
Materials and Methods: The ex-vivo model consists of the subcutaneous
foot vein from freshly slaughtered cows. Using this model primary and acute
effects and initial mechanisms on vein vessel could be studied. In this study
different energy sources (laser and radiofrequency generator), different energy
application parameters were compared. Then the harvested tissues were
processed for histology. Additionally, before and immediately after treatment
the study segments were examined by optical coherence tomography (OCT)
using a prototype rotating endoluminal device. OCT-cross sections and HEstained tissue slices were correlated and the induced effects are described.
Results: By means of this ex-vivo model it becomes possible to create
reproducible conditions for the application of endoluminal thermal energy in
each specimen.
Radiofrequency treated veins showed no macroscopic evidence of thermal
lesions other than a discrete induration and thickening of the vessel wall and
a contraction of vessel lumen. After Laser treatment in-situ dissection of
treated veins showed various blood deposits in perivascular tissue and
transmural thermal lesions. Pre-interventional endovenous OCT cross
sections reproducibly show different layers of the of vessel wall. A good
correlation between OCT and histological cross section could be
demonstrated. Furthermore the Rf and laser induced tissue damage could
be quantified.
Conclusion: These preliminary ex-vivo experiments indicate that the
application for endovenous laser treatment should be modified and needs
treatment standardization to ensure a controlled homogenous circumferential
thermal damage of wall layers, avoiding perforation and alteration of
perivascular structures. The ex-vivo model is suitable for reproducible
scientific evaluation of endovenous treatment modifications under
standardized conditions with established macroscopic and histologic criteria.
OCT seem to be a practicable addition to macroscopic and histological
evaluation. OCT may prove a viable alternative to complex histological testing
in subsequent series.
6632-09, Session 2
The effects of intense pulsed light on blood
vessels investigated by mathematical modeling
W. Bäumler, Univ. Regensburg (Germany); G. Shafirstein, Univ. of
Arkansas for Medical Sciences (USA)
Intense pulsed light (IPL) sources have been successfully used for coagulation
of blood vessels in clinical practice. However, the broadband emission of
IPL hampers the clinical evaluation of optimal light parameters. We describe
a mathematical model in order to visualize the thermal effects of IPL on skin
vessels, which was not available, so far.
One IPL spectrum was shifted towards the near infrared range (near IR shifted
spectrum: NIRSS) and the other was heavily shifted toward the visible range
(visible shifted spectrum: VSS). The broadband emission was separated in
distinct wavelengths with the respective relative light intensity. For each
wavelength, the light and heat diffusion equations were simultaneously solved
with the finite element method. The thermal effects of all wavelengths at the
given radiant exposure (15 or 30 J/cm(c)˜) were added and the temperature
in the vessels of varying diameters (60, 150, 300, 500 µm) was calculated for
the entire pulse duration of 30 ms.
VSS and NIRSS both provided homogeneous heating in the entire vessel.
With the exception of the small vessels (60 µm), which showed only a
moderate temperature increase, all vessels exhibited a temperature raise
within the vessel sufficient for coagulation with each IPL parameter. The time
interval for effective temperature raise in larger vessels (diameter \> 60 µm)
was clearly shorter than the pulse duration. In most instances, the vessel
temperature was higher for VSS when compared to NIRSS.
6632-10, Session 2
Interaction of a dual-wavelength laser system
with cutaneous blood vessels
B. B. Majaron, M. Milanic, Institut JoÏef Stefan (Slovenia); S. J. Nelson,
Univ. of California/Irvine (USA)
In laser therapy of port wine stain (PWS) birthmarks, epidermal heating due
to melanin absorption limits the clinically applicable radiant exposure, thus
impairing the therapeutic success in many patients. Our working hypothesis
was that a dual-wavelength Nd:YAG laser, emitting simul¬taneously at 1064
and 532 nm, may induce stronger heating of PWS blood vessels relative to
the epidermis than the customary KTP laser.
We have measured laser-induced temperature depth profiles in vivo using
pulsed photothermal radiometry. A PWS lesion on a volunteer patient was
irradiated with a single laser pulse at a sub-therapeutic radiant exposure.
The transient change of IR emission was recorded by an InSb camera at a
rate of 1000 fps, and the initial temperature profile reconstructed using a
custom iterative algorithm. The results indicate that extending the pulse
European Conferences on Biomedical Optics 2007 •
duration from 1 ms to 20-25 ms and increasing the radiant exposure (from 12 to 3-4 J/cm2) resulted in a more favorable ratio of dermal vs. epidermal
heating in both dual-wavelength and customary KTP laser. Significant heating
deeper than 1 mm was observed with the dual-wavelength irradiation in one
lesion, but also with PDL in another one, suggesting that gross interaction
features are highly lesion-specific.
We have also irradiated one PWS lesion and one healthy skin site with the
dual-wavelength laser at increasingly higher radiant exposures (1-5 J/cm2).
Reconstruction and analysis of the resulting temperature profiles is under
way. We hope it will reveal whether the hypothesized conversion of
hemoglobin to met-hemoglobin, with the associated increase in NIR
absorption, has taken place in the target blood vessels.
6632-11, Session 3
A novel 3D modeling and simulation technique in
thermotherapy predictive analysis on biological
tissue
F. Fanjul-Vélez, J. L. Arce-Diego, Univ. de Cantabria (Spain); O. G.
Romanov, A. L. Tolstik, Belarusian State Univ. (Belarus)
Optical techniques applied to biological tissue allow the development of new
tools in medical praxis for particular diseases, either in tissue characterization
or treatment. Belonging to the last case, we could mention Photodynamic
Therapy (PT) or Low Intensity Laser Treatment (LILT), and also a promising
technique called thermotherapy, that tries to control temperature increase in
a pathological tissue in order to reduce or even eliminate disease effects.
The application of thermotherapy requires a previous analysis in order to
avoid collateral damage to the patient, and also to choose the appropriate
optical source parameters.
There are different implementations of complex opto-thermal models, that
simulate laser-tissue interaction. Here the one used takes into account a
RTT (Radiation Transport Theory) model solved via a numerical Monte Carlo
method for the optical part, and a bio-heat equation, that models heat
transference, with conduction, convection, radiation, blood perfusion and
vaporization depending on the specific problem, solved via a numerical
spatial-temporal explicit finite difference approach, for the thermal part. From
temperature data in tissue, thermal damage can be studied, based on an
Arrhenius analysis, as a way of predicting harmful effects. The usual drawback
of the numerical method of the thermal model is that convergence constraints
make spatial and temporal steps very small, with the natural consequence of
slow processing. In this work, a new algorithm implementation is used for
the bio-heat equation solution, in such a way that the simulation time
decreases considerably. Such a speed improvement allow a quicker
thermotherapy prediction and so the consideration of several sources can
be taken in a reasonable time period. The complete model can be used for
concrete treatment proposals, as a way of predicting treatment effects and
consequently decide which optical source parameters are appropriate for
the specific disease, mainly wavelength and optical power, with reasonable
security margins in the process, and in a appreciably minor computational
time.
6632-13, Session 3
Space-time modeling of the photon diffusion in a
three-layered model: application to the study of
muscular oxygenation
C. Mansouri, Groupe ISAIP-ESAIP (France); J. L’huillier, Ecole Nationale
Supérieure d’Arts et Métiers (France); A. Humeau, Groupe ISAIP-ESAIP
(France)
The application of the lasers in the medical field provides new non-invasive
techniques supporting the diagnosis of major tissue structures. In this context,
the combined action of the absorption and diffusion coefficients of tissues
modulates the penetration of the radiation in the structures to be explored.
In addition it was shown that radiation wavelengths spread out between the
limits fixed by the interval of the therapeutic window (0.6 µm-1 µm), allow
access to structures located at depths of several centimeters of the explored
medium.
Among the various techniques implemented and aiming at apprehending
the optical parameters, the temporal method offers certain assets related to
the simplicity of extraction of the latter.
This work presents results on the modelling of the photons diffusion in a
model with 3 layers: the skin (thickness 1mm), the fat (thickness within 2 to
15 mm) and the muscle. The Finite Element method (FEM) was performed in
order to calculate the temporal response of the above mentioned structure,
with a luminous impulse of 1 ps.
For a distance between the source and the receiver (located on the surface)
fixed at 30 mm, various simulations reveal that the decreasing part of the
temporal response contains the information correlated with the absorption
coefficient of the third layer (muscle).
It is shown, in addition, that according to this configuration, it is possible to
recover with a good precision this coefficient and this until a thickness of the
layer of fat not exceeding 5 mm. Beyond this limit a correction is proposed in
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67
Conf. 6632: Therapeutic Laser Applications and Laser-Tissue Interactions
order to make measurements coherent. The field of application of this method
could be extended to other more complex models like the brain for example.
6632-14, Session 3
S. Firdous, Sr., Pakistan Institute of Engineering and Applied Sciences
(Pakistan)
We demonstrate significant differences in the propagation of polarized laser
light through biological tissue phantom. The Stokes vectors along with degree
of linearly and circularly polarized light were measured with stokes polarimetry
techniques. The measurements were performed on dense and diluted tissue
phantoms that consisted of soybean oil interloped. Liquid crystal variable
retarder (LCVR) Stokes polarimeter is used for either rotating the major axis
of elliptically polarized light or for converting an input linearly polarized beam
into an arbitrary elliptically polarized beam. This system makes possible a
direct measurement of a component of the Stokes vector with phase change
detection of polarization modulation for polarimetric measurements of turbid
media and biological tissue.
6632-15, Session 4
Oxygen consumption in photodynamic
inactivation of bacteria: the role of singlet
oxygen
T. Maisch, J. Baier, B. Franz, R. Szeimies, M. Landthaler, W. Bäumler,
Univ. Regensburg (Germany)
To optimize photodynamic inactivation of bacteria, it is a major goal to
understand the generation and decay of singlet oxygen in bacteria. Singlet
oxygen can be visualized by measuring its luminescence at 1270 nm directly
in solvents and in living cells. Experiments were carried out with S. aureus
and E. coli as representative Gram-positive, Gram-negative bacteria species
and Photofrin a clinical approved photosensitizer. At low S. aureus
concentrations, time resolved luminescence of singlet oxygen showed a decay
time of 6±2µs, which is an intermediate time of singlet oxygen decaying in
phospholipids (14±2µs) of membranes and in the surrounding water
(3.5±0.5µs). Adding the quencher Sodium azide, the luminescence decay
time was shortened (3±1µs). Singlet oxygen had sufficient access to water
outside of S. aureus by diffusion at low bacteria concentration. Thus, singlet
oxygen seems to be generated in the outer cell wall areas or adjacent
cytoplasm membranes of S. aureus. In contrast, no singlet oxygen
luminescence was detected in E. coli. At higher concentrations of S. aureus,
the decay time significantly increased up to 40µs due to oxygen depletion at
high bacteria density. There is remarkable oxygen consumption at high
bacteria density due to the generation of singlet oxygen in S. aureus leading
to a decrease of oxygen concentration in the bacteria. This observation is
important since oxygen supply is a crucial factor in the efficacy of
photodynamic inactivation of bacteria, in particular when using this new
approach against multi-resistant bacteria growing as biofilms or agglomerates.
6632-16, Session 4
Photodynamic therapy combined with an
antiseptic for treatment of local infections
H. C. Diddens, Univ. zu Lübeck (Germany)
Microbial resistance to antibiotics is currently the most serious problem in
the treatment of bacterial and fungal infections. Furthermore, side-effects
caused by systemic application of antibiotics and by local application of skin
disinfectants also necessitate the development of alternative antimicrobial
strategies. We pursue a novel approach for selective destruction of
microorganisms by combining the application of a local wound antiseptic
Octenisept with antimicrobial photodynamic therapy (PDT) using the
photosensitizer toluidine blue O, which involves the killing of target cells by
light in the presence of a photosensitizing agent and oxygen. While the
antiseptic offers good results in a broad spectrum of indications, its
applicability is limited by cytotoxicity which may result in retarded wound
healing.
We found that the combination of PDT with Octenisept results in prominent
synergistic effects leading to highly efficient killing of native and antibioticresistant pathogenic bacteria. The interaction between PDT and Octenisept
is much less pronounced with respect to cytotoxic damage of human skin
fibroblasts, thus indicating good selectivity for microorganisms vs. host tissue.
TBO-PDT plus Octenisept may have potential for decontamination of
extensive wounds and large areas of damaged tissue, like burns. Due to the
mechanisms involved in PDT, the emergence of resistance is unlikely to
develop. The mode of action of PDT is independent of acquired antibiotic
resistance mechanisms, thus, this concept may be very useful in treatment
of lesions contaminated with antibiotic-resistant strains and may be a valuable
alternative to antibiotic therapy.
European Conferences on Biomedical Optics 2007 •
Investigations on the laser light induced
decomposition of indocyanine green (IGC)
W. Bäumler, E. Engel, R. Schraml, R. Vasold, Univ. Regensburg
(Germany)
Laser stokes polarimetry for the
characterization of bio-materials using liquid
crystal variable retarders
68
6632-17, Session 4
Indocyanine green (ICG) is widely applied for diagnostic reasons. Moreover,
it has been shown that photoactivated ICG kill cells involving the generation
of singlet oxygen. This was confirmed indirectly with quenchers like sodium
azide, which suggests a photodynamic reaction of type II. However, a direct
proof of singlet oxygen by its luminescence at 1270 nm had failed.
Therefore, the interaction of ICG with light was investigated in more detail.
ICG in solution was irradiated with a diode laser at 810 nm and analyzed by
HPLC DAD technology using also LC/MS online coupling. The results show,
that the decomposition by laser irradiation is induced by singlet oxygen
(photodynamic type II reaction). Within this self-sensitized photo oxidation
dioxetanes are generated by 2+2 cycloaddition of singlet oxygen. These
dioxetanes thermally decompose into different carbonyl compounds. This
can be proved by the mass of the laser-induced products of ICG, as well as
by the inhibition of the decomposition of ICG when adding sodium azide a
quencher of singlet oxygen.
After that, ICG was irradiated with a diode laser at 810 nm to generate these
carbonyl compounds yielding the typical yellow colour of the solution. When
incubating colonic cancer cells with this solution for two hours, the
mitochondrial activity of the cells decreased by about 33 % as compared to
untreated control cells. Obviously, these decomposition products of ICG
damaged the cells. Thus, the incubation of cells with ICG and a subsequent
irradiation mimic a photodynamic effect of ICG.
6632-18, Session 4
Frequency domain, time-resolved, and
spectroscopic investigations of photosensitizers
encapsulated in liposomal phantoms
O. Mermut, J. Bouchard, J. Cormier, I. Noiseux, M. L. Vernon, Institut
National d’Optique (Canada); K. R. Diamond, M. S. Patterson,
McMaster Univ. (Canada)
A broadband frequency domain fluorescence lifetime system (from ns to ms
time scale) has been developed to study the photochemical and
photodynamic behavior of model, well-controlled photosensitizerencapsulating liposomes. These liposomal phantoms are efficient and
selective photosensitizer drug delivery vesicles, although their effects on the
photochemical properties of the photosensitizer are not well characterized.
The physical and chemical properties of liposomes can be highly tailored,
making them suitable tissue-like model systems. The liposomes employed
in this study (both blank and photosensitizer-containing) were characterized
using dynamic light scattering, scanning electron microscope, optical
microscopy, and spectrofluorometry. The fluorescence decay of the
encapsulated photosensitizer, either a metallophthalocyanine tetrasulfonate
or 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide (HPPH), has been
examined as a function of the liposome’s physical properties, such as sizescale (0.1µm to 1 µm), size distribution, degree of lamellarity, concentration
and the photosensitizer spatial confinement. The ionic strength of the solution,
and the chemical properties of the liposome, and photosensitizer were varied
to study these effects on fluorescence decay of the capsulated
photosensitizer. The emission decay of PDT-encapsulated liposomes in
deoxygenated environments, relevant to pathway I phototoxicity, was also
probed in the frequency-domain. Fluorescence lifetime measurements were
performed using the broadband frequency-domain instrument as well as a
time-domain system for comparison and confirmation. Additional
measurements on the model photosensitizers in solu were obtained to verify
the consistency of the two systems. To examine spectral shifts related to
the photosensitizer encapsulation and confinement, or the formation of
photosensitizer aggregates within the liposomes, spectrophotometric
measurements were also acquired.
6632-19, Session 5
Photodynamic therapy of non melanoma skin
cancer murine model by topical application of a
novel mTHPC liposomal formulation
E. Alexandratou, M. Kyriazi, National Technical Univ. of Athens (Greece);
T. A. Trebst, CeramOptec GmbH (Germany); S. Gräfe, biolitec AG
(Germany); D. M. Yova, National Technical Univ. of Athens (Greece)
Photodynamic therapy (PDT) has been used so far in the treatment of various
skin diseases including non melanoma skin carcinomas (NMSC). However,
until now there are no publications concerning the efficacy of PDT after topical
application of m-THPC.
Although topical photosensitizer application presents many advantages over
systemic drug administration, ALA-induced protoporphyrin IX is the only
sensitizer topically used so far.
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Conf. 6632: Therapeutic Laser Applications and Laser-Tissue Interactions
In the present study photodynamic efficacy of the highly potent sensitizer
meso-tetra(hydroxyphenyl)chlorin (mTHPC), supplied in a novel liposome
formulation is investigated after topical application in hairless SKH-HR1 mice,
bearing non melanoma skin carcinomas. The drug was applied topically for
drug- light interval of 4 hours. The fluence rates were 100 and 50 mW/cm2
and two total energy doses, 10 J/cm2 and 100 J/cm2 were studied in groups
of 5 animals. Three PDT sessions were performed in each animal, once every
7 days. The final evaluation of PDT effects was performed 14 days after the
3rd PDT treatment by measuring the geometrical characteristics of tumors.
The groups treated with 100 mW/cm2 presented a higher complete tumor
remission than the group of 50 mW/cm2 but an unusual high mortality. In the
group of 50 mW/cm2 and 100 J/cm2, although the complete tumor remission
percentage is poor, the tumor growth rate was decreased. No lesion,
papilloma, or tumor was observed in the treated area even four month after
tumor remission. Furthermore tumours up to 7 mm were achieved to be
treated, indicating that this novel mTHPC formulation could be used for deeper
and not only superficial carcinomas or lesions.
6632-23, Session 5
Interstitial PDT of glioblastoma with 5-ALA:
clinical studies and method for measurement of
sensitizer concentration
6632-21, Session 5
Photodynamic therapy of bladder cancer: a
phase I study using hexyl-aminolevulinate
M. J. Bader, D. Zaak, Ludwig-Maximilians-Univ. München (Germany);
M. Ehlers, M. Kriegmair, MTC GmbH (Germany); T. Pongratz, W. Beyer,
C. G. Stief, H. G. Stepp, Ludwig-Maximilians-Univ. München (Germany)
Introduction and objectives: The hexyl-derivative of 5-aminolevulinic acid (hALA, Hexvix(r)) has recently gained approval for bladder cancer (BC) detection
by fluorescence imaging. The fluorescent compound Protoporphyrin IX (PPIX)
is intracellularly synthesized upon intravesical instillation of its precursor hALA. As PPIX is also a photosensitizer, irradiation of the bladder wall with
visible light upon sensitization with h-ALA might represent a treatment method
for bladder cancer. The objective of this prospective controlled study was to
assess feasibility and safety of h-ALA together with whole bladder wall
irradiation using white light and a newly developed catheter.
Material and methods: In 14 pts. with known high grade or frequently recurring
superficial BC PDT was applied in general anesthesia in 3 sessions with 6
week intervals. Irradiation was performed with a high-power white light source
via a 1.5 mm diameter quarzfiber with a spherically diffusing tip fixed in a
flexible irrigation catheter. 100 J/cm(c)˜ were applied during approx. 1 hour
irradiation time. Instillation of h-ALA (16 and 8 mM, 50 ml) was performed
2+-1 hour prior to PDT. The bladder volume was adjusted to maintain a
distended bladder wall without folds and controlled in 10 min intervals by
ultrasound. Median irradiation time was 72 minutes (range: 52-100
min).Immediately before and after PDT, a standard fluorescence inspection
of the bladder wall was performed.
Results: Assessment of effectiveness of the PDT was not a primary study
aim, preliminary evaluation shows an initial complete response of all 12 pts.
having finished all three PDT-sessions after the first (9 pts) or second (3 pts)
session. No tumor recurrence was observed in 5 of these patients so far.
PDT was performed without any complications Output power of the fiber as
measured after PDT was 3.4 W (median, range: 1.6 W to 4.6 W). Complete
bleaching of the PPIX-fluorescence could be achieved in all cases as intended.
Most prominent side effects were postoperative urgency and bladder pain,
all symptoms being more severe after 16 mM h-ALA.
Conclusions: White-light PDT with the special flexible catheter system is
technically feasible and safe. Initial data on effectiveness suggest an irradiation
protocol comprising of 2 PDT sessions 6 weeks apart followed by
maintenance sessions in longer intervals.
Future work is directed towards reducing postoperative symptoms and
towards avoiding general anesthesia.
W. Beyer, T. J. Beck, R. Sroka, J. Mehrkens, W. Rachinger, LudwigMaximilians-Univ. München (Germany); W. Stummer, Heinrich-HeineUniv. Dusseldorf (Germany); F. Kreth, R. Baumgartner, H. G. Stepp,
Ludwig-Maximilians-Univ. München (Germany)
Introduction: Due to the disturbed blood brain barrier systemic 5-ALA
application results in a high PPIX contrast between normal and tumor tissue,
making interstitial PDT to a promising approach. In a pilot study therapyrelated side-effects have been investigated and the clinical impact has been
evaluated. For precise dosimetry it would be helpful to know the sensitizer
concentration. A method to measure this concentration absolutely by an
interstitial fiber was developed, by analyzing the conditions for which the
influence of optical tissue properties on the detected fluorescence signal is
minimized.
Materials and Methods: 10 patients with recurrent maligant glioma with a
diameter smaller than 3cm have been treated after oral application of 20mg/
kg 5-ALA. 2 to 6 cylindrical light diffusers have been implanted stereotactically
and a light dose of 720 J/cm with 200 mW/cm at 633 nm was applied.
Expected light and temperature distributions were studied by numerical
simulation. Follow-up MRI was performed at 24h, 4 weeks and then in 3
month intervals post treatment. Fluorescence induced and detected by the
same interstitial fiber has been studied by Monte Carlo simulations and
compared with measurements on tissue phantoms.
Results: Early contrast enhanced MRI showed CR for 70% and PR for 30%
of the patients. While no treatment-induced edema was observed, a transient
treatment-related morbidity occurred for 2 patients. The survival ranged from
4 to 49 months with a mean of 18 months. The fluorescence signal of a
detecting fiber is proportional to the sensitizer concentration with only minor
dependence on the tissue parameters, if the fiber radius r follows 0.1 < µs’ *
r < 3 and the ratio of absorption and effective scattering is µa/µs’ ˜ 0.1, a
typical value for many tissue types.
Conclusion: Due to the encouraging results of the clinical pilot study a phase
I/II study has been initiated based on the same clinical protocol. 8 of 15
patients have been included so far. Well adjusted single fiber fluorescence
detection for determination of absolute sensitizer concentration promises to
be sufficiently quantitative for most applications.
6632-24, Session 5
Spectroscopic monitoring of topically applied
temoporfin for photodynamic therapy
N. Bendsoe, K. Svanberg, S. Andersson-Engels, Lund Univ. Hospital
(Sweden)
6632-22, Session 5
Photodynamic therapy for the treatment of
Crohn’s disease: preclinical and clinical results
L. Favre, D. Vekub, Ctr. Hospitalier Univ. Vaudois (Switzerland); F. Borle,
École Polytechnique Fédérale de Lausanne (Switzerland); D.
Bachmann, Ctr. Hospitalier Univ. Vaudois (Switzerland); T. Gabrecht,
École Polytechnique Fédérale de Lausanne (Switzerland) and Ctr.
Hospitalier Univ. Vaudois (Switzerland); H. Bouzourene, Ctr. Hospitalier
Univ. Vaudois (Switzerland); G. A. Wagnières, H. van den Bergh, École
Polytechnique Fédérale de Lausanne (Switzerland); P. Michetti, M.
Ortner, Ctr. Hospitalier Univ. Vaudois (Switzerland)
Photodynamic therapy (PDT) may modify the mucosal immune response and
thus serve as a therapy for Crohn’s disease (CD). The safety and effect of
“low dose” PDT was studies in a SCID mouse colitis model.
Materials and Methods: Safety: 5-aminolaevulinic acid (5-ALA) was
administered orally in BALB/c mice. Irradiation (635 nm) of the colon was
performed after 3h with 5 J/cm2 or 10 J/cm2. Body weight, overall wellness,
histology, and cytokine expression index (CEI) were measured at 74h post
irradiation. Effect: An endoscopic idex of colitis (EIC) was established. SCID
mice with moderate or marked colitis were randomised in a PDT group and
European Conferences on Biomedical Optics 2007 •
a control group. The PDT mice were irradiation with 10 J/cm2 after oral
administration of 15 mg/kg. EIC, CEI, weight and length of the colon, and
histology were evaluated.
Results: Safety: PDT in the BALB/c mice did not influence body weight, overall
wellness, histology, or CEI. Effect: EIC was improved in mice with moderate
colitis 1 week after PDT. In mice with marked colitis, healing was observed at
3 days after PDT. The EIC correlated with the CEI of IFN-c (R=0.77; p<0.0001),
TNF-a (R=0.62; p=0.0033) and IL-10 (R=0.79; p<0.001). The expression
indices of these cytokines were lower in the PDT group than in the control
group.
Conclusions: Low dose PDT downregulated the pro-inflammatory immune
response and improved colitis in a mouse model. This makes low dose PDT
a promising approach for the treatment of CD. First clinical results are
presented.
Fluorescence monitoring of a topically applied liposomal Temoporfin
formulation and photodynamic therapy of non-pigmented skin malignancies
Niels Bendsoe1, Linda Persson2, Ann Johansson2, Johan Axelsson2, Jenny
Svensson2, Susanna Gräfe3, Tilmann Trebst3, Stefan Andersson-Engels2,
Sune Svanberg2 and Katarina Svanberg*4
1Department of Dermatology, Lund University Hospital, Lund, Sweden,
2Department of Physics, Lund University, Lund, Sweden, 3Research &
Development, Biolitec AG, Jena, Germany, 4Department of Oncology, Lund
University Hospital, Lund, Sweden, *E-mail: Katarina.Svanberg\@onk.lu.se
ABSTRACT Meso-tetra(hydroxyphenyl)chlorin (mTHPC) (INN: Temoporfin) is
a potent photodynamically active substance in clinical use today. Usually
the substance is given systemically and a known drawback with this
administration route is a prolonged skin light sensitisation. For the first time
to our knowledge, a liposomal Temo-porfin gel formulation for topical
application was studied in connection with photodynamic therapy (PDT) of
non-pigmented skin malignancies in humans. Intervals of 4 hours between
drug administration and light irradiation were used. Sensitiser distribution
within tumor and surrounding normal skin was investigated by means of
point-monitoring and imaging fluorescence spectroscopy before, during and
after PDT, showing high tumor selectivity. Furthermore, the bleaching of
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Conf. 6632: Therapeutic Laser Applications and Laser-Tissue Interactions
Temoporfin was studied during the PDT procedure by monitoring the
fluorescence following excitation by using the therapeutic light. A 30-35%
light-induced photo metabolisation was shown. No pain occurred during or
after treatment. It was also observed that the treated area did not show any
swollen tissue or reddening as is often seen in PDT using topical d-aminolevulinic acid. On controlling the patients one week after treatment, healing
progress was observed in several patients and no complications were
registered.
6632-25, Session 6
Temperature control during diode laser welding
in a human cornea
F. Rossi, P. Matteini, R. Pini, Istituto di Fisica Applicata Nello Carrara
(Italy); L. Menabuoni, Azienda USL 4 (Italy)
Low power diode laser welding is a technique used to join biological tissues:
it was proposed in ophthalmic surgery (cataract surgery and penetrating
keratoplasty), in order to induce immediate sealing of clear corneal wounds.
The welding effect is achieved after staining the cut walls with a water solution
of Indocyanine Green and then irradiating it with a low power diode laser
(12.5 W/cm2 \@810 nm). The resulting thermal effect induces structural
modifications in the stromal collagen, followed by a welding effect upon
cooling. In this work we present the first attempt to study temperature
dynamics developing during welding in a human eye. An experimental
measurement session was set up during surgery: an infrared thermocamera
was used to study superficial temperature dynamics, assuring a non-contact
direct detection of thermal effect on the external cornea surface. The
experimental data were used as a starting point for a theoretical investigation
of the temperature rising inside the ocular structures during laser procedure:
we developed a mathematical model based on the bio-heat equation and
solved by the use of the Finite Element Method (FEM). The predictive accuracy
was verified by comparing the temperature post-processing description with
the results obtained from the thermographic data. The model was then used
to study the temperature rise and heat propagation inside the eye.
Experimental results and model analysis were in good agreement, indicating
heat confinement during treatment procedure, a modest temperature
enhancement (about 55°C inside the laser treated wound), thus evidencing
the safety of the procedure in clinical applications.
6632-26, Session 6
Femtosecond laser keratoplasty: reducing side
effects and improving penetration depth
K. Plamann, V. Nuzzo, O. Albert, G. A. Mourou, École Nationale
Supérieure de Techniques Avancées (France); M. Savoldelli, Hôpital
Hôtel Dieu (France); D. Donate, Hôpital Hôtel Dieu (France) and Hôpital
Édouard Herriot (France); J. Legeais, Hôpital Hôtel Dieu (France)
Femtosecond lasers are currently in the process of replacing the mechanical
microkeratome in the laser in situ keratomileusis (LASIK) procedure in
refractive surgery. Current research focuses on the application of these lasers
to glaucoma and cataract surgery as well as corneal transplant procedures.
Some state-of-the art commercial systems already offer pre-programmed
routines for certain types of corneal transplant operations.
While the laser-tissue interaction process may be well controlled at high
numerical apertures and when working on clear tissue, the situation is more
complex in the case of pathological corneas. Light diffusion and aberrations
induced by tissular irregularities broaden the point spread function and may
considerably reduce the effective numerical aperture. This, in turn, not only
reduces the energy density in the depth of the tissue, but also changes the
regime of the laser-tissue interaction: at lower numerical apertures,
filamentation processes are likely to compete with the optical breakdown of
the tissue.
To avoid unwanted tissue damage it is therefore important to maintain a
working energy just above the ablation threshold independently of the local
and global tissue properties. We report on a method to quantify and
compensate for the laser attenuation by measuring the optical emission of
the tissue generated by second harmonic generation in situ. Experiments
were performed on human cornea presenting different degrees of oedema
using a variety of numerical apertures and pulse energies including optimised
energy adapted to the tissular properties. The quality of the incisions was
examined by histology and ultrastructural analysis by transmission electron
microscopy.
6632-27, Session 6
Femtosecond refractive eye surgery: study of
laser parameters for even more efficiency and
safety
R. Le Harzic, Fraunhofer-Institut für Biomedizinische Technik (Germany);
C. Wüllner, Wavelight Laser Technologie AG (Germany); D. Bruneel,
Univ. Jean Monnet Saint-Etienne (France); C. Donitzky, Wavelight Laser
Technologie AG (Germany); K. König, Fraunhofer-Institut für
Biomedizinische Technik (Germany)
70
European Conferences on Biomedical Optics 2007 •
Studies on corneal surgery and flap processing on enucleated porcine eyes
have been performed using a dedicated 100 kHz femtosecond laser source
based on Ytterbium technology. The influence of laser parameters such as
wavelength, energy, repetition rate and numerical aperture has been studied.
Best parameters for ocular femtosecond laser surgery are discussed in terms
of process efficiency and safety aspects. Flaps with a diameter of 6 mm and
150 µm thick have been performed in less than 1 min. The transmittance of
ultraviolet, visible and near infrared femtosecond laser pulses through the
ocular media of porcine eyes has been measured for a collimated beam and
during flap processing using an integrating sphere. Spectral measurements
and have also been performed. More than 25 % of energy is transmitted
through the whole eye at the retina during IR pulses flap processing.
Concerning UV pulses low transmissions can be detected at the retina (less
than 2%). The majority of UV radiations are absorbed by the lens. For IR
pulses visible light generation is detectable. A relative high transmission
towards the retina of visible light centred on 440 nm was found for UV pulses.
To minimize the risks and undesirable effects or post operative problems,
the tendency in femtosecond refractive surgery is to perform flaps faster by
improving the repetition rate of the lasers and in the same time by reducing
the energy per pulse to ensure an even safer procedure
6632-28, Session 6
Retinal temperature determination during laser
photocoagulation
R. Brinkmann, Univ. zu Lübeck (Germany); J. U. Stalljohann, B. Weber,
Medizinisches Laserzentrum Lübeck GmbH (Germany); K. Schlott, J.
Kandulla, R. Birngruber, Univ. zu Lübeck (Germany)
Introduction:
Retinal laser photocoagulation is an established therapy for a variety of retinal
diseases. The extent of the coagulation depends on the temperature increase
and the time of elevated temperature during the irradiation period. The
temperature rise does not only depend on the laser settings but also on the
retinal/choroidal pigmentation and the transmission characteristics of the
radiation through the whole eye, which are both unknown. So far, clinical
dosimetry is performed post irradiation by estimating appropriate powertime settings for the next spot in order to achieve whitish lesions. Due to
intraocular changes in pigmentation and transmission, often too large burns
are produced, which can lead to extended scotoma and bleedings in the
worst case. This work investigates a non-invasive technique for an online
temperature monitoring during photocoagulation. Far aim of the research is
an automatic on-line dosimetry system by means of regulating the treatment
laser onto minimal invasive and well defined coagulations.
Methods:
Optoacoustic techniques are used to determine the temperature increase
during photocoagulation. Therefore, Q-switched Nd:YLF-laser pulses (527
nm, 200 ns, 200 Hz) are applied to excite the emission of pressure waves
from the retina, which are detected with an ultrasonic transducer embedded
in the contact lens. The pressure amplitude can be used to calculate the
temperature. The ns-pulses are transmitted to the eye via the same slitlamp
and fiber as the treatment laser radiation (cw Nd:YAG-laser, 532 nm).
Experiments are performed on enucleated pig globes.
Results:
Irradiation with a constant power of 175 mW onto 400 µm spots of medium
pigmented eyes lead to a temperature rise of 28 K after 500 ms with an
approximately logarithmic temperature rise over time as expected from the
heat diffusion theory. Applying pulses of 200 ms with different powers, we
found a temperature rise of 0.13 K/mW at maximum at the end of the
irradiation period. At the onset of retinal denaturation, a change in the pressure
transients is observed. From the measured temperature/time history at
threshold, the Arrhenius constants for retinal tissue denaturation can be
calculated.
Conclusions:
These first retinal temperature measurements demonstrate the possibility of
a non-invasive real time monitoring of laser photocoagulation. The data
achieved show very promising towards realization of an automatic dosimetry
control almost independent of the individual eye transmission and
pigmentation.
6632-29, Session 6
Interferometric optical online dosimetry for
selective retina treatment (SRT)
H. Stoehr, L. Ptaszynski, A. Fritz, R. Brinkmann, Univ. zu Lübeck
(Germany)
The selective retina treatment (SRT) is a new laser based method to treat eye
diseases associated with disorders of the retinal pigment epithelium (RPE).
This most likely accounts for retinal diseases as e.g. early stages of age
related macular degeneration (AMD) and diabetic maculopathy (DMP), which
belong to the most common origins of blindness. Selective RPE cell damage
is achieved by applying a train of 1.7µs laser pulses at 527nm, which cause
heat-induced transient microbubbles at the strongly absorbing RPE
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Conf. 6632: Therapeutic Laser Applications and Laser-Tissue Interactions
melanosomes. Cell membrane disruption caused by the associated volume
increase is expected to be the origin of the angiographically observed RPE
leakage.
Due to the high variability of the optical absorption of RPE cells and the
unvisibility of the effects at the slitlamp, a real-time dosimetry control is
required to ensure the selective treatment effect. For this purpose we
investigate the detection of transient microbbubles by optical interferometry.
The signal transients contain informations about the bubble lifetimes and
surface velocities. Previous experiments with single pulse irradiation of porcine
RPE cells in vitro have demonstrated the suitability of the observed MHz
interferometric transients for microbubble detection and SRT real-time
dosimetry. Currently we are developing a dosimetry system for clinical
application based on a fiber interferometer operated at 830nm. We present
first results with this system on porcine RPE explants in vitro, complete porcine
eye globes ex vivo and rabbits in vivo. We examine suitable threshold criteria
for cell death and possible data analyzing schemes of the interferometer
transients for SRT online dosimetry.
6632-57, Poster Session
Mechanisms in photodynamic therapy:
photosensitizers and cellular localization on
K562 cells
R. Ion, Institutul National de Cercetare (Romania) and Valahia Univ.
(Romania); M. Neagu, G. Manda, C. Constantin, Victor Babes National
Institute (Romania); M. Calin, National Institute of R&D for Optoelectronics (Romania)
The use of non-toxic dyes or photosensitizers (PS) in combination with visible
light that is known as photodynamic therapy (PDT) has been known for over
a hundred years, but is only now becoming widely used. The most important
factor governing the outcome of PDT is how the PS interacts with cells in the
target tissue or tumor, and the key aspect of this interaction is the subcellular
localization of the PS. This paper was designed to explore the pattern of
lymphoblastic cell line K562 cells death, the effects on their cell cycle induced
by 5,10,15,20-tetra-p-sulphonato-phenyl-porphyrin-based photodynamic
therapy (TSPP-PDT). Flow cytometry combined with Annexin V-FITC/PI
labeling was used to detect the pattern of K562 cells’ death induced by
TSPP-PDT. These effects frequently lead to induction of apoptosis by the
mitochondrial pathway involving caspases. The transmission electron
microscope (TEM) and confocal laser scanning microscopy (CLSM) were
used to detect the localization and time-biodistribution of sensitizers in the
cells. After 1 h of TS4PP administration, the sensitizer shows an non-uniform
distribution, following that after 4h of administration, the sensitizer to be
localized in some cellular targets and an increased fluorescence intensity is
being detected. After 8 h and 24 h post-administration, the sensitizer is
released from the cells and the light-irradiation (He-Ne laser, l=632,8 nm)
could start. Immediately after irradiation, many typical apoptotic bodies were
seen in the cells treated. Most of the cells treated were necrotic at 24 hours
following irradiation.
6632-58, Poster Session
Photodynamic therapy as a method of choice in
the treatment of multifocal oral leukoplakia
A. Z. Kawczyk-Krupka, W. Latos, A. Kosciarz-Grzesiok, A. Misiak, A. E.
Ledwon, S. Kwiatek, A. Sieron, Medical Univ. of Silesia, Katowice
(Poland)
In our study we assess the effectiveness and possibilities of Photodynamic
Therapy in the treatment of multifocal oral leukoplakia basing it on a case
description of patient treated in our Center for a histopathologically
confirmed glossal leukoplakia. The changes for the last 12 months were
accompanied by burning sensation and glossalgia . At the admission,
autofluorescence was carried out ( Xillix Onco LIFE System) - it revealed
pathological autofluorescence (maximum Numerical Color Value: 1,62) . 1,5
hour after application of 20% delta-aminolevulenic acid , the changes were
irradiated in local anaesthesia with the PDT Diomed Laser. Three PDT
sessions were performed with the total dose of 120J/cm(c)˜ each, in three
weeks’ intervals. During the therapy no complications were observed except
from mild pain after first 2 procedures . After third session, the changes’
regression was seen macroscopically and in the white light diagnostics.
The patient reported subsidence of the symptoms.
On the ground of our Center’s experience in the treatment of oral leukoplakia,
we may say that PDT is a method of choice especially in the therapy of
multifocal changes that could not be as successfully treated by other
methods. The superiority of PDT bases on such features as the lack of
scarring, good cosmetic and functional effects, precise localization of
destroyed epithelium, out-patient mode of treatment in local anaesthesia,
noninvasiveness, safety, being well tolerated by patients. Additionally, it should
be underlined that PDT allows to treat multifocal lesions during one
session with narrow range of possible complications
European Conferences on Biomedical Optics 2007 •
6632-59, Poster Session
Real-time evaluation of tissue properties for
feed-back dosimetry in interstitial photodynamic
therapy
J. Axelsson, A. Johansson, Lunds Tekniska Högskola (Sweden); J.
Swartling, T. Johansson, Spectracure AB (Sweden); S. Pålsson, Lunds
Univ. (Sweden); J. Stensson, Spectracure AB (Sweden); K. Svanberg, N.
Bendsoe, Lund Univ. Hospital (Sweden); S. Svanberg, S. AnderssonEngels, Lunds Tekniska Högskola (Sweden)
Prostate cancer treatment utilizing photodynamic therapy (PDT) has been
reported to induce tissue necrosis and decrease in prostate specific antigen.
The treatment response show large variations possibly due to biological
variations. Our group is developing an instrument for interstitial PDT capable
of delivering light into the prostate. The system utilizes real-time treatment
feedback which relies on light transmission measurements conducted during
the treatment session. The prostate geometry is imaged using ultrasound
which renders a three-dimensional representation of the target volume. The
optical fibers are then positioned using a iterative random-search algorithm
to ascertain that the whole prostate can be treated. Before the treatment
starts an optimization algorithm is run to predict individual fiber irradiation
times. During the treatment the light irradiation halts during predefined timeintervals and the light transmission measurements are performed. The system
can measure the treatment light transmission, nir-light transmission and
photosensitizer fluorescence. The measurements are then used to assess
the effective attenuation coefficient, by means of spatially resolved
spectroscopy, for the treatment light which forms the input to the optimization
algorithm. Hence, the irradiation times for individual fibers are updated
throughout the treatment in order to compensate for the influence of changes
in tissue composition on the light distribution at the therapeutic wavelength.
6632-60, Poster Session
Antimicrobial activity of water-soluble cationic
porphyrins
G. V. Gyulkhandanyan, Institute of Biotechnology (Armenia); R. K.
Ghazaryan, Yerevan State Medical Univ. (Armenia); A. Hovsepyan, M.
Paronyan, S. S. Ghambaryan, Institute of Biotechnology (Armenia); A.
G. Tovmasyan, Yerevan State Medical Univ. (Armenia); A. G.
Gyulkhandanyan, Yerevan State Univ. (Armenia)
Nowadays the obtaining of new preparations showing high efficiency against
pathogenic microorganisms is an important and actual problem [1].
Photodynamic antimicrobial chemotherapy utilizes photosensitizers and light
to give a phototoxic response via oxidative damage [2, 3]. In the present
work the efficiency of 3 water-soluble cationic porphyrins (meso-tetra[4-N(2?-oxyethylpyridyl)]-porphine and its Zn, Ag metallocomplexes) against
various strains of Gram (+) and Gram (-) microorganisms and also fungi has
been investigated. It has been shown, that Ag-containing porphyrin showed
higher toxicity on Gram (-) microorganisms (E. coli, Salmonella sp.) and fungi
(Candida albicans), than Zn-containing and metal-free porphyrins. At the same
time metal-free porphyrin showed higher toxicity in comparison with
metalloporphyrins on Gram (+) microorganisms (Stphylococcus aureus and
Stphylococcus epidermidis). Also it has been shown, that Zn-containing and
metal-free porphyrins demonstrated significantly higher phototoxic influence,
than Ag-containing porphyrin. The results show high antimicrobial efficiency
and perspectivity of investigated porphyrins.
1. Neu HC. The crisis in antibiotic resistance. Science (1992) 257: 10641073.
2. Jori G. Photodynamic therapy of microbial infections: state of the art and
perspectives. Journal of Environmental Pathology, Toxicology, and Oncology
(2006) 25: 505-519.
3. Wainwright M. Photodynamic antimicrobial chemotherapy (PACT). Journal
of Antimicrobial Chemotherapy (1998) 42: 13-28.
6632-61, Poster Session
Synthesis and anticancer activity of new watersoluble cationic (metallo)porphyrins
A. G. Tovmasyan, R. K. Ghazaryan, L. Sahakyan, Yerevan State Medical
Univ. (Armenia); G. Gasparyan, N. Babayan, Yerevan State Univ.
(Armenia); G. V. Gyulkhandanyan, Institute of Biotechnology (Armenia)
Now a wide-range of research is directed to application of photodynamic
activity of a variety of new (metallo)porphyrins against harmful microorganisms
[Stojiljkovic I. et al., 2001; Reddi E. et al., 2002; Lambrechts S. et al., 2005;
Jori G., 2006], cell malignant growth [Dougherty T.J. et al, 1998; Ohse T. et
al., 2001; Tome J. et al., 2004, Berg K. et al., 2005], human immunodeficiency
virus type 1 [Vzorov A. et al., 2002]. In the present study new, water-soluble
cationic porphyrin (meso-tetra(4-N-allylpyridyl)-porphine [TAll4PyP]) and its
Zn, Ag, Co, Fe metallocomplexes were synthesized as novel
chemotherapeutics. The structures of all synthesized porphyrins were
characterised by the methods of NMR and electronic absorption
spectroscopy. Dark- and photo-toxic influence of the new (metallo)porphyrins
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Conf. 6632: Therapeutic Laser Applications and Laser-Tissue Interactions
were tested in vitro by vital dye (trypan blue) exclusion test. On the base of
obtained data, the investigated (metallo)porphyrins may be arranged by their
toxic influence in the following order: Ag-TAll4PyP\>Co-TAll4PyP\>ZnTAll4PyP\>Fe-TAll4PyP\>TAll4PyP. Investigation of phototoxicity of
synthesized (metallo)porphyrins showed, that free porphyrin and its Zn
metallocomplex have significantly higher photo-influence, then Ag, Fe, Co
metallocomplexes of TAll4PyP. Thus, i) the presence of the central metal atom
in porphyrin ring is responsible for the cytotoxic action of porphyrins and ii)
mechanism of cytotoxic influence of various metalloporphyrins includes not
only the ways PDT does.
6632-62, Poster Session
Aqueous gel as effective delivery system of 5aminolevulinic acid
V. M. Negrimovsky, N. A. Sakharova, Organic Intermediates and Dyes
Institute (Russia); N. I. Kazachkina, A. A. Pankratov, R. I. Yakubovskaya,
P. A. Hertzen Moscow Research Oncological Institute (Russia); E. A.
Lukyanets, G. N. Vorozhtsov, Organic Intermediates and Dyes Institute
(Russia)
The new aqueous gel compositions based on ALA for fluorescent diagnostics
and photodynamic therapy of superficial diseases have been elaborated.
Biodegradable polymer was used as gel-forming component, and some
additives - solubilizer, emulgator etc - were used to improve distribution
uniformity and penetration ability. These new compositions represent colorless
and transparent gels which are long-term stable at the storage temperature
of =5°?.
The effectiveness of aqueous gels as ALA delivery systems is demonstrated.
After topical administration of the gel at the skin with Ehrlich tumor inoculated
subcutaneously, ALA effectively induces the synthesis of PPIX in the skin
and in the tumor. Intensity of ALA-induced PPIX fluorescence grows with
increasing ALA concentration in the gel and time of gel application. The
deepness of ALA penetration in the tumor reaches 5-6 mm after 4 h gel
application.
A distribution of ALA-induced PPIX in mouse tumor tissue after 4 h gel
application depends on ALA concentration in gel. Intensity of PPIX
fluorescence in deep-located part of tumor was substantially higher with gel
containing 20% ALA than with ones containing 10% or 5% ALA.
Photodynamic therapy for the choroidal
neovascularization
M. Budzinskaya, T. Kiseleva, S. Shevchik, V. B. Loschenov II, A.M.
Prokhorov General Physics Institute (Russia); S. G. Kuzmin, G. N.
Vorozhtsov, Organic Intermediates and Dyes Institute (Russia)
Purpose: To report our experience with photodynamic therapy (PDT) with
«Photosens»for patients with choroidal neovascularization (CNV).
Material and methods: 18 patients with subfoveal CNV in age-related macular
degeneration (AMD), 24 patients with subfoveal CNV in pathological myopia
(PM) and 4 patients with subfoveal CNV associated with toxoplasmic
retinochoroiditis were observed. CNV was 100% classic in all study patients.
Standardized protocol refraction, visual acuity testing, ophthalmologic
examinations, biomicroscopy, fluorescein angiography, and ultrasonography
were performed before treatment and 1 month, 3 months, 6 months, and 1
year after treatment; were used to evaluate the results of photodynamic
therapy with «Photosens» (0.02% solution of mixture sulfonated aluminium
phtalocyanine 0.05 mg/kg, intravenously). A diode laser («Biospec», Inc,
Moscow) was used operating in the range of 675 nm. Need for retreatment
was based on fluorescein angiographic evidence of leakage at 3-month followup intervals.
Results: At 3, 6, 9 month 26 (56.5%) patients had significant improvement
in the mean visual acuity. At the end of the 12-month minimal fluorescein
leakage from choroidal neovascularization was seen in 12 (26.1%) patients
and the mean visual acuity was slightly worse than 0.2 which was not
statistically significant as compared with the baseline visual acuity. Patients
with fluorescein leakage from CNV underwent repeated PDT with
«Photosens». 3D-mode ultrasound shown the decreasing thickness of
chorioretinal complex in CNV area.
Conclusions. Photodynamic therapy with «Photosens» can safely reduce the
risk of severe vision loss in patients with predominantly classic subfoveal
choroidal neovascularization secondary to AMD, PM and toxoplasmic
retinochoroiditis.
6632-64, Poster Session
Adjuvant photodynamic therapy (PDT) with
photosensitizer photosens for superficial
bladder cancer
O. Apolikhin, I. Chernyshev, D. Altunin, Ministry of Health (Russia); S. G.
Kuzmin, Organic Intermediates and Dyes Institute (Russia); G. N.
Vorozhtsov, I.M. Sechenov Moscow Medical Academy (Russia)
European Conferences on Biomedical Optics 2007 •
Materials and methods: 14 patients with transional-cell bladder cancer in
stage T1N0M0G2 after transurethral bladder resection were offered adjuvant
treatment with PDT. The patients were informed about possible side effects
of the treatment. The patients with hereditary acute porphyria, skin
photosensitivity, renal or hepatic insufficiency were excluded from the trial.
Adjuvant PDT was performed 1-1.5 months after transurethral bladder
resection for superficial bladder cancer. Prior to PDT conventional and
fluorescent cystoscopy were performed. If necessary, a biopsy was
performed. In the absence of inflammation and after full epitalisation of
postoperative wound a session of therapy was performed. 24 hours prior to
PDT-session photosensitizer Photosens was injected intravenously in the
dose of 0.8 mg per kg of body weight. Prior to PDT local anesthesia of urethra
with lidocain-gel was performed. Cystoscopy was carried out. The cavity of
a bladder was filled up with physiological saline 0.9 %. The volume of a
bladder was fixed in the report (mean volume 150-200 ml). PDT was performed
with laser „Biospek”. During the session the place of standing diffuser and
the volume of a bladder were controlled.
Light dose was 15 J/cm2 per session. The mean time of session was 22
minutes. 12 patients underwent 1 session of PDT, 2 underwent 2 sessions of
PDT. In the subsequent time every 3 months in all the patients control
sonography and both conventional and in blue-violet light cystoscopy was
performed. If necessary biopsy was carried out.
Results:. After 7 months of observation no tumor recidives were observed.
Registered side effects were not life-threatened. 5 patients (35,7%) had pain
or discomfort in suprapubic area, ceasing spontaneously or requiring
administration of analgetics. No systemic side-effects or allergic reactions
were observed.
Conclusion. The method can be used in out-patient practice. Absence of
early recidives shows efficiency of PDT in the treatment of superficial bladder
cancer. Further study is necessary to estimate optimal regimen of PDT. The
further controlling of condition on the patients in this group is required.
6632-65, Poster Session
Results of photodynamic therapy in the
combined treatment of the choroidal metastasis
V. Likhvantseva, E. Osipova, M. Petrenko, O. Merzlyakova, Russian
Academy of Sciences (Russia); S. G. Kuzmin, G. N. Vorozhtsov, Organic
Intermediates and Dyes Institute (Russia)
6632-63, Poster Session
72
Purpose of the research: to estimate effectiveness of PDT with photosensitizer
Photosens as adjuvant therapy of superficial bladder cancer.
Background: Choroidal metastasis (CM) are more and more spreading type
of eye’s neoplasma. The frequency of CM is increasing with prolonging of
cancer patients’ life. And it makes worse the quality of their life because
blindness. Photodynamic therapy (PDT) is very delicate modality, which can
be used for this aim.
Aim: To open the possibility and to determine the efficacy of photodynamic
therapy (PDT) in the treatment of patients with CM.
Material and methods: PDT was performed simultaneously with standard
chemotherapy in 8 oncological patients with CM. We used photosensitizer
Photosens in doses of 0.3 mg/kg and light doses 150 J/cm2 (675 nm). PDT
was performed in the some séances. Its are ranged from 7 to 10. Complete
tumor regression was achieved in 6 cases (75 % response). The high retina
ablation was developed in one case. And in one case effect was not complete:
tumor size reduced from 5 mm to 3 mm of thickness. We didn’t notice any
recurrence for 6-18 months follow-up.
Conclusions: PDT is modality that could to be used in the in the combined
treatment of the CM. PDT was demonstrated a high efficacy (75 %) in the
combined treatment of the CM with tumor control during 6 - 18 month’s of
follow-up.
6632-66, Poster Session
Mid-infrared porcine cornea ablation
measurements and the role of water absorption
E. Spyratou, M. I. Makropoulou-Loukogiannaki, C. Bacharis, A. A.
Serafetinides, National Technical Univ. of Athens (Greece)
The most common cornea application in human ophthalmology, for the last
two decades, is the refractive surgery with the 193-nm excimer laser. However,
a few characteristic problems of the excimer laser (e.g. toxic gases, high
manufacture and maintenance costs) and the incidence of some refractive
errors, (e.g. myopia over or under correction, high order aberrations,
keratectacia, etc.), stimulates further research for alternative laser sources.
The achievement of precise cornea laser reshaping requires the use of laser
wavelengths possessing a small optical penetration depth in cornea that
serves to confine the energy deposition to a small volume. Because
ophthalmic tissues are consist of 25%-90% water, the use of Er:YAG laser is
indicated for ablation due to high water absorption coefficient at 2.94 µm. In
this work, ablation experiments of ex vivo porcine cornea samples were
conducted with a Q-switching Er:YAG laser. In order to investigate the role of
water absorption properties in the laser ablation efficiency, the cornea samples
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were divided in two groups. In the first group, the porcine cornea samples
were directly irradiated by Er:YAG laser and in the second group were first
immersed in a D2O solution for two hours and then were irradiated with the
same conditions. All the cornea ablation rates were measured for fluences
lower than 200mJ/cm2. The surface roughness and the collateral damage
were investigated by scanning electron microscopy and histological analysis
respectively. As the H2O and D2O mid-infrared optical properties differ, the
cornea ablation efficiency (both quantitative and qualitative results) differs
accordingly.
ACKNOWLEDGEMENTS: The project described in this article is co-funded
by the European Social Fund (75%) and National Resources (25%) Operational Program for Educational and Vocational Training II (EPEAEK II)
and particularly the Program Pythagoras II (Project: “Laser beam and
ophthalmic tissue interactions - correlation with the physical parameters of
the radiation”).
6632-67, Poster Session
Optoacoustic online temperature determination
during retinal laser photocoagulation
K. Schlott, Univ. zu Lübeck (Germany) and Medizinisches Laserzentrum
Lübeck GmbH (Germany); J. U. Stalljohann, B. Weber, Medizinisches
Laserzentrum Lübeck GmbH (Germany); J. Kandulla, K. Hermann, R.
Birngruber, R. Brinkmann, Univ. zu Lübeck (Germany)
Introduction:
The retinal photocoagulation is an established treatment of different diseases
at the fundus of the eye. The extent of the coagulation is besides other
parameters mostly dependent on the effected temperature. So far the laser
dosage is based on empirical values as well as visible whitish lesions on the
retina post irradiation. In this work an optoacoustic technique is presented,
which allows a temperature monitoring during photocoagulation. Aim of the
work is to probe the temperature with two different laser wavelengths.
Methods:
The photocoagulation is performed on enucleated pig globes with a Nd:YAG
laser at 532 nm/cw. The temperature is determinated by analysing the pressure
waves following absorption and thermal expansion during irradiation with a
pulsed Nd:YLF laser (527 nm/200 ns) and a Nd:YAG-laser (1064 nm, 1 ns)
with a pulse repetition rate of 200 Hz.
The waves are detected by an ultrasonic transducer, which is embedded in
an ophthalmological contact lens.
Results:
The temperature rise for a treatment time of 200 ms, a spot diameter of 400
µm and different powers was found to be approximately 0,13 °C/mW using
527 nm and 0,18 °C/mW using 1064 nm. The maximum temperature value is
proportional to the power of the treatment laser. During the onset of retinal
denaturising a change in the transients was found for both probe wavelengths.
Conclusion:
The presented method is suitable for the determination of the temperature at
the fundus during retinal photocoagulation.
Due to the different spectral absorbtion, the results achieved by the green
wavelength represent the temperature at the retinal pigment epithelium and
those archieved by the IR-wavelength the temperature at the choroidea.
6632-68, Poster Session
Dynamics and detection of laser induced
microbubbles in the retinal pigment epithelium
(RPE)
A. Fritz, L. Ptaszynski, H. Stoehr, R. Brinkmann, Univ. zu Lübeck
(Germany)
Transient microbubbles in the retinal pigment epithelium (RPE) are expected
to be the origin of cell damage due to irradiation with short laser pulses. The
bubbles emerge at the strongly absorbing RPE melanosomes. Cell membrane
disruption caused by the associated volume increase is expected to be the
origin of the angiographically observed RPE leakage.
This is utilized by the selective retina treatment (SRT) which is a new method
to treat eye diseases associated with disorders of the RPE. Selective RPE
cell damage is achieved by applying a train of 1.7 µs laser pulses at 527 nm.
The treatment of retinal diseases as e.g. age related macular degeneration
(AMD) and diabetic maculopathy (DMP), which belong to the most common
origins of blindness, is currently investigated within clinical studies.
Previous research about RPE microbubble dynamics has shown that shorter
pulses could be used for SRT. Therefore we investigate microbubble formation
and dynamics in porcine RPE using irradiation pulse durations of 10 ns and
150 ns.
We use laser interferometry at 830 nm for the microbubble detection and
lifetime measurement.
Additionally high speed digital imaging with nanosecond flashes is used.
Beside interferometry we investigate alternative optical microbubble detection
techniques, e.g. utilizing reflected light of the irradiation pulse.
European Conferences on Biomedical Optics 2007 •
6632-69, Poster Session
Influence of choroidal perfusion on retinal
temperature increase during retinal laser
treatments
K. Herrmann, Univ. zu Lübeck (Germany); C. Flöhr, Univ. Eye Hospital
(Germany); J. U. Stalljohann, Medizinisches Laserzentrum Lübeck
GmbH (Germany); G. Apiou-Sbirlea, Air Liquide (France); J. Kandulla,
Univ. zu Lübeck (Germany); R. Birngruber, R. Brinkmann, Medizinisches
Laserzentrum Lübeck GmbH (Germany)
Abstract:
Purpose: In most retinal laser treatments the therapeutic effect is initiated by
a transient temperature increase at and around the retinal pigment epithelium
(RPE). The temperature increase depends on many individual parameters.
Especially in long exposure time treatments like Transpupillary Thermotherapy
(TTT) choroidal perfusion has a strong influence on the realised temperature
at the fundus. The fundus blood circulation and therefore the heat dissipation
is influenced by the intraocular pressure (IOP). The poster presents the
behaviour of temperature increases generated by IOP changes during invivo retinal cw laser treatment in rabbit eyes.
Methods: To reduce the choroidal perfusion, the IOP is increased by injection
of physiological saline solution into the eye of anaesthetised rabbits.
Radiation (irradiation = 3.64 W/cm(c)˜) of a TTT-laser (? = 810 nm) is applied
for t = 60 s causing a retinal temperature increase. Realtime temperature
determination of the irradiated spot is achieved by a non-invasive
optoacoustic technique.
Results: In earlier comparative experiments between pre (full perfusion) and
post mortem (no perfusion) measurements, the fundus temperature increases
up to 114 %.
By alteration of the IOP we achieved temperature data in between those two
statuses. Further experiments are in progress.
Conclusion: Previous results have shown that perfusion has a strong influence
on temperature increase during retinal laser treatments.
Due to the fact, that the intraocular pressure is individual from human to
human, the need of an online temperature determination during long time
retinal laser treatments seems necessary.
6632-70, Poster Session
Optical interferometric online dosimetry system
for selective retina treatment (SRT)
L. Ptaszynski, A. Fritz, H. Stoehr, R. Brinkmann, Univ. zu Lübeck
(Germany)
The selective retina treatment (SRT) is a new laser based method to treat eye
diseases associated with disorders of the retinal pigment epithelium (RPE).
This most likely accounts for retinal diseases as e.g. early stages of age
related macular degeneration (AMD) and diabetic maculopathy (DMP), which
belong to the most common origins of blindness. Selective RPE cell damage
is achieved by applying a train of 1.7µs laser pulses at 527nm, which cause
heat-induced transient microbubbles at the strongly absorbing RPE
melanosomes. Cell membrane disruption caused by the associated volume
increase is expected to be the origin of the angiographically observed RPE
leakage.
Due to the high variability of the optical absorption of RPE cells and the
unvisibility of the effects at the slitlamp, a real-time dosimetry control is
required to ensure the selective treatment effect. It has been shown that for
this purpose the detection of the transient microbubbles by optical
interferometry is suitable and the threshold for RPE cell damage can be
determined.
We develop a dosimetry system for clinical application which is based on a
fiber interferometer and operated with a laser diode at 830nm. We describe
the optical system, the slitlamp adaptation and the data acquisition and
analysis of the MHz signal transients. Also we discuss noise limitations of
the system. First measurement results obtained with complete porcine eyes
ex vivo are shown.
6632-72, Poster Session
Cationic colloidal gold assisting delivery of
macromolecular fluoresceins into target CHOK1 cells by focused femtosecond laser
Z. Li, Z. Zhang, Xi’an Jiaotong Univ. (China); G. Hüttmann, Univ. zu
Lübeck (Germany)
We describe a new method for delivering macromolecules into the target
cells based on light-absorbing cationic colloidal gold nanoparticles that are
irradiated by focused femtosecond laser pulses. Cationic colloidal 15nm gold
particles which were made by conjugation with poly-L-Lysine, were attached
on the anionic sites, especially on the membrane, of CHO-K1 cells because
of their strong positive charge at physiological pH. Target cells labeled with
cationic gold nanoparticles were imaged under two-photon fluorescence
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Conf. 6632: Therapeutic Laser Applications and Laser-Tissue Interactions
microscopy, and lifetime images of the same targets were taken by TCSPC
technique in order to verify the fluorescence of the marker and the
luminescence of the gold particles.
A macromolecular 10k Dalton fluorescein isothiocyanate dextran (FITC-D),
was added into the sample and the focused femtosecond laser of two-photon
fluorescence microscopy was employed to scan the target cells layer by
layer. Typical laser power level used in biological imaging is about 3-5 mW.
Here the laser power of scanning was below 5 mW in order to prevent
photochemical damage of the fs-pulses alone and to localize effects to the
nanoparticles on a nano-scale. After scanning the target cells under stack
mode, macromolecular fluoresceins surrounding the cells was observed to
cross the membrane and to diffuse in the cytoplasma. Comparing with the
images before scanning, the two-photon fluorescence and fluorescence
lifetime images revealed the delivery of FITC-D into target cells.
6632-30, Session 7
Ophthalmic drug delivery utilizing two-photon
absorption: a novel approach to treat posterior
capsule opacification
H. Kim, J. K. Träger, M. Zorn, N. Haberkorn, N. Hampp, Philipps-Univ.
Marburg (Germany)
Intraocular lens (IOL) implantation is the standard technique to treat cataract.
Despite recent progress in surgical procedures, posterior capsule opacification
is one of the sill remaining postoperative complications of cataract surgery.
We present a novel strategy to reduce the incidence of posterior capsule
opacification. A drug delivery polymer suitable for manufacturing intraocular
lenses has been developed which enables repeated drug release in a noninvasive and controlled manner. The therapeutic molecules are attached
through a UV light sensitive linkage to the polymer backbone which is mainly
responsible for the optical properties of the intraocular lenses. However, UV
light can not trigger the release of drug from the polymer due to the high
absorption of the cornea. We developed linkers which enable drug release
by two-photon absorption induced cleavage of the linker structure. Since
the two-photon absorption requires high photon densities, this does not occur
in any lighting conditions in daily life, but is easily triggered by focused laser
beams from a pulsed laser. In this proof-of-principle study we have employed
a cyclobutane-type linker and investigated the properties of the therapeutic
system with approved drugs, 5-fluorouracil and chlorambucil. The controlled
drug delivery was successfully demonstrated in vitro and additional cell tests
confirmed that the device itself shows no cytotoxicity until photochemical
stimulation. This presented concept can provide a powerful method in
ophthalmic drug delivery.
6632-31, Session 7
Materials for intraocular lenses enabaling photocontrolled tuning of focal length in vivo
J. K. Träger, H. Kim, Philipps-Univ. Marburg (Germany); N. Hampp,
Philipps-Univ. Marburg (Germany) and University of Marburg (Germany)
Typical postoperative complication in cataract surgery is that refractive power
and curvature of the implanted intraocular lens (IOL) do not have optimum
values for the patient which requires to wear viewing aids. This is mainly
because biometric data relevant for calculation of the IOL’s shape cannot be
determined with the required precision. Hence, there is a need for methods
to tune the focal length postoperatively in a non-invasive manner.
We have developed polymers where we can induce a change in refractive
index by linking or cleaving bonds between a sufficiently large number of
side groups of the polymer main chain in a photoinduced cyloaddition or
cycloreversion reaction, respectively. These photoreactions lead to a change
in refractive index great enough to be interesting for the concept of in vivo
tunable IOL’s. The photochemical reaction can be triggered by a two-photon
process (TPA) using a pulsed laser system, i.e. the energy required for bond
breaking is provided by two photons in the visible range. This is important
because light in the UV cannot induce undesired changes of the refractive
index owing to the strong UV-absorption of the cornea. Undesired changes
due to light in the visible range of the spectrum are unlikely to happen because
photon density of sun light is much too low for TPA. Due to the excellent
spatial resolution that can be achieved with two-photon processes one cannot
only modify the refractive index of the entire lens but also selectively in well
defined areas enabling to correct for aberrations such as astigmatism.
Here, we present new polymers that do not only exhibit a photoinduced
change of refractive index great enough to induce a change of focal length of
more than 2 diopters in a standard IOL. These new polymers have also
significantly improved material properties with respect to the fabrication of
the IOL and the TPA-sensitivities and the light energy required to induce the
refractive index change.
74
European Conferences on Biomedical Optics 2007 •
6632-32, Session 7
Fs-Lentotomie: changing the accommodation
amplitude of presbyopic human crystalline
lenses by fs laser pulses
S. Schumacher, Laser Zentrum Hannover e.V. (Germany); U. Oberheide,
Laserforum Koln e.V. (Germany); H. Theuer, M. Fromm, T. Ripken, Laser
Zentrum Hannover e.V. (Germany); G. Gerten, Laserforum Koln e.V.
(Germany); W. A. Ertmer, Univ. Hannover (Germany); H. Lubatschowski,
Laser Zentrum Hannover e.V. (Germany)
According to Helmholtz’ theory of accommodation one of the mayor reasons
for the development of presbyopia is the increasing sclerosis of the lens.
One concept to overcome this hardening of the lens is to regain its flexibility
by inducing gliding planes inside the lens. Femtosecond laser pulses are a
suitable tool for this treatment. Showing in former work that we could increase
the flexibility of enucleated porcine (ex vivo) lenses up to 25 %, we focused
our recent work on human autopsy lenses. The age of the human donors
ranged between 20 and 70 years. For an evaluation of the gain in flexibility
the lens’ thickness was measured undertaking the Fisher’s spinning test before
and after laser treatment. Depending on the age and the quality of applied
cutting pattern the lens thickness increased after treatment up to 0.4 mm
leading to an theoretical increase of several dioptres of optical power. The
flexibility could be increased up to 70 % compared to the measurements
before treatment. Since the age of the human donors had a broad range,
leading to different degrees of lens hardening, the variance of the measured
flexibility changes was up to 30%. An addition the influence of the laser
treatment onto the lens on the accommodation amplitude will be shown in a
three dimensional finite-element simulation.
6632-33, Session 7
Femtosecond laser-induced cavitations in the
lens of the human eye
L. Kessel, J. Nymand, M. Harbst, Copenhagen Univ. Hospital Glostrup
(Denmark); M. v. d. Poel, Danmarks Tekniske Univ. (Denmark); M.
Larsen, Univ. of Copenhagen (Denmark) and Kennedy Institute National Eye Clinic (Denmark)
Ultrafast femtosecond lasers are used increasingly for a wide range of medical
purposes. Experimental and accidental tissue exposure has demonstrated
an immediate tissue response to pulses above a variable threshold that induce
optical breakdown, which is often visible as gas-filled cavities that persist for
some time after the event. Associated opacification of the surrounding tissue
can subside after some time, suggesting that the use of such effects may
have a therapeutic potential. Nevertheless, there may be reason to suspect
that at some level above the cavitation threshold, the lens will be permanently
damaged and optically deficient.
In the present study, we attempted to define the cavitation threshold in the
human lens in vitro using multiphoton effects based on radiation from a
femtosecond 800 nm Ti:Sapphire laser.
Cavitations were observed from peak intensities of 5-10 mJ/cm2, but only
after several minutes of exposure and not as a result of a single laser pulse.
This suggests that cavitation was caused by a process that differs from the
one-shot cavitation that is seen at higher intensities. To evaluate whether the
release of gas was caused by ionization and plasma formation or by thermal
effects, we introduced pauses into the pulse train, which did not change the
exposure time to cavitation/gas formation. This suggests that local heating
did not play a role in producing the observed phenomenon. Similar effects
were observed in animal lenses (porcine and canine lenses), but at slightly
higher intensities, possibly because these lenses are clearer than the much
older human lenses.
In conclusion, cavitation and/or gas release was observed in both human
and animal lenses at much lower intensities than reported for other biological
media. The mechanism of cavitation appears not to involve thermal effects
and may be unrelated to plasma formation, suggesting that photochemical
reactions may play a role. These results suggest that there are several types
of ultrafast laser effects in the lens that with a potential for therapeutic
application and treatment of eye disease.
6632-34, Session 8
Principles of laser catapulting of live cells
A. Vogel, N. Linz, V. Horneffer, Univ. zu Lübeck (Germany)
Separation and transport of living cells by laser microdissection (LMD) and
subsequent laser pressure catapulting (LPC) is of interest for stem cell
research, organ culture, and tissue engineering.
Cells were cultured in a dish consisting of a 25 µm thick Teflon membrane for
mechanical support and a 1.35 µm thick Polyethylene naphthalate (PEN) foil
conditioned with polylysine mounted above the Teflon membrane. Before
LMPC with UV-A ns pulses, the culture medium was almost completely
removed such that only a thin (up to 40 µm) layer of liquid remains above the
cells. Then the region of interest was dissected, and the dissectat of cells
and PEN foil catapulted by a single laser pulse into the cap of a microfuge
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tube wetted with culture medium. The original protocol [1] involves the use
of laser pulses focused at the specimen periphery for catapulting but we
also performed series of experiments with defocused pulses because we
hoped that this would minimize bending of the specimen and shear stresses
on the cells. In some experiments a thin (30-100 µm) layer of culture medium
was injected between Teflon membrane and PEN foil since we had observed
that this enhances catapulting. To test cell viability after LMPC, the cells
were tansferred into 12-well plates and recultivated. The dynamics of LMD
was investigated by flash photography through the microscope, and the
kinetics of LPC of specimens with 100 µm diameter was analyzed by flash
photography in side view.
The working mechanisms of LMD is plasma-mediated ablation. Since the
plasma was confined by the surrounding liquid, transient cavitation bubbles
with a lifetime of a few microseconds were formed around the laser focus.
The shear stress exerted by the oscillating bubbles swept cells off the PEN
foil up to a distance of 20-30 µm from the laser cut, when cell adhesion was
weak, but with strong adhesion the damage range was only 5-8 µm.
When the catapulting laser pulse was focused into the periphery of the
specimen, 16 % of the specimens (n = 60) missed the cap but out of the
specimens that could be transferred into the 12-well plate, 98% (all besides
one) could be recultivated. By contrast, with defocused catapulting using a
spot diameter of 50 µm, all specimens (60 out of 60 %) arrived in the cap, but
to our surprise the majority of the cells had been swept off the foil, and
recultivation was possible only in 4 cases. Time resolved photography
revealed that in defocused catapulting strong shear forces originate from the
flow of the thin layer of culture medium covering the cells. By contrast, pulses
focused at the periphery of the specimen cause a fast rotational movement
that makes the specimen wind its way out of the culture medium, thereby
undergoing much less shear stresses.
[1] Stich et al. (2003) Pathol. Res. Pract. 199, 405-409.
6632-35, Session 8
Laser microbeams as versatile tools for for stem
cell purification and clonal expansion
A. Buchstaller, Ludwig-Maximilians-Univ. München (Germany); Y. Niyaz,
P.A.L.M. Microlaser Technologies GmbH (Germany); S. Soria-Lopez,
Ludwig-Maximilians-Univ. München (Germany); K. Schütze, P.A.L.M.
Microlaser Technologies GmbH (Germany)
In the emerging field of regenerative medicine and tissue engineering
embryonic and adult stem cells are used as a renewable source of replacement
cells for diseased or injured organs. Stem cells can also serve as target cells
for drug screening and as vehicles for cell-based gene and tumor therapies.
To this purpose stem cells need to be isolated from embryos or adult
organisms, recultivated, differentiated in vitro, and re-administered to the
patient.
Laser Microdissection and Pressure Catapulting (LMPC) is a non-contact
laser-based, fast and reliable method that enables the identification of specific
cells in their microenvironment under microscopic high-resolution sample
control followed by pure and homogeneous cell preparation. We used LMPC
for the isolation of life stem cells according to cell morphology and marker
expression. In a first experiment we isolated single cells from CD34+ and
CD34- murine adult stem cell lines. We then proceeded to sort out and clonally
expand single adherent fibroblast-like CD29+/CD34+ mesenchymal stem cells
from a complex mixture of bone marrow-derived cells. Single cells were
microdissected using various microscope objectives, then catapulted in
Eppendorf caps and transferred in 24-well cell culture plates filled with a
mixture of fresh and conditionned media. In 90% of the experiments, the
cells adhered well and proliferated quickly. In a different set of experiments
we isolated and recultivated single murine embryonic carcinoma cells as
well as large P19 embryoid bodies and also separated undifferentiated
embryonic carcinoma cells from their differentiated progeny. Again the
recultivated clonal cells expressed the same stem cell-specific markers as
the originating cells and maintained the same morphology.
In summary LMPC opens new approaches in establishing homogenous cell
populations from adherent cell and tissue cultures in order to characterize
and expand defined cell types.
6632-83, Session 8
Laser micromanipulation of cells and tissue
K. Schütze, P.A.L.M. Microlaser Technologies GmbH (Germany)
Ultrashort laser pulses recently became an important tool in biophotonics
and micro machining of materials. Focused inside the bulk of transparent
materials ultrashort pulses provide intensity sufficient to initialize nonlinear
ionization.
A plasma is generated at the site of the focus eventually resulting in optical
breakdown and manipulation of the material.
To gain better spatial resolution, applications such as cell surgery have recently
evolved strongly towards tight focusing of ultrashort pulses using microscope
objectives of high numerical aperture (NA) as focusing units.
The pulse energy required to generate optical breakdown was thus reduced
to nanojoules.
The mechanical effects subsequent to plasma generation are minimized to
the very focus, enabling to precisely ablate single cell organelles without
hazardous effects observable to the surroundings or the entire cell.
The nonlinear interaction of ultrashort laser pulses with transparent media is
numerically investigated at focusing conditions of high numerical aperture.
A nonlinear Schrödinger equation of both nonparaxial and vectorial character
is derived to account for ultrashort pulse propagation at high NA and the
interaction with the generated plasma in the focus.
A multi rate equation model for dielectrics is used to simultaneously calculate
the nonlinear ionization.
Initial conditions are provided using nonparaxial and vectorial diffraction
theory.
Numerical calculations based on this model are used to understand the
dependence between size, geometry and density of optical breakdown
plasmas at various focusing conditions of high NA.
It is shown that breakdown plasmas of axial and transversal size below the
diffraction limit can be generated, when a certain numerical aperture is
exceeded. This is in contrast to weak focusing conditions, where due to
strong nonlinear interactions, relatively large and spatially asymmetric plasmas
were calculated in the focus.
Within this work the code is applied to water as a model substance to
biological soft tissue and cellular constituents, but it can also be applied to
arbitrary isotropic dielectrics, such as fused silica.
6632-37, Session 9
Femtosecond laser-induced nanocavitation
N. Linz, S. Freidank, Univ. zu Lübeck (Germany); G. Paltauf, KarlFranzens-Univ. Graz (Austria); A. Vogel, Univ. zu Lübeck (Germany)
We showed recently that femtosecond-laser-induced nanocavitation is the
working mechanism of cell surgery with fs laser pulses at low and moderate
repetition rates up to 1 MHz. At the same time, it is the most important
mechanism for collateral damage.
In the present study we present breakdown thresholds for 315-fs laser pulses
with wavelengths of 1040 nm, 520 nm and 347 nm using bubble formation in
water as breakdown criterion. Unlike previous data, these threshold values
are neither influenced by nonlinear propagation artifacts (because they were
obtained at large NA) nor are they distorted by optical aberrations (because
the pulses were focused through UV-VIS-IR water immersions microscope
objectives built into the cuvette wall).
Investigation of fs-laser-induced optical breakdown thresholds in water at
large NA is challenging, because the breakdown threshold energies are only
a few nanojoules and the size of the transient laser-produced bubbles is well
below the optical resolution limit. Therefore, we developed a probe beam
scattering method in which the bubble size is inferred from the bubble
oscillation time. We were able to detect bubbles with a maximum radius as
small as 150 nm and an oscillation time of 15 ns and investigated also the
dependence of bubble size on the laser pulse energy. We found that this
dependence is considerably weaker for UV wavelengths than for IR
wavelengths, as expected from previous numerical calculations on plasma
formation.
Close to threshold, we identified a regime with very small conversion efficiency
of laser pulse energy into bubble energy, ranging from 0,0002 % up to about
0,01 %. This regime, which is broadest for UV-wavelengths, is very suitable
for cellular nanosurgery. The mechanism of bubble formation is thermoelastic
rupture of the liquid at temperatures below the critical point. The agreement
between theoretically predicted and experimentally determined bubble sizes
is excellent. With increasing pulse energy, we found a steep increase of the
conversion efficiency into bubble energy, indicating a similarly steep increase
of the plasma energy density.
No abstract available
6632-38, Session 9
6632-36, Session 9
Simulation of ultrashort pulse induced optical
breakdown plasmas generated at high
numerical aperture focusing
C. L. Arnold, Laser Zentrum Hannover e.V. (Germany); W. A. Ertmer,
Univ. Hannover (Germany); H. Lubatschowski, Laser Zentrum Hannover
e.V. (Germany)
European Conferences on Biomedical Optics 2007 •
Luminescent high-energy density femtosecond
plasmas in bulk aqueous materials
A. Vogel, N. Linz, S. Freidank, Univ. zu Lübeck (Germany); G. Paltauf,
Karl-Franzens-Univ. Graz (Austria)
Fs laser pulses focused at large NA into nominally transparent materials are
used for cellular nanosurgery but can also produce well localized effects of
considerably larger extent when the laser pulse energy is increased. This
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Conf. 6632: Therapeutic Laser Applications and Laser-Tissue Interactions
differs strongly from fs breakdown at small NAs for which an increase of the
pulse energy leads to an ever more delocalized energy deposition owing to
nonlinear beam propagation effects including plasma defocusing and
filamentation that limit the maximum possible plasma energy density.
6632-77, Session 9
Femtosecond laser nanoprocessing for
manipulation of stem cells
K. König, IBMT St. Ingbert (Germany)
We characterized fs breakdown in water at large NA by measuring the energy
dependence of plasma transmission T and cavitation bubble size Rmax. The
bubble size was determined by means of a sensitive probe beam scattering
technique. For E/Eth = 7 (Eth = bubble formation threshold), we observed
plasma luminescence on photographs of the focal region that allowed to
determine the plasma size. From plasma absorption A = 1-T and plasma
volume V we determined the energy density e = Eabs / V. Knowledge of e,
the equation of state of water, and the Grüneisen coefficient allows to assess
the temperature T after equilibration of electron and ion temperatures and
the resulting thermoelastic pressure. The data on plasma volume (= initial
cavitation bubble volume) and maximum bubble radius Rmax were
implemented into the Gilmore model of cavitation bubble dynamics and used
to calculate the initial plasma pressure averaged over the period of stress
confinement and the time after relaxation of the thermoelastic stress.
We found that for large NAs and well above the breakdown threshold the
energy density amounts to about 24 kJ cm^-3, which is comparable to ns
plasmas. Owing to stress confinement an ultrashort (sub-nanosecond)
thermoelastic stress wave is produced that exhibits a peak pressure which
is larger in ns plasmas. However, the thermoelastic wave will be rapidly
damped, and the time-averaged pressure during the first nanoseconds
amounts to 13.5 kbar, which is similar to the case of ns pulses. These results
are in striking contrast to recent claims (Juodkazis et al., PRL 96, 2006) that
multimegabar pressures are achieved during fs breakdown in solid bulk media.
Nevertheless, the number density r must be considerably larger than the
critical free electron density of rho = 10^21 cm-3. to account for the large
volumetric energy density of 24 kJ cm^-3 because only one set of free
electrons is produced in fs breakdown. This outcome raises interesting
questions about the laser plasma coupling in fs breakdown because it is not
consistent with the common view that plasma becomes completely reflective
at the critical free-electron density.
Thus, fs optical breakdown phenomena in bulk aqueous media at large NA
span a large range: from nanocavitation induced by a temperature rise of
less than 200 °C for energies at the bubble formation threshold to plasma
energy densities 10 times larger than the vaporization enthalpy of water. This
has important consequences for laser surgery of cells and tissues.
6632-39, Session 9
Effects of pulse duration and pulse energy on
laser microbeam-induced cell lysis and
membrane permeabilization
A. N. Hellman, Univ. of California/Irvine (USA); K. R. Rau, Tata Institute
of Fundamental Research (India); P. A. Quinto-Su, V. Venugopalan, Univ.
of California/Irvine (USA)
Time-resolved imaging was used to examine pulsed laser microbeam
irradiation for cell lysis and transient cell membrane permeabilization. Lysis
was achieved through the delivery of 532 nm laser pulses with pulse durations
in the sub-nanosecond to nanosecond regime via a 40x, 0.8 NA objective to
a location 10 microns above confluent monolayers of PtK2 cells. These studies
have demonstrated that while the process is initiated by laser-induced plasma
formation, cell lysis is produced by the fluid shear stress produced during
the cavitation bubble expansion [1,2]. The dynamics of laser-induced cell
lysis caused by 6 ns pulses have been studied and we have developed
physical models of cell lysis based on experimental data [3]. Here, we study
the process dynamics using pulse durations ranging from 180 ps to 6 ns and
pulse energies corresponding to 1x, 2x, 3x, and 5x the threshold for plasma
formation. The cell lysis process was imaged at times of 0.5 ns to 50 us after
laser pulse delivery to visualize the plasma formation, pressure wave
propagation, and cavitation bubble dynamics. The spatial extent of cell lysis
increased with pulse duration and pulse energy. Hydrodynamic analysis
indicated that cells subject to transient shear stresses in excess of a critical
value were lysed while cells exposed to lower shear stresses remained
adherent and viable. Fluorescence assays are used to correlate the physical
effects with the subsequent biological response, and cell viability, transient
membrane permeabilization, apoptosis, and cytoskeletal integrity are
assessed.
References:
1. K. Rau, A. Guerra III, A. Vogel, and V. Venugopalan, Appl. Phys. Lett. 84,
070415 (2004)
2. V. Venugopalan, A. G. Guerra III, K. Nahen and A. Vogel, Phys. Rev. Lett.
88 078103 (2002)
3. K. Rau, P. Quinto-Su, A. Hellman, and V. Venugopalan, Biophysical Journal.
91 (2006)
76
European Conferences on Biomedical Optics 2007 •
No abstract available
6632-82, Session 9
Laser nanosurgery for stem cell research
A. Heisterkamp, Laser Zentrum Hannover e.V. (Germany)
No abstract available
6632-84, Session 9
Laser micromachining in living cells
F. S. Pavone, Univ. degli Studi di Firenze (Italy)
No abstract available
6632-199, Session 9
Influence of laser parameters on femtosecond
near-infrared opto-injection of living cells
C. Peng, R. E. Palazzo, I. Wilke, Rensselaer Polytechnic Institute (USA)
No abstract available
6632-40, Session 10
Mechanisms of selective nanophotothermolysis
with gold nanoparticles
V. K. Pustovalov, Belarussian Institute of System Analysis (Belarus); A.
S. Smetannikov, A.V. Luikov Heat and Mass Transfer Institute (Belarus);
V. P. Zharov, Univ. of Arkansas for Medical Sciences (USA)
Modeling of mechanisms of selective nanophotothermolysis of cells with
nano- and picosecond laser pulses and gold nanoparticles (GNs) are
performed. Mechanisms of selective nanophotothermolysis with GNs with
sizes 10-100 nm and short laser pulses with duration 10-6 = tp = 10-12 s
were theoretically investigated in this paper. Seven possible physical
processes were discussed which can be proposed for selective laser damage
of cancer cells: 1) thermal denaturation of proteins around GNs; 2) thermal
expansion of GNs with generation of acoustic wave; 3) explosive evaporation
of water around GNs and bubble formation and dynamics; 4) melting of GNs
with increasing its radius, 5) evaporation GNs with formation of gold vapor
bubble, 6) explosion of GNs with formation of small particles and 7) optical
breakdown with formation and dynamics of plasma cavity and shock wave.
The more detailed results were obtained for first three phenomena 1), 2), and
3).
Characteristic parameters of these processes such as the temperature levels,
threshold of bubble formation, acoustic pressure, among others are
determined. Irradiation of GNs with sizes 10-100 nm by nano-, pico- and
femtosecond laser pulses with different wavelengths could result in thermal
and (or) mechanical intracellular effects with spatial confinement of about or
less than 10-100 nm. It is possible to relate the physical processes of heating,
heat transfer, thermochemical transformations of proteins and explosive
vaporization near GNs to the specific nanoeffects of laser pulses in cells.
Comparison of therapeutic efficiency of different mechanism allowed making
recommendation for laser parameters.
6632-41, Session 10
Selective protein knockout by laser-induced
heating of gold nanoparticles
M. Bever, Univ. zu Lübeck (Germany); R. Rahmanzadeh, Research Ctr.
Borstel (Germany); G. Hüttmann, Univ. zu Lübeck (Germany)
Irradiation of spherical gold nanoparticles with a laser beam results in
nanoscale thermophysical effects around the nanoparticle that include
heating, formation of microbubbles, or photochemical reactions . These
effects can be used for manipulating the proximate environment of the
nanoparticles. Thus, laser-induced heating of gold nanoparticles that have
been coupled to specific target proteins is an efficient method to produce a
selective protein denaturation in a multi-protein environment, e.g. cells. The
inactivation of a proteins catalytic function resulting from denaturation is of
fundamental interest for understanding the specific function of the targeted
proteins as well as for future development in medical application.
Experiments were done with the well-known proteins alkaline phosphatase
(AP) from bovine intestinal mucosa and horseradish peroxidase (HRP) from
amoracia rusticana. Both proteins have a high catalytic activity. AP was
coupled either directly or via antibody to 6-, 15-, and 30-nm gold
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Conf. 6632: Therapeutic Laser Applications and Laser-Tissue Interactions
nanoparticles. HRP was coupled only directly to 15-nm nanoparticles. The
samples were irradiated with 527-nm pulses of 35 ps duration and with 532nm pulses of 9 ns duration.
The irradiation caused an exponential decrease of the activity of both proteins
with increasing radiant exposure. Using the same pulse energy, ps-pulses
were significantly more efficient because heat conduction of the gold
nanoparticles into the surrounding aqueous medium is less effective with
shorter puls duration. The inactivation characteristics of 15-nm gold
conjugates were similar to those of 30-nm conjugates. However, the protein
binding stability of the 15-nm gold particles was much better. We found that
irradiation of 15-nm gold conjugates with 527-nm ps-pulses is an efficient
way to produce a selective thermophysical knockout of a microscopic
biological system that is coupled to a gold nanoparticle. Moreover, using
two different antibody species coupled to the nanoparticles, we were able to
show that the irradiation effect is spatially restricted and does not affect nonbound proteins.
6632-42, Session 10
Cell and protein inactivation with optical
absorbers
R. Rahmanzadeh, J. Gerdes, T. Scholzen, Research Ctr. Borstel
(Germany); G. Hüttmann, Univ. zu Lübeck (Germany)
Optical absorbers in combination with light irradiation are valuable tools for
cell and protein inactivation with high spatiotemporal precision. These
structures absorb light of a certain wavelength stronger than their environment.
With strongly absorbing gold nanoparticles a destruction of cells and proteins
is possible, here this approach is named nanoparticle-assisted laser
inactivation (NALI). If dye molecules are used as absorbers, single proteins
can be inactivated by photochemical reactions. This approach is known as
chromophore-assisted laser inactivation (CALI) and has been successfully
employed for several years. With gold particles a highly selective destruction
of cells was observed after laser irradiation by coupling the particles through
antibodies to the cell membrane. The specificity of the damage was
demonstrated with mixed cell cultures at high cell concentrations, where
selectively the targeted cell type was damaged to nearly 100 %, whereas the
untargeted cells were barely affected. At the energy levels used, the formation
of cavitation bubbles is expectable and in consequence most likely a
mechanical damage of the cell membranes.
In vitro studies on protein inactivation using 15 nm goldparticle-antibodyconjugates showed a fragmentation of the target protein pKi-67 upon
irradiation, while irradiation of FITC- and Alexa 488-labeled antibodies led to
specific crosslinking of the target protein. With the help of dye labeled
antibodies, a successful inactivation of pKi-67 was found in living cells on
sub-nuclear level. The resulting inhibition of polymerase I-dependant rRNAsynthesis inside the nucleoli represents the first functional evidence for the
physiological role of pKi-67 in living cells.
6632-43, Session 10
Laser-activated nanoparticle-directed cell
elimination
F. Levold, A. Limmer, Univ. Bonn (Germany); G. Hüttmann, Univ. zu
Lübeck (Germany); E. Endl, Univ. Bonn (Germany)
The use of nanoparticles in cancer cell diagnostics, drug delivery and
therapeutics is an active field of research in medicine. Nanoparticle mediated
targeting and precise depletion of a specific cell type without any side effect
to non-target cells might be suited to modulate immunological processes on
a cellular level. An innovative technique for this approach is the use of gold
nanoparticles, which can be navigated onto target cells by coating of the
particles with monoclonal antibodies. Subsequent deposition of high energy
by pulsed laser irradiation results in physical phenomena which are lethal for
target cells and can therefore be used for selective depletion of cells. We
name this method laser-activated nanoparticle-directed cell-elimination
(LANCE). We can show, that LANCE is highly effective and selective in cell
depletion in mixed cell suspension, whereas non-target cells are left unaffected
and completely retain their immunological functions.
We eliminated B220+ cells out of splenic white blood cells of OTIxB6 mice
ex vivo by LANCE using B220 immunogold conjugates. The selective cell
killing efficiency was more than 98%. After the elimination of B220+ cells,
the treated and untreated cells were incubated for 72h with soluble OVA to
stimulate the remaining Ovalbumin (OVA) specific CD8+ T cells. Subsequently
we analysed the proliferation of the OVA-specific CD8+ cells and the cytokine
production in the samples. The Division Index received from the CFSE
proliferation pattern) and the percentage of Ki-67+ cells in the CD8+ cell
population of treated and control samples were similar. This demonstrates
that in the LANCE treated sample the OVA was still incorporated, processed
and presented by APCs and that the antigen specific CD8+ T cells responded
with proliferation as known.
We have developed and demonstrated the laser-activated nanoparticledirected cell elimination(LANCE) as a gentle method with high efficancy and
high yield for ex vivo cell depletion. One of the major application for LANCE
will be the selective and effective elimination of residual tumor cells in human
European Conferences on Biomedical Optics 2007 •
bone marrow and stem cells extracted from blood. This purification is an
essential step towards Autologous stem cell transplantation, a promising
method in the treatment of leukemia.
6632-44, Session 10
Progress in gene transfection by the use of
laser-induced stress wave
S. Sato, National Defense Medical College (Japan); M. Terakawa, M.
Obara, Keio Univ. (Japan)
We have demonstrated efficient, targeted gene transfer to rat skin and mouse
brain in vivo by the use of laser-induced stress wave (LISW). In this method,
plasmid DNA is injected into targeted tissue, on which a laser target composed
of a light absorption layer (black rubber disk) and a transparent layer
(polyethylene terephthalate sheet) for plasma confinement is placed. The
target is irradiated with nanosecond laser pulses (532 nm, 6 ns) to produce
plasma, its expansion creating high-peak-pressure compressive waves which
can modify cell membranes for macromolecules to enter the cytoplasm.
Experiments using reporter genes, such as EGFP (enhanced green fluorescent
protein) gene, showed highly site-specific and tissue-specific gene expression
in the tissues. Recently, we applied this technique to skin grafting with the
objective of enhancing adhesion of the grafts, for which a therapeutic vector
construct carrying hepatocyte growth factor (HGF) was delivered to skin grafts
of rats by using LISW. Angiogenesis was accelerated significantly in the
grafted skins with gene transfection when compared with in normal grafted
skins, suggesting the efficacy of this method for improveing the outcome of
tissue transplatation. Development of fiber-based gene delivery system will
also be described.
6632-45, Session 10
Towards a selective photochemical inactivation
of the progesterone receptor
W. S. L. Strauss, Univ. Ulm (Germany); K. Raunegger, C. Hoedl, E.
Haslinger, Karl-Franzens-Univ. Graz (Austria); R. W. Steiner, Univ. Ulm
(Germany); H. W. Schramm, Karl-Franzens-Univ. Graz (Austria)
Chromophore- and fluorophore-assisted laser inactivation (CALI / FALI) have
been suggested to be appropriate techniques for a transient knock-down or
knock-out of proteins in living cells. Selectivity of these probes to the target
proteins is usually mediated by antibodies. However, targeting of intracellular
proteins is expected to be less effective as compared to membraneassociated proteins, since cellular uptake of chromophore- / fluorophoreantibody conjugates might be impaired. Thus, development of cell-permeant
CALI- / FALI-probes seems to be desirable; these probes mediate selectivity
to the target proteins by small ligands of low molecular weight.
The present work aimed to develop cell-permeant CALI- / FALI-probes
directed against the progesterone receptor (PR), which is a ligand-activated
nuclear transcription factor in various tissues. Chemical synthesis of the
desired probes started from the well-known PR-antagonist mifepristone.
Interaction of the dye-mifepristone conjugates as well as of its precursors
with PR was determined in cell culture (alkaline phosphatase assay in T47D
breast cancer cells). Antiprogestagenic activity of the intermediates was
comparable to that of the parent compound. Even after attachment of the
bulky dye moieties (malachite green or fluorescein), considerable
antiprogestagenic activity was maintained. In case of the fluoresceinmifepristone conjugate, microscopic studies revealed that fluorescence of
the probe was almost confined to the nuclei of steroid hormone receptorpositive cells, whereas the nuclei of steroid hormone receptor-negative cells
remained unstained. These results are encouraging to further study the
photochemical and photobiological properties of these conjugates upon light
exposure.
6632-46, Session 10
Efficacy of a single high dose versus multiple
low doses of lllt on wounded skin fibroblasts
D. H. Hawkins, H. Abrahamse, Univ. of Johannesburg (South Africa)
Background/purpose: In vivo studies have demonstrated that phototherapy
accelerates wound healing in the clinical environment; however the exact
mechanism is still not completely understood. The main focus of this study
was to use in vitro laboratory results to establish an effective treatment regimen
that may be practical and applicable to the clinical environment. This in vitro
study aimed to compare the cellular responses of wounded fibroblasts
following a single exposure of 5J/cm2 or multiple exposures of low doses
(2.5J/cm2 or 5J/cm2) on one day of the week to a single application of a
higher dose (16J/cm2) on day 1 and day 4.
Methods: Cellular responses to Helium-Neon (632.8nm) laser irradiation were
evaluated by measuring changes in cell morphology, cell viability, cell
proliferation, membrane integrity and DNA damage.
Results: Wounded cells exposed to 5J/cm2 on day 1 and day 4 showed an
increase in cell viability, increase in the release of bFGF, increase in cell density,
decrease in ALP enzyme activity and decrease in caspase 3/7 activity
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Conf. 6632: Therapeutic Laser Applications and Laser-Tissue Interactions
indicating a stimulatory effect. Wounded cells exposed to three doses of 5J/
cm2 on day 1 showed a decrease in cell viability and cell proliferation and an
increase in LDH cytotoxicity and DNA damage indicating an inhibitory effect.
Conclusion: Results indicate that cellular responses are influenced by the
combination of dose administered, number of exposures and time between
exposures. Single doses administered with sufficient time between exposures
is more beneficial to restoring cell function than multiple doses within a short
period. Although this work confirms previous reports on the cumulative effect
of laser irradiation it provides essential information for the initiation of in vivo
clinical studies.
6632-85, Session 10
Lab-on-a-chip: The future of single cell analysis?
E. Eriksson, M. Goksör, Göteborg Univ. (Sweden)
No abstract available
6632-47, Session 11
Laser-mediated perforation of plant cells
M. M. Wehner, Fraunhofer-Institut für Lasertechnik (Germany); H.
Schinkel, Fraunhofer Institut Molekularbiologie und Angewanate
Oekologie (Germany); P. Jacobs, Fraunhofer-Institut für Lasertechnik
(Germany); S. Schillberg, Fraunhofer Institut Molekularbiologie und
Angewanate Oekologie (Germany)
The functional analysis of plant cells at the cellular and subcellular levels
requires novel technologies for the directed manipulation of individual cells.
Lasers are increasingly exploited for the manipulation of plant cells, enabling
the study of biological processes on a subcellular scale including
transformation to generate genetically modified plants. In our set-up either a
picosecond laser operating at 1064 nm wavelength or a continuous wave
laser diode emitting at 405 nm are coupled into an inverse microscope. The
beams are focused to a spot size of about 1.5 µm and the tobacco cell
protoplasts are irradiated. Optoporation is achieved when targeting the laser
focal spot at the outermost edge of the plasma membrane. In case of the
picosecond laser a single pulse with energy of about 0.4 µJ was sufficient to
perforate the plasma membrane enabling the uptake of dye or DNA from the
surrounding medium into the cytosol. When the ultraviolet laser diode at a
power level of 17 mW is employed an irradiation time of 200 - 500 milliseconds
is necessary to enable the uptake of macromolecules. In the presence of an
EYFP encoding plasmid with a C-terminal peroxisomal signal sequence in
the surrounding medium transient transformation of tobacco protoplasts could
be achieved in up to 2% of the optoporated cells. Single cell perforation
using this novel optoporation method shows that isolated plant cells can be
permeabilized without direct manipulation. This is a valuable procedure for
cell-specific applications, particularly where the import of specific molecules
into plant cells is required for functional analysis.
6632-49, Session 11
Cost-effective generation of nano- and
microeffects in cells and tissues by ns laser
pulses
A. Vogel, N. Linz, S. Freidank, Univ. zu Lübeck (Germany); G. Paltauf,
Karl-Franzens-Univ. Graz (Austria)
Nanosecond optical breakdown in commonly believed to be associated with
bright plasma luminescence. However, we found experimentally that for VIS
and UV laser pulses with Gaussian temporal shape ns-breakdown is a two-step
process. In the first step, a non-luminescent low-density plasma is formed the
expansion of which creates minute bubbles (R = 500 nm - 10 µm, depending on
pulse energy). The bubble size was experimentally determined by means of a
sensitive probe beam scattering technique. Numerical investigations revealed
that the bubble formation threshold corresponds to the onset of a phase exlosion
of the superheated water in the focal volume. In this regime, electron-hole
recombination limits the free-electron density to values of up to 10^20 cm^-3,
and the conversion efficiency of laser energy into bubble energy is, at threshold,
as small as 0.00003%. For energies 10-30 times above the bubble formation
threshold (for 532 nm and 355 nm, respectively), plasma suddenly assumes a
much larger size (1600 times the focal volume), bright luminescence is observed,
and large bubbles (R \> 200 µm) are produced. This plasma inflation is attributed
to a thermal ionization runaway that overcomes recombination and results in
high plasma densities reaching full ionization. Plasma energy density is limited
by energy transport out of the absorbing region by UV radiation and/or ejection
of fast electrons. When the laser energy is further increased, the plasma grows
along thin streaks into the cone angle. The bright and strongly scattering streaks,
which resemble Lichtenberg figures, are the origin of the diffuse plasma
luminescence observed in a much larger volume. For IR breakdown, the
thresholds for bubble and luminescent plasma formation coincide, and
breakdown is thus a one-step process.
Plasma models considering only the interplay of multiphoton ionization and
avalanche ionization do not portray the two steps in the optical breakdown
dynamics. If additionally recombination is taken into account, the formation of
non-luminescent low-density plasmas is correctly predicted but not the jump in
free-electron density resulting in brightly luminescent plamas. A realistic
description of the formation of the luminescent high-density plasmas requires
consideration of thermal ionization. We established a rate equation model
including thermal ionization that predicts the two steps of ns breakdown in
excellent agreement with our experimental findings.
The discovery that minute plasma-mediated effects can be produced not only
with femtosecond pulses but also with VIS and UV nanosecond pulses is of
particular importance for cost-effective cell surgery and corneal refractive surgery.
6632-78, Session 11
Optical knocking out of single cells in tumor
spheroids
6632-48, Session 11
A. A. Uchugonova, Fraunhofer-Institut für Biomedizinische Technik
(Germany)
Dosimetry in cellular optoperforation by realtime monitoring of bubble formation
No abstract available
N. Linz, V. Horneffer, S. Freidank, A. Vogel, Univ. zu Lübeck (Germany)
6632-79, Session 11
Gentle optoperforation of cells is of great interest for gene transfer, and also
for other biological applications, such as the transport of antibody-conjugated
nanoparticles into the cell. Because the cell membrane exhibits only poor
linear absorption for UV-A, visible or near infrared wavelengths, plasma
formation is usually required to achieve localized energy deposition. With
pulses from a fs oscillator, membrane permeabilization can be achieved by
the cumulative chemical action of a series of about 1 million laser pulses [1].
By contrast, optoperforation by single pulses or with pulse series in the kHz
regime requires plasma densities leading to the formation of minute cavitation
bubbles [2]. When these bubbles become too large, they will lead to unwanted
cell rupture and cell death. Therefore, it is necessary to adjust the laser pulse
energy to a level, where the induced cavitation bubble only perforates the
cell membrane without killing the cell.
For real-time monitoring of the laser induced bubble formation, we coupled
a nIR cw probe laser into the microbeam system used for cell perforation.
The probe laser is aligned collinearly with the “pump” laser (fs or ns laser).
The light transmitted through the focusing objective for cell perforation is
collected by the condenser of the microscope and reflected by a dichroic
mirror onto an AC-coupled high speed photoreceiver. Small bubbles produced
in the focal volume yield a characteristic forward scattering signal that allows
to determine the bubble oscillation time and thus the bubble size. The
minimum detectable bubble radius was 150 nm. A controlled increase of the
energy of subsequent pump laser pulses while monitoring the actual realtime bubble size enables us to realize an online dosimetry for cellular
optoperforation.
[1] Tirlapur, König Nature 418, 290-291 (2002)
[2] Vogel et al. Appl Phys B 81, 1015-1047 (2005)
3D-Laser assisted processing of biocompatible
polymers for biomedical applications on the
cellular level
M. Stark, IBMT St. Ingbert (Germany)
No abstract available
6632-80, Session 11
Laser assisted processing of cross-linked alginat
hydrogel
F. Ehrhart, IBMT St. Ingbert (Germany)
No abstract available
6632-81, Session 11
New developments in femtosecond laser corneal
refractive surgery
R. LeHarzic, JenLab (Germany)
No abstract available
6632-86, Session 11
Femtosecond laser scanning microscopy and
surgery of epiretinal membranes
M. Krause, Universitaetskliniken Homburg (Germany)
No abstract available
78
European Conferences on Biomedical Optics 2007 •
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Conference 6633: Biophotonics 2007: Optics in Life Science
Room BO.R2 • Monday-Wednesday 18-20 June 2007
Part of Proceedings of SPIE Vol. 6633 Biophotonics 2007: Optics in Life Science
6633-01, Session
6633-03, Session 1
Breaking the barrier: fluorescence microscopy
with diffraction-unlimited resolution (Keynote)
Approaches to quantitative in vivo studies by
single-molecule fluorescence spectroscopy
S. W. Hell, Deutsches Krebsforschungszentrum (Germany)
H. Ta, C. M. Roth, P. I. Heinlein, D. Herten, Ruprecht-Karls-Univ.
Heidelberg (Germany)
In 1873, Ernst Abbe discovered that the resolution of focusing (‘far-field’)
optical microscopy is limited to ~ 200 nm which has been the practical
resolution limit ever since. In this lecture we discuss concepts that, by
exploiting selected molecular transitions, neutralize the resolution-limiting
role of diffraction in fluorescence microscopy<sup\>1</sup\>. The first viable
concept of this kind is Stimulated Emission Depletion (STED)
microscopy<sup\>2</sup\> which uses a focused excitation beam along
with a red-shifted doughnut- beam to switch off fluorescence by stimulated
emission. The doughnut confines the fluorescence near its central zero in
such a way, that the effective fluorescence spot (point spread function) can
be arbitrarily reduced in size<sup\>3,4,5</sup\>. The concept underlying
STED microscopy can be expanded by employing other molecular transitions
that switch fluorescence emission: (i) shelving the fluorophore in a metastable
triplet state<sup\>4,6</sup\>, and (ii) photoswitching optically bistable
markers between a ‘fluorescence on’ and a ‘fluorescence off’ conformational
state<sup\>3</sup\>. Examples for the latter include organic compounds
and fluorescent proteins which undergo a photoinduced cis-trans
isomerization or cyclization reaction. Due to their optical bistability, these
molecules entail low saturation intensities, meaning that the diffraction barrier
can be broken at low intensity values. By providing appropriate bistable
molecular markers, organic chemistry and protein biotechnology play a key
role in overcoming the diffraction barrier<sup\>3</sup\>. Finally, we discuss
recent work showing that the advent of far-field ‘nanoscopy’ solves
fundamental problems in biology.<P\></P\>
1) S. W. Hell, Opt. Commun. 106, 19 (1994).<BR\>
2) S. W. Hell and J. Wichmann, Opt. Lett. 19 (11), 780 (1994).<BR\>
3) S.W. Hell, Nature Biotechnol. 21 (11), 1347 (2003).<BR\>
4) S. W. Hell, in Topics in Fluorescence Spectroscopy, ed. by J.R. Lakowicz
(Plenum, NY, 1997), 5, pp. 361.<BR\>
5)M. Dyba and S.W. Hell, Phys. Rev. Lett. 88, 163901 (2002); V. Westphal
and S.W. Hell, Phys. Rev. Lett. 94, 143903 (2005); G. Donnert, et al., Proc
Natl Acad Sci 103, 11440 (2006).<BR\>
6) S. W. Hell and M. Kroug, Appl. Phys. B 60, 495 (1995).<BR\>
7) K. I. Willig, S. O. Rizzoli, V. Westphal et al., Nature 440 (7086), 935 (2006).
6633-02, Session 1
Optical and chemical switches: key molecules
for improved fluorescence imaging and tracking
with high optical resolution
M. Sauer, Univ. Bielefeld (Germany); K. H. Drexhage, Univ. Siegen
(Germany); J. Mattay, P. Tinnefeld, Univ. Bielefeld (Germany)
A molecular photoswitch exhibits two stable and selectively addressable
states, a fluorescent state and a non-fluorescent one, which can be reversibly
interconverted upon irradiation with different wavelengths of light. Efficient
molecular optical switches are strongly desired for improved protein tracking
in living cells. In contrast to photobleaching techniques of fluorescently labeled
proteins in living cells the application of photoswitchable molecules enables
the direct visualization of the movement of individually addressable
intracellular proteins. Furthermore, photoswitchable molecules are potentially
useful for far-field fluorescence imaging with improved resolution. Because
suitable photoswitches reversibly undergo light-induced transitions between
two thermally stable states, and the transitions are saturable, a spatial intensity
distribution of two laser wavelengths (one to switch off the chromophore
and another to reactivate fluorescence) featuring a local minimum might allow
fluorescence imaging at the nanoscale. Conceptually similar to stimulatedemission depletion microscopy molecular optical switches promise similar
spatial resolution, but require much lower saturation of switching intensities.
We present our recent collaborative efforts to design and synthesize water
soluble and functionalized molecular photoswitches based on organic dyes
in combination with diarylethenes and spiropyranes and discuss their
spectroscopic performance (switching efficiency, thermal recovery,
photostability) under ensemble as well as single-molecule conditions. In
addition, we present first results towards the development of molecular
chemical switches, that is, fluorescent molecules whose fluorescence is
switched on only after specific conjugation to certain target functional groups,
e.g. thiol groups. Through the combination of optical and chemical switchable
properties we hope to realize the ideal photoswitch for improved fluorescence
tracking and imaging with high optical resolution.
European Conferences on Biomedical Optics 2007 •
The approach of modeling intracellular networks of biochemical reactions in
systems biology demands novel methods suited for acquiring quantitative
data about transport and interaction of proteins and metabolites within the
heterogeneous environment of living cells. Single-molecule fluorescence
spectroscopy (SMFS) has proven a valuable tool for investigating complex
structures and processes in biochemistry and molecular biology providing a
rich set of methods for in vitro studies of protein/protein and protein/DNA
interactions. Although especially designed to reveal spatial and temporal
heterogeneities, very few applications of SMFS to living cells were reported.
Recently we developed methods based on spectrally-resolved fluorescence
lifetime imaging microscopy (SFLIM) aiming at single molecule studies in
living cells. Herein we show that diffusion imaging microscopy (DIFIM) resolves
spatial heterogeneities in the diffusion of fluorescently labeled probes, based
on single photon correlation in individual pixels of an raster scan image
acquired on our SFLIM setup. Although we found that DIFIM can be applied
to monitor spatial heterogeneities in diffusion and transport in living cells it is
clear that parallel methods are demanded for real-time monitoring in highly
dynamic biological systems. By application of more sophisticated data
analysis schemes based on single-photon detection by two avalanche photo
diodes (APD), we are able to determine the number of chromophores present
in the confocal detection volume. Although limited to a maximum number of
three chromophores, we demonstrated that this method can be applied to
fixed and living cells. Recent simulations demonstrated that in theory the
method should be able to resolve more than three individual chromophores
when the number of parallel APDs is extended to four.
6633-04, Session 1
Fluorescence imaging of cholesterol and
temperature dependent cell membrane
dynamics
P. Weber, M. Wagner, Fachhochschule Aalen (Germany); W. S. L.
Strauss, Univ. Ulm (Germany); H. Schneckenburger, Fachhochschule
Aalen (Germany)
Cholesterol content is an important factor for membrane dynamics of living
cells. With well defined protocols of cholesterol depletion and enrichment
we are able to quantify this effect by fluorescence microscopy. In addition,
we determined the cholesterol content with biochemical methods. Changes
of cholesterol amounts in cell membranes have previously been related to
specific disease and may have some influence on the uptake of
pharmaceutical agents.
A combination of conventional and total internal reflection fluorescence
microscopy was applied to the fluorescence marker laurdan, a polaritysensitive probe, whose electronic excitation energy is different in polar and
non-polar environment. Once incorporated into cell membranes, the
fluorescence of laurdan shows a spectral shift towards longer wavelength
when its molecules get into contact with adjacent water molecules, e.g. when
a phase transition from the tightly packed gel phase to the liquid crystalline
phase of membrane lipids occurs. The generalized polarization (GP,
characterizing this spectral shift) as well as the fluorescence lifetime (t) of
laurdan revealed to be appropriate measures for membrane stiffness and
fluidity. GP generally decreased with increasing temperature and was always
higher for the plasma membrane than for intracellular membranes.
Enrichment of cholesterol caused a pronounced increase, whereas depletion
of cholesterol caused a decrease of GP. In addition pronounced changes of
the fluorescence lifetime pattern occurred in the subnanosecond range. GP,
and t were determined as integral values of single cells or small cell collectives
and were also displayed as microscopic images.
6633-05, Session 1
Fluorescence tomography of biological tissue
based on ultrasound tagging technique
M. Kobayashi, T. Mizumoto, D. Q. Trinh, Tohoku Institute of Technology
(Japan); M. Takeda, Tohoku Univ. (Japan)
We report a study for the development of tomographic imaging technique of
fluorescence in biological tissue for assays of biological function. Ultrasonic
modulation of light based on the acousto-optic effect (so called ultrasound
‘tagging’) is applied for imaging of fluorescence distribution in the lightscattering media. Sound-field characteristics that affect the light by
modulating its amplitude through variation of the refractive index in the
medium were determined. With using focused ultrasound, selectively
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Conference 6633: Biophotonics 2007: Optics in Life Science
modulated fluorescence on a depth-axis of the medium can be detected.
Ultrasound tagging technique applied measuring the optical absorption in
light scattering media is well known, and it is principally based on the
modulation of speckle pattern. On the contrary, in the case of fluorescence,
displacement of scattering particles and variation of the refractive index that
is induced by density distribution in a sound field might produce the intensity
modulation of scattered light. We have experimentally shown that ultrasound
tagging technique is also available for fluorescence measurement. In this
paper, we demonstrate the result of tomographic images of fluorescence in
dense scattering media using biological tissue, porcine muscle and bovine
adipose. Tissue samples had the dimension of 40 x 40 mm in section and
fluorescent region which had the 3mm size was embedded in the center of
the tissue. The image of the fluorophor was determined with the spatial
resolution of focus size of the ultrasound, suggesting the applicability of this
technique for visualization of fluorescent probes in deep portion of living
body. Reference: M. Kobayashi, et al., Appl. Phys. Lett. 89, 181102 (2006)
6633-06, Session 1
Imaging growth of thick engineered tissues with
fluorescence diffuse optical tomography
Y. Bérubé-Lauzière, J. Desrochers, P. Vermette, R. Fontaine, Univ. de
Sherbrooke (Canada)
Current efforts in tissue engineering (TE) are directed towards growing volumes
of tissues in 3D on the order of cubic centimetres. Yet there are no noninvasive instruments to image bio-molecular processes occurring at the cell
level within thick engineered tissues. Optical microscopy (OM) is currently
the tool of choice in TE for imaging these processes. However, it does not
allow deep (=1cm) and non-invasive 3D imaging of tissues during growth
and requires histological cuts, thereby destroying the costly tissue. To address
this problem, we are working on an optical fibers-based Fluorescence Diffuse
Optical Tomography (FDOT) system to image directly into the bio-reactor
where tissues are grown. This avenue has not yet been explored and we aim
to eventually monitor non-invasively and continuously control tissue growth.
FDOT offers the possibility to exploit the well established and large spectrum
of fluorescent markers developed for OM. We are currently developing a
technique to image in 3D, via fluorescence labelling, the formation of microblood vessels in tissue cultures grown on biodegradable scaffolds in
bioreactor conditions. We give experimental results showing the capability
of our approach to localize a fluorophore-filled 500µm capillary immersed in
an absorbing and scattering medium contained in a cylindrically shaped
bioreactor; these conditions being representative of experiments to be carried
on real tissue cultures.
6633-07, Session 2
Metal-enhanced fluorescence
J. Enderlein, Eberhard Karls Univ. Tübingen (Germany)
We present an overview of the concept of metal-enhanced fluorescence (MEF)
and its manifold applications in biophotonics. Different aspects of MEF are
highlighted: enhancement of excitation by local field amplification,
enhancement of radiative transition, and reorganization of the angular
distribution of radiation. In particular, surface-plasmon coupled emission in
planar systems, and fluorescence enhancement in core-shell nanocavities
are discussed in detail.
6633-08, Session 2
Axially resolved polarization microscopy of
membrane dynamics in living cells
M. Wagner, P. Weber, H. Schneckenburger, Fachhochschule Aalen
(Germany)
Membrane dynamics has a large impact on cellular uptake and release of
various metabolites or pharmaceutical agents. For a deeper understanding
of the cellular processes involved, we used U373-MG human glioblastoma
cells as a model system. As conventional microscopy does not permit to
investigate individual layers in living cells, we used structured illumination
techniques and total internal reflection fluorescence microscopy (TIRFM) to
analyse the plasma membrane and intracellular membranes of living cells
selectively. The optically sectioned images provide a high resolution and the
possibility of 3D reconstruction.
Membranes of living cells were characterized by the membrane marker 6dodecanoyl-2-dimethylamino naphthalene (laurdan). Due to its spectral and
kinetic properties this fluorescence marker appears appropriate for measuring
membrane stiffness and fluidity. After excitation with linearly polarized laser
pulses, membrane fluidity of human glioblastoma cells was determined by
measurements of steady-state and time-resolved fluorescence anisotropy
r(t), since with increasing viscosity of the environment, the rotation of an
excited molecule is impeded. The corresponding time constant tr of molecular
relaxation decreased with temperature and increased with the amount of
cholesterol. In addition, fluorescence anisotropy r(t) values of the plasma
membrane were larger than the values of intracellular membranes for all
temperatures in the range of 16°C < T < 41°C.
80
European Conferences on Biomedical Optics 2007 •
6633-09, Session 2
Direct detection of singlet oxygen generated by
UVA irradiation in phospholipids, human cells,
and skin
J. Baier, T. Maisch, W. Bäumler, Univ. Regensburg (Germany)
UVA light produces deleterious biological effects in which singlet oxygen
plays a major role. These effects comprise a significant risk of carcinogenesis
in the skin and the cataract formation of the eye lens. Singlet oxygen is
generated by UVA light absorption in endogenous molecules present in the
cells. To elucidate the primary processes and sources of singlet oxygen in
tissue, it is a major goal to uncover the hidden process of singlet oxygen
generation, in particular in living tissue.
Singlet oxygen can be directly detected by its luminescence at 1269 nm.
When exposing keratinocytes, HT29 cells, pig skin (ex vivo) or human skin
(in vivo) to UVA laser light (355 nm, 6 J/cm(c)˜), we measured a clear
luminescence signal of singlet oxygen. This is a positive and direct proof of
singlet oxygen generation in cells and skin by UVA light.
Moreover, when exposing pure phosphatidylcholine in an aqueous
suspension to 355nm laser light, singlet oxygen is clearly detected by its
luminescence. This provides evidence that phosphatidylcholine can contribute
to the generation of singlet oxygen when irradiated by UVA light. This is a
very striking result in light of the oxidative damage and gene regulations in
cells caused by singlet oxygen in vitro and in vivo.
6633-10, Session 3
Improvements of laser biomedical spectroscopy
and imaging
V. V. Tuchin, Saratov State Univ. (Russia)
Fundamentals and advances of tissue optical properties controlling as a novel
modality for the improvement of laser biomedical spectroscopy and imaging
will be presented. As a major technology the optical immersion method at
usage of exogenous optical clearing agents (OCAs) will be discussed. Water
transport in a tissue, tissue swelling and hydration at its interaction with an
OCA will be considered. Optical clearing properties of fibrous and cellstructured tissues will be analyzed in the framework of receiving of more
valuable information from spectroscopic and polarization measurements,
confocal microscopy and OCT, as well as from nonlinear spectroscopy, such
as two-photon fluorescence and SHG. In vitro, ex vivo, and in vivo
spectroscopic studies of a variety of human and animal tissues, such as eye
sclera, skin, cerebral membrane (dura mater), gastric tissue, tendon, blood
vessels and blood, will be presented. OCA delivery, tissue permeation and
skin reservoir function will be discussed. Improvements of tissue, cell and
cell flows imaging at optical clearing will be also shown. Some important
applications of tissue immersion technique, such as glucose sensing will be
demonstrated.
6633-11, Session 3
High throughput high content live cell screening
platform
R. Uhl, TILL Photonics GmbH (Germany); H. Harz, S. Neogy, LudwigMaximilians-Univ. München (Germany)
The goal was to develop a light-microscope platform concept which allows
to characterize live cells in microtiterplates with a speed, sensitivity and
versatility unattainable so far.
The goal was achieved by combining several novel technological concepts:
a model-based digital control for a voice coil focus drive, scanner technology
to follow a continuously moving sample during image acquisition, thus
avoiding the usual stop&go, fast sectioning capabilities by using slit-scan
confocal concepts, motorized dual emission image registration, and integrated
environmental control.
The novel platform is a highly compact, highly rigid structure which could
well become a new industry standard.
6633-12, Session 3
Techniques and applications of digital
holographic microscopy for life cell imaging
B. Kemper, P. Langehanenberg, J. Schnekenburger, G. von Bally, Univ.
Münster (Germany)
In connection with microscopy, digital holography provides contact-less,
marker-free, quantitative phase-contrast imaging for modular Integration into
commercial microscopes [1]. In this way, digital holographic metrology
facilitates a combination with established microscopy techniques like Laser
Scanning Microscopy or Fluorescence imaging as well as with optical Laser
micromanipulation methods. Particularly, the feature of (subsequent)
numerical auto focus adjustment enables applications in the field of life cell
analysis. Here, prospects for long term time-lapse investigations in toxicology
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Conference 6633: Biophotonics 2007: Optics in Life Science
and cancer research [2] as well as for monitoring of fast dynamic processes
like shape variations are opened up. The evaluation of the obtained
quantitative phase contrast provides data for thickness monitoring and cell
tracking as well as for the observation of cell swelling kinetics due to osmotic
stimulation and optical micro manipulation. Furthermore, the integral refractive
index of cells and its statistics can be determined. Results from investigations
on toxin induced reactions of cancer cells like apoptosis, cellular refractive
index measurements, the cell response to optical manipulation, shape
variations of human erythrocytes and reactions of epithelia cells due to
different substrates demonstrate digital holographic microscopy application
fields for quantitative life cell imaging. [1] G. von Bally et al.: New ways for
marker-free life cell and tumor analysis, in: J. Popp, M. Strehle (Eds.):
Biophotonics: visions for a better health care, Wiley, 301-360, 2006. [2] B.
Kemper, D. Carl, J. Schnekenburger, I. Bredebusch, M. Schäfer, W. Domschke,
G. von Bally, Investigation of living pancreas tumor cells by digital holographic
microscopy, J. Bio. Opt. 11 034005 (2006).
6633-13, Session 4
6633-16, Session 4
Multicolor single molecule spectroscopy for the
study of complex interactions and dynamics
Autofocus algorithms for digital-holographic
microscopy
P. Tinnefeld, D. Fetting, R. Kasper, Bielefeld Univ. (Germany)
P. Langehanenberg, B. Kemper, G. von Bally, Univ. Münster (Germany)
Digital-holographic metrology enables quantitative phase contrast microscopy
of reflective and (partially) transparent samples. In this way new applications
are opened up for non-destructive investigations of technical samples as
well as for marker-free and time-resolved analysis of cell biological processes
[1].
Especially studies on long-term biological processes require permanent focus
position readjustment to maintain an optimum image quality. With digital
holographic microscopy subsequent digital holographic focusing by variation
of the propagation distance of the reconstructed data is made possible. Here,
besides an optimization of the reconstruction parameters [2], the
determination of the optimal propagation distance is of particular importance.
This is performed by evaluation of the image definition as a function of the
propagation distance.
At the Laboratory of Biophysics different image definition quantification
algorithms were adapted to the requirements of digital holographic
microscopy. The object-dependent optical absorption properties are taken
into consideration in order to obtain robust and reliable algorithms.
Automatic focus tracking is demonstrated on investigations with digital
holographic microscopy on both reflective technical objects and (partially)
transparent cell biological probes.
[1] G. von Bally, B. Kemper et al., “New Methods for Marker-Free Live Cell
and Tumor Analysis (MIKROSO)” in: J. Popp, M. Strehle (Eds.): Biophotonics.
Visions for better Health Care, Wiley, 301 - 360, 2006
[2] D. Carl, B. Kemper, G. Wernicke, G. von Bally, “Parameter-optimized digital
holographic microscope for high resolution living cell analysis”, Appl. Opt.,
43, 6536-6544, 2004
6633-14, Session 4
Analysis of cellular structure and dynamics with
digital holography microscopy
P. P. Marquet, Ctr. Hospitalier Univ. Vaudois (Switzerland); T. Colomb, F.
Charrière, École Polytechnique Fédérale de Lausanne (Switzerland); J.
G. Kühn, Ecole Polytechnique Fédérale de Lausanne (Switzerland); Y.
Emery, Lyncée Tec SA (Switzerland); B. Rappaz, P. Jourdain, P. J.
Magistretti, École Polytechnique Fédérale de Lausanne (Switzerland)
No abstract available
6633-15, Session 4
Dynamic in vivo analysis of drug induced actin
cytoskeleton degradation by digital holographic
microscopy
J. Schnekenburger, I. Bredebusch, W. Domschke, G. von Bally, B.
Kemper, Univ. Münster (Germany)
Background: The actin cytoskeleton mediates a variety of crucial cellular
functions as migration, intracellular transport, exocytosis, endocytosis and
force generation. The highly dynamic actin fibers are therefore targets for
several drugs and toxins. However the study of actin interfering processes
by standard microscopy techniques fails in the detailed resolution of dynamic
spatial alterations required for a deeper understanding of toxic effects. Here
we applied digital holographic microscopy in the online functional analysis
of the actin cytoskeleton disrupting marine toxin Latrunculin B.
Methods: For scanning electron microscopy (SEM) PaTu 8988S pancreas
tumor cells were fixated with 1 % glutaraldehyde. Dehydrated and dried cells
were coated with platinum and carbon and analyzed using a standard SEM.
Fluorescence microscopy was performed by staining fixated cells with
fluorescein labelled phalloidin. For digital holographic investigations the cells
were trypsinized, seeded sub confluent on glass slides or tissue culture plates,
European Conferences on Biomedical Optics 2007 •
cultured for 24 hrs and analyzed at room temperature and normal atmosphere.
Results: SEM and fluorescence microscopy showed a rapid Latrunculin B
induced cell flattening and actin fiber degradation in pancreas tumor cells.
The digital holographic in vivo analysis of the drug dependent cellular
processes demonstrated a collapse of the cell within 3 min. The spatial
resolution of the morphological alterations revealed an unequal degradation
of the actin cytoskeleton. The collapsing cells developed rapidly membrane
covered impressions indicating a delayed collapse along stronger actin
bundles.
Conclusions: The marker free, non-destructive online analysis of cellular
morphology and dynamic spatial processes in living cells by digital holography
offers new insights in actin dependent cellular mechanisms. Digital
holographic microscopy was shown to be a versatile tool in the screening of
toxic drug effects.
Most biological processes are governed by assemblies of several dynamically
interacting molecules. We have developed confocal multicolor single-molecule
spectroscopy with optimized detection sensitivity on three spectrally distinct
channels for the study of biomolecular interactions and FRET between more
than two molecules. Using programmable acousto-optical devices as
beamsplitter and excitation filter, we overcome some of the limitations of
conventional multichroic beamsplitters and implement rapid alternation
between three laser lines. This enables to visualize the synthesis of DNA
three-way junctions on a single-molecule basis and to resolve seven
stoichiometric subpopulations as well as to quantify FRET in the presence of
competing energy transfer pathways. A merit of the method is the ability to
study correlated molecular movements by monitoring several distances within
a biomolecular complex simultaneously. Finally, a new strategy to improve
observation times by reducing photobleaching will be presented.
6633-17, Session 4
High resolution spectral optical coherence
microscopy assists diabetes research
R. A. Leitgeb, M. L. Villiger, T. Lasser, École Polytechnique Fédérale de
Lausanne (Switzerland); P. Meda, Univ. de Genève (Switzerland); W.
Pralong, École Polytechnique Fédérale de Lausanne (Switzerland)
Spectral optical coherence microscopy (SOCM) merges the advantages of
spectral OCT with respect to imaging speed and sensitivity with high resolution
of microscopy. OCT tomograms exhibit high contrast that can be compared
to images of stained histology. The drawback of SOCT of loosing depth range
using microscope objectives for high resolution imaging has recently been
overcome by employing novel confined illumination geometries. 3D cell
imaging with high contrast and high imaging speed along an extended focal
depth range is demonstrated. The use of an axicon lens and a telescopic
imaging system creates a cylindrically symmetric interference pattern with a
strong central lobe that serves as laterally highly confined illumination needle.
Nearly constant transverse resolution of ~1.5µm along a focal range of 200µm
is experimentally verified with a maximum sensitivity of 105dB. A broad
bandwidth Ti:Sapphire laser allowes for an axial resolution of 3µm in air. The
xf-FDOCM system is applied to the imaging of mouse and rat pancreas. The
amount and functionality of Langerhans-islets in the pancreas are of particular
interest in diabetes research. They contain the secretory ß-cells which produce
insulin. The possibility to localize and characterize these islets within an
unprepared pancreas sample is a first promising step towards in-vivo
diagnosis of ß-cell functionality. The results demonstrate the high potential
of FDOCM for small animal imaging, since no sample labeling, or staining,
nor sample slicing is needed.
6633-18, Session 4
Ultrafast dynamics in a live cell irradiated by
femtosecond laser pulses
H. Kawano, C. Hara, The Institute of Physical and Chemical Research
(Japan); T. G. Etoh, Kinki Univ. (Japan); A. Miyawaki, The Institute of
Physical and Chemical Research (Japan)
An ultrafast video microscope (UVM), the frame rate of which reaches one
million per second has been developed. Our UVM system provides pictures
with high-contrast and high-resolution by using special devices for differential
interference contrast (DIC), phase contrast, or dark field imaging. It allows
us to observe fast events which occur in live cells when irradiated by ultrashort
laser pulses. Femtosecond laser pulses can be used to manipulate, stimulate,
and destroy specific cells and organelles under the microscope. The
irradiation of such an intense laser immediately results in some physical
events, such as shock wave generation, micro cavitation, and photoporation.
We investigate biophysical mechanisms underlying the ultrafast processes.
Our data will contribute to development of new bio-imaging modalities, which
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Conference 6633: Biophotonics 2007: Optics in Life Science
implement cell surgery. We also present a new method to observe side views
of live cells on a substrate. We used a polymer material CYTOP as the
substrate for HeLa cells. CYTOP has a refractive index of 1.34, which is
close to 1.33 of water. When the CYTOP substrate was set perpendicular to
the sample stage of the microscope, the organelles of HeLa cells were clearly
seen in DIC images by using a water-immersion 60X objective with a
sufficiently long working distance at a rate of one million frames per soecond.
We investigate generation of microbubbles beneath the plasma membranes
with a time resolution of one microsecond for the purpose of improving the
efficiency of photoporation.
6633-19, Session 4
Non-linear and ultra high-speed imaging for
explorations of the murine and human heart
L. Kaestner, P. Lipp, Univ. des Saarlandes (Germany)
Atrial fibrillation is the most common type of cardiac arrhythmias, it causes
stroke and therefore morbidity is an issue. The pathopysiology of these
arrhythmias is complex and still far from being comprehensively explored.
Atrial fibrillation is caused by an ectopic excitation in cardiac myocytes, which
are triggered by spontaneous Ca2+-relase of the sarcoplasmic reticulum.
Once initiated, electrical and structural remodelling causes the persisting of
atrial fibrillation. Examples for structural remodelling are hypertrophy and
fibrosis. With recent developments of optical technologies the phenomena
mentioned above can be visualised: The cause for the “cellular arrhythmias”,
namely Ca2+-sparks and Ca2+-waves leading to delayed after depolarisations
or early action potentials, are best acquired by subcellular calcium-imaging
with framerates between 200 and 400 Hz on isolated cardiac myocytes. In
contrast, the structural remodelling can be observed in unstained tissue of
murine and human hearts by visualising extra cellular matrix components
(e.g. collagen) with second harmonic generation (SHG) imaging. SHG-imaging
can be combined with the detection of tissue auto-fluorescence in a second
channel. It is thus possible to simultaneously gain information about the
amount of the extracellular matrix and the condition of the cells by observing
the mount of cellular flavoprotein to test for apoptotic activity. The ultra highspeed as well as the non-linear imaging techniques provide in their
combination an extremely powerful tool to solve essential physiological and
pathophysiological questions in animal models as well as in the human patient.
6633-20, Session 4
Improving the optical contrast of backscattering
signal in reflectance-based imaging with gold
nanoshells
J. C. Y. Kah, National Univ. of Singapore (Singapore); T. Chow, Nanyang
Technological Univ. (Singapore); M. C. Olivo, National Cancer Ctr.
Singapore (Singapore); B. Ng, Nanyang Technological Univ. (Singapore);
C. J. R. Sheppard, National Univ. of Singapore (Singapore)
The application of gold nanoparticles as contrast agent in optical bioimaging
is well appreciated, but limited to a narrow excitation range due to its rather
invariable optical resonance typically at 520 nm. Compared to gold
nanoparticles, the optical response of gold nanoshells can be tuned to match
the higher excitation wavelength of many clinical imaging modalities such as
the OCT. In this study, we demonstrate the tunability of gold nanoshells to
improve the optical contrast of backscattering signal by synthesizing them
in two different size configurations with optical responses matched to
operating wavelength of two reflectance-based imaging modalities. The gold
nanoshells were synthesized and conjugated to antibodies for in vitro
demonstration of their selective optical contrast in cancer cells over normal
cells under the confocal reflectance microscopy. The OCT signals from these
particles were analyzed with their optical scattering properties extracted and
compared to other particle scatterers and intrinsic tissue scattering using
appropriate phantom models. We have shown that gold nanoshells were
able to elicit an optical contrast to discriminate between cancerous and normal
cells under the confocal reflectance microscopy. Compared to other particle
scatterers of similar sizes, gold nanoshells possess higher backscattering
coefficients and hence result in a much higher backscattering signal under
OCT. The backscattering signals were such that the optical contrast of these
nanoshells, when embedded in tissue phantoms, was strong enough to be
visible under OCT. The optical tunability of gold nanoshells thus enables
them to improve the optical contrast of backscattering signals in various
optical imaging modalities with different operating wavelengths of light.
6633-21, Session 5
Tip-enhanced Raman scattering: pushing the
limits of structural analysis
V. Deckert, Institute for Analytical Sciences Dortmund (Germany)
No abstract available
82
European Conferences on Biomedical Optics 2007 •
6633-22, Session 5
Wide field surface plasmon-enhanced total
internal reflexion fluorescence microscopy:
application to live cell imaging
V. Studer, Y. Goulam-Houssen, E. Le Moal, A. Simon, Z. Lenkei, E. Fort,
École Supérieure de Physique et de Chimie Industrielles (France)
Total internal reflection fluorescence microscopy (TIRFM) employs the unique
properties of an induced evanescent wave to selectively illuminate and excite
fluorophores in a restricted specimen region immediately adjacent to the
glass coverslip interface. TIRFM is employed to investigate the interaction of
molecules with surfaces, an area that is of fundamental importance to a wide
spectrum of disciplines in cell and molecular biology. Living cells in particular
provide excellent candidates for TIRFM investigations to study membranes
phenomena and adhesion interactions. Today, the technique is gaining
popularity in part because new high numerical aperture microscope objective
lenses have been developed (NA?1.45).
We will present an alternative technique to Total Internal Reflection
Fluorescence Microscopy (TIRFM) which takes advantage of surface plasmon
(SP) properties of a metallic thin-film [1]. SP cross-emission and near-field
coupling to fluorophores provide many advantages: enhanced fluorescence
signal, increased confinement, and reduction of the photobleaching as well
as of the background noise.
One major limitations of standard TIRFM in cell imaging come from the
scattering of the excitation light by the sample. The resulting fluorescence
from areas outside the surface can obscure important fluorescent information
concerning membranes phenomena. Transmission through a metallic thin
film mediated by SP permits both excitation as well as fluorescence detection
confinement and filtering resulting in an unmatched signal to noise ratio.
We will present applications of this technique to live cell imaging.
6633-23, Session 5
Surface-enhanced Raman scattering substrates
based on nanometre scale structures on
butterfly wings
J. J. Moger, N. L. Cornes, G. Winter, P. Vukusic, C. P. Winlove, The Univ.
of Exeter (United Kingdom)
Surface-enhanced Raman scattering (SERS) has received a great deal interest
as an analytical tool due to its potential for single molecule characterisation.
Many methods for preparing SERS-active substrate have been reported.
These range from nano-particle based methods, which lack reproducibility,
to highly reproducible nano-arrays requiring time consuming and costly
preparation. We show that highly reproducible SERS can be achieved by
applying a metallic coating to the brightly coloured regions of the graphium
butterfly wing. Electron microscopy reveals the wing exhibits nanostructures
with comparable dimensions to the roughness scale of SERS substrates.
SERS measurements performed on wings coated with 60 nm of silver display
enhancement factors of six orders of magnitude with no apparent background
contribution from the wing. Immunoassaying using our preparation coated
with a monoclonal antibody demonstrates very high sensitivity and
reproducibility.
6633-24, Session 6
Recent progresses in optical trap-and-stretch of
red blood cells
A. E. T. Chiou, G. B. Liao, Y. Chen, A. V. Karmenyan, C. Lin, National
Yang-Ming Univ. (Taiwan)
The study of visco-elastic properties of cellular membranes and of living
cells as a whole to correlate with their biochemical and physiological functions
has been a subject of great interest in recent years. Of the several methods
to apply external forces to a cell either locally or distributed over the whole
cell to probe its mechanical response and the associated cellular mechanosignal transduction, optical techniques have been studied intensively because
optical forces can be applied to a living cell non-invasively without any
mechanical (or physical) contact.
A popular approach is the optical stretcher with the aid of a counterpropagating dual-beam trap first demonstrated by Guck et al. in 2001 to
study the elasticity of red blood cells (RBCs) osmotically swollen into spherical
shape. A major advantage of this approach is the potential for high-throughput
measurement by incorporating an appropriate micro-fluidic system to control
the flow of each sample cell into the trapping zone. The disadvantage of the
approach is the requirement of fairly high optical power (~ hundreds of mW);
besides, all the optical stretching of RBCs in a counter-propagating dualbeam optical stretcher reported to date were accomplished with osmotically
swollen RBCs in spherical shape, rather than in their physiological bo-concave
shape. More recently Bronkhorst et al. applied multiple focal beam-spots to
trap and bend (or fold) bo-concave human red blood cells and measured
their recovery time.
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In this paper, we briefly review the earlier approaches and results of optical
stretching of RBCs and present some preliminary experimental data on optical
trap-and-stretch of human bo-concave RBCs by two optical approaches,
namely the counter-propagating dual-beam trap and the oscillatory optical
tweezers. Potential advantages and disadvantages of these approaches in
comparison with other approaches will be discussed.
6633-25, Session 6
Lasers as unique tools for cell manipulation
K. Schütze, P.A.L.M. Microlaser Technologies GmbH (Germany)
No abstract available
6633-26, Session 6
Studying red blood cell agglutination by
measuring electrical and mechanical properties
with a double optical tweezers
A. Fontes, H. P. Fernandes, A. A. de Thomas, L. C. Barbosa, M. d. L.
Barjas-Castro, C. L. Cesar, Univ. Estadual de Campinas (Brazil)
The red blood cell (RBC) viscoelastic membrane contains proteins and
glycolproteins embedded in, or attached, to a fluid lipid bilayer and are
negatively charged, which creates a repulsive electric (zeta) potential between
the cells and prevents their aggregation in the blood stream. The basis of the
immunohematologic tests is the interaction between antigens and antibodies
that causes hemagglutination. The identification of antibodies and antigens
is of fundamental importance for the transfusional routine. This agglutination
is induced by decreasing the zeta-potential through the introduction of artificial
potential substances. This report proposes the use of the optical tweezers to
measure the membrane viscosity, the cell adhesion, the zeta-potential and
the size of the compact layer of charges (CLC) formed around the cell in an
electrolytic solution. The adhesion was quantified by slowly displacing two
RBCs apart until the disagglutination. The CLC was measured using the force
on the bead attached to a single RBC in response to an applied voltage. The
zeta-potential was obtained by measuring the terminal velocity after releasing
the RBC from the optical trap at the last applied voltage. For the membrane
viscosity experiment, we trapped a bead attached to RBCs and measured
the force to slide one RBC over the other as a function of the relative velocity.
After we tested the methodology, we performed measurements using antibody
and potential substances. We observed that this experiment can provide
information about cell agglutination that helps to improve the tests usually
performed in blood banks. We also believe that this methodology can be
applied for measurements of zeta-potentials in other kind of samples.
6633-27, Session 6
Automated microinjection system for adherent
cells
S. Youoku, Y. Suto, M. Ando, A. Ito, Fujitsu Labs. (Japan)
We have developed an automated microinjection system that can handle
more than 500 cells an hour.
Microinjection injects foreign agents directly into cells using a micro-capillary.
It can randomly introduce agents such as DNA, proteins and drugs into various
types of cells. However, conventional methods require a skilled operator and
suffer from low throughput.
The new automated microinjection techniques we have developed consist
of a Petri dish height measuring method and a capillary apex position
measuring method.
The dish surface height is measured by analyzing the images of cells that
adhere to the dish surface. The contrast between the cell images is minimized
when the focus plane of an object lens coincides with the dish surface. We
have developed an optimized focus searching method with a height accuracy
of ±0.2 µm.
The capillary apex position detection method consists of three steps: rough,
middle, and precise. These steps are employed sequentially to cover capillary
displacements of up to ±2 mm, and to ultimately accomplish an alignment
accuracy of less than one micron.
Experimental results using this system we developed show that it can
introduce fluorescent material (Alexa488) into adherent cells, HEK293, with
a success rate of 88.5%.
6633-49, Poster Session
Evaluation of drug release from PLGA
nanospheres containing betametazon
been focused on the use of particles prepared from polyesters like PLGA,
due to their biocompatibility and resorbability through natural pathways [4,5].
In this research poly (d,l-lactide-coglycolide acid) (PLGA) as polymeric
nanospheres, poly(vinyl alcohol) (PVA) with 87-89% hydrolysis degree as
surfactant and distilled water as suspending medium were used. The
encapsulated drug was Betametazone.
The nanospheres were prepared by an emulsion-solvent evaporation method.
A solution of 40 mg of PLGA and 20 mg of betametazon in 4 mL of dichloro
methane, were mixed with 10mL of 0.5, 1, 2 and 3% PVA aqueous solution
separately. These mixtures were then homogenized for 2 minutes by 24000
rpm vortex and then sonicated using an ultrasound micro tip probe with an
output power of 55W for 5 minutes. The nanospheres were first recovered
by ultracentrifugation, washed twice with water and then freeze-dried.
The nanospheres characterized by photon correlation spectroscopy (PCS)
and scanning electron microscopy (SEM) (Fig. 1). The amount of drug release
was determined by HPLC. In emulsion-solvent evaporation technique, time
of ultrasound exposure, surfactant content in the formulation and evaporation
rate of organic solvents were considered as formulation variables.
The results showed that the increase in the exposure time leads to a reduction
in the nanosphere’s mean diameter. It was also observed that the
granulometric distribution became narrower as the amount of PVA was
increased and smaller mean diameter was achieved due to faster solvent
evaporation rate (Fig 2). The amount of emulsifier concentration and particle
size affect release time from nanospheres. While about 55% of drug from
3% PVA concentrated nanospheres were released after approximately 250
h, only 35% and 17% of betametazone were released from nanospheres
with 1 and 0.5% PVA containing particles, respectively (Fig 3).
6633-50, Poster Session
A field test study of our non-invasive thermal
image analyzer for deceptive detection
S. Sumriddetchkajorn, A. Somboonkaew, National Electronics and
Computer Technology Ctr. (Thailand); T. Sodsong, I. Promduang, N.
Sumriddetchkajorn, Office of the Council of State (Thailand)
We have developed a non-invasive thermal image analyzer for deceptive
detection (TAD2) where far-infrared data around the periorbital and nostril
areas are simultaneously. Measured change in maximum skin temperature
around two periorbital regions is converted to a relative blood flow velocity.
A respiration pattern is also simultaneously determined via the ratio of the
measured maximum and minimum temperatures in the nostril area. In addition,
our TAD2 employs a simple normalized cross correlation scheme to
independently track locations of the two periorbital and nostril areas. Our
field case study from 7 subjects based on two real crime scenes and with
the use of our baseline classification criteria shows two-fold improvement in
classification rate compared to our analysis using either the periorbital or
nostril area alone.
6633-51, Poster Session
Singlet oxygen luminescence reveals oxygen
depletion in albumin suspension
J. Baier, M. Loibl, J. Regensburger, T. Maisch, W. Bäumler, Univ.
Regensburg (Germany)
The direct detection of singlet oxygen can be performed by measuring timeresolved its luminescence at 1269 nm. However, the shape of the
luminescence signal is critically affected by the oxygen concentration, which
can decrease in case oxygen is consumed due to oxidative reactions with
lipid or proteins.
Singlet oxygen was generated by exciting a photosensitizer (TMPyP) in
aqueous solution (H2O or D2O) of bovine serum albumin. The luminescence
signal of singlet oxygen significantly changed with irradiation time. The longer
the exposure to laser light the shorter the rise time and the longer the decay
time. A sensor for oxygen concentration revealed a rapid decrease of oxygen
concentration (oxygen depletion). The extent and time course of oxygen
depletion in aqueous albumin solution depends on the amount of light energy
and the solvent. In H2O the oxygen depletion was achieved after about 450
seconds and for D2O after about 50 seconds. Prior to irradiation,
chromatography showed that most of the sensitizer molecules were not bound
to albumin. Thus, the luminescence signal was predominantly due to singlet
oxygen generated by unbound TMPyP in H2O or D2O, whereas in D2O the
chemical quenching of singlet oxygen is enhanced by the long lifetime of
about 68 µs. The results in solutions with albumin were idenatical to
experiments in solution, where oxygen was consecutively replaced by
nitrogen.
Oxygen consumption should be considered when evaluating the course of
singlet oxygen luminescence, in particular in vitro and in vivo.
M. E. Khosroshahi, J. Tavakoli, M. Enayati, S. Shafiei, Amirkabir Univ. of
Technology (Iran)
Biodegradable colloidal particles have received considerable attention as a
possible means of delivering drugs and genes [1-3]. Special interest has
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Conference 6633: Biophotonics 2007: Optics in Life Science
6633-52, Poster Session
Development and performance characteristics
of flash lamp pumped Yb:YAG, Cr:Tm:Ho:YAG,
Er:Tm:Ho:YLF laser sources and investigation of
their potential biological applications
A. A. Serafetinides, D. N. Papadopoulos, N. K. Karadimitriou, B. J.
Klinkenberg, National Technical Univ. of Athens (Greece)
Laser ablation for the formation of apodized patterns on intraocular lenses,
instead of the conventional injection molding, has been proved to be a very
promising new technique. For the precise lenses ablation, the use of suitable
laser wavelength and pulse duration, resulting in a small optical penetration
depth in lens and confinement of the energy deposition in a small volume, as
well as the reduced thermal damage to the surrounding tissue, is essential.
Mid-infrared laser wavelengths, at which the organic simulator absorption
coefficient is large, meet well the above conditions. Towards the complete
understanding of the intraocular lens ablation procedure and therefore the
choice of the optimum laser beam characteristics for the most accurate,
efficient and safe surgical application, the comparative study of various midinfrared laser sources is of great interest.
In this work we investigate the potential of the development of three different
mid-infrared laser sources, namely an Yb:YAG, a Cr:Tm:Ho:YAG and an
Er:Tm:Ho:YLF laser oscillator, operating at 1029 nm, 2060 nm and 2080 nm
respectively and their ability in forming patterns on biomaterials. Pumping
was achieved with conventional Xe flash lamps in a double elliptical pump
chamber. A properly designed Pulse-Forming-Network capable of delivering
energy up to 800 J, in variable lamp illumination durations is used. Several
hundreds of mJoules were achieved from the Yb:YAG oscillator and several
Joules from the Ho:YAG and Ho:YLF oscillators. Free running and Q-switched
laser operation studies and preliminary experiments on laser and biomaterials
(biopolymers and animal tissues) interactions will be reported.
ACKNOWLEDGEMENTS: The project described in this article is co-funded
by the European Social Fund (75%) and National Resources (25%) Operational Program for Educational and Vocational Training II (EPEAEK II)
and particularly the Program Pythagoras II (Project: “Laser beam and
ophthalmic tissue interactions - correlation with the physical parameters of
the radiation”).
6633-53, Poster Session
A fiber optic sensor for measuring respiratory
changes in chest-circumference
Fluctuations in photon emission in the course of 24 h period were
demonstrated for each location of the hand. Mean photon emission over the
24 h period differed both between subjects and hand locations. To detect a
pattern in the fluctuations, the mean value for each location in each
experimental session was utilized for normalization of the corresponding data
of that time course and to calculate fluctuations during the course of 24 h for
each anatomical location.
Fluctuation in the intensity of photon emission in the course of 24 h was
more at dorsal sides of hands than palms. Fluctuations in both palms were
highly and significantly correlated. The fluctuations in dorsal locations also
were highly and significantly correlated. In contrast, correlations between
fluctuations in palm and dorsal sides were rather low.
During the 24 h period a change in left-right symmetry occurred both for the
dorsal sides and the palms. Photon emission at the left locations was
predominantly during night, while the right locations emitted more during the
day.
It is concluded that intensity as well as left-right symmetry vary diurnally,
suggesting similar fluctuations in endogenous pro- and anti-oxidative
capacities. Such diurnal rhythms have been estimated for rats. Lipid
peroxidation levels increased progressively during the night and started to
decline in the morning. In future studies the influence of other factors like
hand temperature are related to the observed rhythms.
6633-55, Poster Session
Spectral analysis of photoinduced delayed
luminescence from human skin in vivo
F. F. Musumeci, Univ. di Catania (Italy) and LNS-INFN (Italy); L. L.
Lanzanò, Instituto Nazionale di Fisica Nucleare (Italy) and Univ. di
Catania (Italy); S. S. Privitera, LNS-INFN (Italy) and Univ. di Catania
(Italy); S. S. Tudisco, Instituto Nazionale di Fisica Nucleare (Italy); A. A.
Scordino, Instituto Nazionale Di Fisica Nucleare (Italy)
The Delayed Luminescence (DL), induced by a Nitrogen-Dye Laser has been
measured in vivo in the forearm skin of healthy volunteers of different sex
and age. To reach this goal an innovative instrument able to detect, in single
photon counting mode, the spectrum and the time trend of the DL emission
has been developed. Age, sex and seasonal variations have been investigated.
The differences encountered between the subjects and the potential
development of a new analysis technique based on this phenomenon are
discussed.
6633-56, Poster Session
M. Pinchas, A. Avraham, A. Babchenko, I. Faib, S. Mizrahi, M. Nitzan,
Jerusalem College of Technology (Israel)
Improving spFRET by confining molecules in
nanopipettes
The origin of the respiratory-induced fluctuations in arterial blood pressure
was attributed either to direct mechanical effect of respiratory-induced
thoracic pressure changes on the arteries or to sympathetic or
parasympathetic tone oscillations. In order to study the temporal relationship
between respiration and peripheral hemodynamics, a novel sensor for the
measurement of the respiratory-induced changes in chest-circumference has
been developed, and was used simultaneously with the
photoplethysmographic (PPG) signal, which reflects the cardiac-induced
increase in the tissue blood volume during systole. The sensor principle is
based on the dependence of light transmission through bent optic-fiber on
its radius of curvature. Some light rays, which are totally reflected in the
core-cladding surface in straight fiber, may escape through the cladding when
the fiber is bent, if the angle to the surface normal becomes lower than the
critical angle. The optic-fiber sensor was connected to an elastic chest belt
which increased its radius of curvature during inspiration, resulting in higher
light transmission. Two PPG devices, composed of infrared LED and
photodetector were attached to a finger and to forehead of several healthy
persons. The PPG baseline, which is inversely related to tissue blood volume,
changed during deep respiration, for both finger and forehead, but the change
was not the same for all examinations, probably due to the different innervation
by the autonomic nervous system.
J. Vogelsang, S. Doose, M. Sauer, P. Tinnefeld, Univ. Bielefeld (Germany)
In recent years Fluorescence Resonance Energy Transfer (FRET) has been
widely used to determine distances, observe distance dynamics, and monitor
molecular binding at the single-molecule level. A basic constraint of singlepair FRET studies is the limited distance resolution owing to low photon
statistics. We demonstrate that by confining molecules in nanopipettes (50100 nm diameter) spFRET can be measured with improved photon statistics
reducing the width of FRET proximity ratio (PR) histograms. This increase in
distance resolution makes it possible to reveal subpopulations and dynamics
in biomolecular complexes. The confinement further allows extending singlemolecule investigations towards weaker interactions since higher
concentrations can be used.
6633-57, Poster Session
Analyzing the influence of contact-induced
quenching processes on Förster resonance
energy transfer
R. Brune, S. Doose, M. Sauer, Univ. Bielefeld (Germany)
6633-54, Poster Session
Spontaneous ultra-weak photon emission from
human hands varies diurnally
M. Cifra, Czech Technical Univ. in Prague (Czech Republic) and Institute
of Photonics and Electronics of Academy of Sciences (Czech Republic);
E. P. A. Van Wijk, International Institute of Biophysics (Germany); R. Van
Wijk, Univ. Utrecht (Netherlands)
Ultra-weak photon emission in the visible range is generally associated with
oxidative metabolism and oxygen radical activity. In this study the emission
was measured on palm and dorsal side of left and right hand by means of a
low noise photomultiplier system. We studied the dynamics of this photon
emission in a 24 h period by recording photon emission with 2 h interval in 5
experimental sessions, utilizing strict protocols for dark adaptation and
recording of subjects.
84
European Conferences on Biomedical Optics 2007 •
Experiments based on Förster resonance energy transfer (FRET) are widely
used to obtain information about conformational dynamics of biomolecular
systems. To reliably measure FRET, accurate knowledge of photophysical
properties of the used fluorophores is indispensable. In high FRET constructs
donor (D) and acceptor (A) fluorophores can often approach each other close
enough for electronic interactions. When separated by distances on the order
of van der Waals radii, photophysical properties can be changed reversibly,
opening new non-radiative relaxation pathways, or irreversibly, chemically
altering the fluorophores. Even transient contacts can thus compromise
accurate FRET measurements.
We investigated various FRET pairs built of commercially available organic
fluorophores commonly used in single-molecule fluorescence spectroscopy.
To study competing processes under D-A contact we labeled TMR and Cy3B
(as D) to the thiol group of cystein (Cys) and Alexa 647 and Atto 647N (as A)
to the amino group of Cys. Absorption spectroscopy, steady-state
fluorescence spectroscopy and time-correlated single-photon counting were
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6633-58, Poster Session
shift are same. The average is 1009cm-1, 1164cm-1 and 1523cm-1. It proved
the same material product the Raman spectrum. Following the time passed
the intensity of Raman peak didn’t decreased. It proved that the wavelength
488.0nm and 514.5nm can’t lead to material decompose obviously which
can product Raman spectrum. The Raman spectrum relative intensity (Ir) is
different after sample (normal and cancers) excited by 488.0nm. The
parameter Ir is 62.9‰, 183‰ and 74‰ of normal, colon cancer and rectum
cancer separately. When sample was excited by 514.5nnm, there is only
change on relative intensity of Raman peak. The parameter Ir is 80.5‰, 112‰
and 55‰of normal, colon cancer and rectum cancer separately. So we can
obtain the parameter ß. It is obvious that ßnormal\>1, ßcolon<1, ßrectum<1.
We often use ß<1 to identification cancer. The parameter ß is a major way
detection cancer and the parameter a and ?? are auxiliary way.
Sonoluminescence from ultrasound contrast
agent microbubbles
6633-61, Poster Session
P. A. Campbell, P. A. Prentice, Univ. of Dundee (United Kingdom)
MUSES: MUlti SEnsors Sphere
Ultrasound Contrast Agents (UCAs) are suspensions of microscopic cavitation
nuclei, typically consisting of a low diffusivity gas core, stabilised by an
encapsulating shell of a biocompatible material such as a phospholipid or
denatured protein. UCAs were originally developed to enhance the
echogenecity of the vascular system to diagnostic ultrasound, but have also
shown promise in the role of intracellular molecular delivery.
In this paper we present preliminary data on ‘Contrast Agent
SonoLuminescence’ or CASL, which involves the conversion of acoustic
energy into light, and its subsequent emission, upon exposure to bursts of
ultrasound. The dependence of the luminescent output on ultrasonic
parameters such as pulse duration and position within the field are observed
and discussed. In particular, a pressure amplitude threshold is identified,
below which no CASL from microbubbles is detected. Additionally, the
containing medium within which the microbubbles are suspended at the time
of insonation, is seen to have a profound impact on the light output.
Further differences in the characteristics of the luminescence derived from
two different types of UCA, specifically OptisonTM and SonoVueTM, under
equivalent conditions are highlighted. We speculate on the underlying reasons
behind these differences and outline future work required to gain further
insight. Possible applications of CASL, such as the monitoring of cavitation
induced bioeffects, are also discussed.
S. S. Tudisco, L. L. Lanzanò, Instituto Nazionale di Fisica Nucleare (Italy)
and Univ. di Catania (Italy); F. F. Musumeci, Univ. di Catania (Italy); S. S.
Privitera, Instituto Nazionale di Fisica Nucleare (Italy) and Univ. di
Catania (Italy); A. A. Scordino, Instituto Nazionale Di Fisica Nucleare
(Italy) and Univ. di Catania (Italy)
6633-59, Poster Session
In the work is realized the detection of proteins structural reconstruction by
studying the emitting deactivation processes of the electronic photoexcitation
energy of luminescent probes, and also triplet-triplet (T-T) energy transfer
between the polar and nonpolar molecules of luminescent probes, connected
with the proteins. The application of luminescent probes, with considerably
higher intensity of luminescence than the chromophores of protein, is
promising for investigating proteins structural dynamics, because it makes it
possible to carry out fastening probes in the interesting regions of protein
globule.
The reagents of the xanthene row were selected as the polar luminescent
probes: eosin, erythrosine. As non-polar luminescent probes was taken
polycyclic aromatic hydrocarbons (PAH): anthracene and pyrene. Selection
of the PAH is determined by the fact that some of these compounds reveal
cancerogenic and mutagenic properties; therefore studies of PAH interaction
with the transport proteins are immediate for medicine. Structural
reconstruction of the human serum albumin and proteins of the blood plasma
was stimulated by the surfactants - sodium dodecylsulfate (SDS), and also
under the action of salts of heavy metals and another denaturants. The studies
of the dependence of intensity and kinetics of the eosin phosphorescence
damping on the concentration SDS made it possible to establish that in the
system protein - eosin are manifested hydrophobic interactions, which change
at the stage of intramolecular structural reconstruction of protein. It is
established that the index of polarity of PAH and pyrene is sensitive to the
processes of the protein denaturation under the SDS action. The index of
polarity was determined by the ratio of the first to the third maximum in the
vibronic structure of the fluorescence spectra of pyrene monomers intensities
and characterized the polarity of the luminescent probe molecules microenvironment. It is shown on the basis of obtained experimental data that T-T
energy transfer between the donor of energy - eosin and the acceptor anthracene is accomplished because of localization of donor and acceptor
of energy on the interface of the polar and nonpolar phases of the protein
globule. The decrease of the probability of the T-T energy transfer with the
insignificant concentrations of SDS is caused by the decrease of hydrophobic
interactions in the protein. As a result intramolecular reconstruction of the
structure of protein is possible. Thus, the luminescence of probes and T-T
energy transfer of the electronic excitation between them possess high
sensitivity to structural reconstruction in the proteins.
used to characterize the various D-Cys-A complexes at the ensemble level.
In addition, we observed single-molecule FRET using alternating-laser
excitation to identify static heterogeneity in low concentrated samples to
exclude intermolecular interactions.
We identified competing quenching processes severely changing D and A
quantum yields upon fluorophore contact and determined their relative
efficiencies. We also observed that a significant fraction of fluorophores is
irreversibly altered resulting in reduced extinction coefficients. These results
are applicable for quantitative analysis of FRET in dynamic molecular systems
that allow transient contact between D and A fluorophores.
Time-resolved diffuse optical spectroscopy of
wood
C. D’Andrea, A. Farina, D. Comelli, A. Pifferi, P. Taroni, G. Valentini, R.
Cubeddu, Politecnico di Milano (Italy)
In this work we propose and experimentally demonstrate that non-invasive
time-resolved optical spectroscopy in the spectral region 700-1040 nm, on a
picosecond scale, is a valuable technique for wood characterization. Two
different wood types have been considered, fir and oak chestnut as an
example of softwood and hardwood, respectively. Both woods have been
measured in three different conditions: dry, wet and degraded by an ozone
treatment. Both types of wood show different absorption and scattering
spectra according to the treatment, revealing both chemical and structural
changes.
6633-60, Poster Session
Discrimination of normal and colorectal cancer
using Raman spectroscopy and fluorescence
Y. Wang, Shenyang Ligong Univ. (China)
Described spectrometer collected the needed spectra and transformed them,
we could observe a spectral band from the fluorescence spectrum in the
PMT system. In the band, region of fluorescence spectrum was wider than
Raman spectrum. The noise mainly composed by system shot noise. After
frequency calibration and spectral correlation in spectrometer system
detection, we could get a relative intensive-wavelength graph.
We performed a series of research and found much useful data for diagnosis
colon cancer and rectum cancer. There are red shift of colon cancer and
rectum cancer obviously , but there is little red shift of 1.7nm to normal. The
red shift (??) is 11.1nm of colon cancer, but the red shift (??) is 34nm of colon
cancer. We select ??\>0.84nm to identification colon cancer, use ??\>16.4nm
distinguish rectum cancer. Generally speaking, the red shift of normal is below
12nm, but the red shift of cancer is beyond 12nm. So we can use ?? to
distinguish cancer. At the same time, we found that the change of native
fluorescence is obvious in range 600nm-640nm of rectum cancer, but there
is a little of change of native fluorescence to colon cancer. It’s proved that
the metabolite content of 600nm-640nm is abundant in rectum. The parameter
a\>1 obviously of normal, but a<1 obvious of rectum cancer.
After sample was excited by 488.0nm and 514.5nm the Raman spectrum
all was product. It proved that the metabolite in serum can product Raman
emission. Raman spectrum all was added to fluorescence, there is strong
fluorescence background, The normal and the cancer’s Raman frequency
European Conferences on Biomedical Optics 2007 •
In this contribution we describe performance and first results of MUSES, a
novel research equipment able to detect and identify photons emitted, after
laser irradiation, from biological samples (like microorganisms and human
cells) for fast ultraweak luminescence analysis.
MUSES has been entirely designed and realised at LNS-Southern National
Laboratory of the Italian INFN-Nuclear Physics National Institute. The excellent
performances in terms of timing, wavelength and angular identification make
this multi-detector a unique device in biophotonics research field.
6633-62, Poster Session
Methods of the probe luminescence in the
detection of the dynamically structured state of
human serum albumin
A. G. Melnikov, Saratov State Univ. (Russia)
6633-64, Poster Session
Raman spectroscopy as an analytic tool for nondestructive investigation
P. Roesch, S. Reitzenstein, M. A. Strehle, D. Berg, Friedrich-SchillerUniv. Jena (Germany); M. Baranska, H. Schulz, E. Rudloff,
Bundesanstalt für Züchtungsforschung an Kulturpflanzen (Germany); J.
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Conference 6633: Biophotonics 2007: Optics in Life Science
Popp, Friedrich-Schiller-Univ. Jena (Germany)
Oilseeds have important worldwide applications in human nutrition and
livestock feeding. They also provide both energy and raw material for manifold
kinds of industrial processes. The utility of an oilseed crop for nutritional as
well as for non-food purposes depends mainly upon the fatty acid composition
of the seed oil. The value of different vegetable oils can mainly be correlated
with a high content of polyunsaturated fatty acids especially omega-3-fatty
acids like linolenic acid because of their contribution to a healthy nutrition.
One expression for the degree of unsaturation is the iodine value normally
measured with gas chromatography. Using Raman spectroscopy allows a
rapid calculation of the iodine value. In addition, only minimal sample volume
is necessary for this investigation. Therefore, this method can be used in
single rape seeds in order to predict the iodine value before harvesting. [1]
Beside the analysis of rape seeds the method can also be used to investigate
the quality of new rapeseed lines. Here, the lipid content and composition of
a plant can be predicted by measuring non-destructively in single germinated
grains. This can be of vital importance in plant breeding, where the demand
exists to have fast, easy and non-destructive analytical methods to
discriminate single seeds regarding their fatty oil profile.
Acknowledgement
Financial support of the Deutsche Forschungsgemeinschaft (DFG) in Bonn,
Germany (grant numbers: Po 563/4-1 and 4-2 as well as Schu 566/7-2) is
gratefully acknowledged.
References:
1) S. Reitzenstein, M. A. Strehle, D. Berg, P. Rösch, M. Baranska, H. Schulz,
E. Rudloff and J. Popp, “Non-destructive analysis of single rape seeds by
means of Raman spectroscopy”, J. Raman Spectrosc. 2007, in print.
6633-65, Poster Session
Raman spectroscopic characterization of
secondary metabolites in plants
K. R. Strehle, P. Roesch, Friedrich-Schiller-Univ. Jena (Germany); H.
Schulz, Bundesanstalt für Züchtungsforschung an Kulturpflanzen
(Germany); J. Popp, Friedrich-Schiller-Univ. Jena (Germany) and Institut
für Physikalische Hochtechnologie e.V. (Germany)
Secondary metabolites are produced in plants but have no elementary
function for the growth or development of the organism. Often these
substances fulfill other functions such as the defense against pathogens or
herbivores, but also to attract pollinators. The function of secondary
metabolites for the human organism is often discussed. They are said to
have a positive influence on the digestion, to be antiseptic and even
anticarcinogenic.
Raman spectroscopy is a powerful technique for the characterization of such
secondary metabolites like for example essential oils.(1) Only little sample
preparation is needed and another advantage is that direct in-situ
measurements are feasible. (2)
Plants belonging to the genus Brassicaceae have developed a special
defending mechanism against herbivores. They produce glucosinolates as
secondary metabolites and when the cell tissue is wounded, the enzyme
myrosinase catalyses the conversion of these glucosinolates to the
representative isothiocyanates. In this contribution we present a Raman
spectroscopic characterization of glucosinolates and isothiocyanates.
Acknowledgement:
Financial support of the Deutsche Forschungsgemeinschaft (DFG; grant
numbers: Po 563/4-1 as well as Schu 577/7-1) is gratefully acknowledged.
References:
1. Strehle, K. R.; Rösch, P.; Berg, D.; Schulz, H.; Popp, J., Quality Control of
Commercially Available Essential Oils by Means of Raman Spectroscopy. J.
Agric. Food Chem. 2006, 54, (19), 7020-7026.
2. Strehle, M. A.; Rösch, P.; Baranska, M.; Schulz, H.; Popp, J., On the way
to a quality control of the essential oil of fennel by means of Raman
spectroscopy. Biopolymers 2005, 77, (1), 44-52.
SERS as analytical tool for detection of bacteria
D. Cialla, P. Roesch, Friedrich-Schiller-Univ. Jena (Germany); J. Popp,
Friedrich-Schiller-Univ. Jena (Germany) and Institute of Photonic
Technology (Germany)
For detection of microbial contaminations Raman spectroscopy has come
to an useful tool in the last few years. The characterization and identification
of single bacterial cells by means of Raman spectroscopy in combination
with support vector machines for classifying the Raman spectra of the bacteria
in cluster is well known [1]. This technique could be included in future in
analytics of bacteria in hospitals, pharmaceutical clean rooms or food
processing.
By the means of SERS (surface enhanced Raman spectroscopy) the detection
of single bacteria should be improved by lowering the acquisition time. As
SERS active substrate nano structured colloids or surfaces consisting of
European Conferences on Biomedical Optics 2007 •
6633-67, Poster Session
Characterization of silver nanoparticles
deposited by an enzyme
T. Schüler, R. Möller, Friedrich-Schiller-Univ. Jena (Germany); A.
Steinbrück, W. Fritzsche, Institut für Physikalische Hochtechnologie e.V.
(Germany); J. Popp, Friedrich-Schiller-Univ. Jena (Germany) and Institue
of Photonic Technology (Germany)
The potential of metal nanoparticles is based on their various interesting
properties regarding electronic, optical, and catalytical applications,
depending on composition, shape, and size of the single particles. Therefore
nanoparticles are utilized in many different approaches such as optics,
magnetics and laser technology.
We present a way for enzymatic deposition of silver nanoparticles and a
bioanalytical application in DNA microarray technology for this method[1].
The technology consists of a microstructured chip with 10µm broad electrode
gaps on the surface and specially designed readout device. In principle we
immobilize gold nanoparticle-labeled DNA in a gap between two electrodes.
Afterwards a silver deposition on the bound gold nanoparticles generates a
conductive layer between the electrodes. The measured drop in the resistance
serves as signal for the chip-based electrical detection of DNA.
To further optimize this system the gold nanoparticles as seed are replaced
by the enzyme horseradish peroxidase. For a better understanding of the
enzymatically silver deposition process the formed silver particles were
analyzed by spectroscopic characterization on a single particle level[2, 3]. A
following investigation of these particles by AFM and SEM should explain
the connection between size/shape and the plasmonic properties at individual
particles.
Acknowledgment:
Funding of research project “Jenaer Biochip Initiative” (JBCI) within the
framework “Unternehmen Region - Inno Profile” from the Federal Ministry of
Education and Research, Germany (BMBF) is gratefully acknowledged.
References:
1. Möller, R., et al., Enzymatic Control of Metal Deposition as Key Step for a
Low-Background Electrical Detection for DNA-Chips. Nano Letters, 2005.
2. Yguerabide, J. and E.E. Yguerabide, Light-scattering submicroscopic
particles as highly fluorescent analogs and their use as tracer labels in clinical
and biological applications. Anal Biochem, 1998. 262(2): p. 157-76.
3. Liz-Marzán, L.M., Tailoring Surface Plasmons trought the Morphology and
Assembly of Meatl Nanoparticles. Langmuir, 2006(22): p. 32-41.
6633-68, Poster Session
6633-66, Poster Session
86
gold or silver are possible [2,3]. Because silver may be harmful for bacterial
cells gold is used as SERS-active substrate. Additionally, applying gold as a
SERS-active substrate the excitation wavelength can be shifted to the red
spectral regions. The SERS spectra of B. pumilus are dominated by
contributions of ingredients of the outer cell wall, e.g. the peptidoglucan layer.
First results have shown that reproducible SERS spectra can be recorded
when using gold colloids for the SERS measurements. To generate more
comparable conditions a lithographically prepared SERS active substrate
for detection of microbial contaminants will be developed.
Acknowledgement:
Funding of research project FKZ 13N8369 (“OMIB”) within the framework
“Biophotonik” and of research project “Jenaer Biochip Innitiative (JBCI)” within
the framework “Unternehmen Region - Inno Profile” from the Federal Ministry
of Education and Research, Germany (BMBF) is gratefully acknowledged.
References:
[1] P. Rösch, M. Harz, K.-D. Peschke, O. Ronneberger, H. Burkhardt, A. Schule,
G. Schmauz, M. Lankers, S. Hofer, H. Thiele, H.-W. Motzkus, J. Popp, Anal.
Chem. 78, 2163 (2006).
[2] W. R. Premasiri, D.T. Moir, M. S. Klempner, N. Krieger, G. Jones II, L. D.
Ziegler, J. Phys. Chem. B 109, 312 (2005).
[3] R. M. Jarvis, A. Brooker, R. Goodacre, Faraday Discuss. 132, 281 (2006).
Towards an understanding of the mode of action
of fluoroquinolone drugs
U. Neugebauer, Friedrich-Schiller-Univ. Jena (Germany); U. Schmid, K.
Baumann, Technische Univ. Braunschweig (Germany); U. Holzgrabe,
Univ. Würzburg (Germany); M. Schmitt, J. Popp, Friedrich-Schiller-Univ.
Jena (Germany)
Fluoroquinolones are important antibacterial drugs. They act bactericidal by
inhibiting the vital bacterial enzyme gyrase. This enzyme introduces negative
supercoils into bacterial DNA which is required for a correct function of many
biological processes such as replication, recombination and transcription.
The fluoroquinolone drugs were found to interfere with the gyrase-DNA
complex; however the detailed mode of action on a molecular level is so far
not understood.
In this contribution Raman spectroscopy is chosen as a non-invasive
technique to first characterize the individual involved components
(fluoroquinolone drugs, and the biological targets DNA and gyrase), and
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Conference 6633: Biophotonics 2007: Optics in Life Science
second to study the influence of the fluoroquinolones on bacteria in in-vivo
experiments. The use of UV resonance Raman spectroscopy with excitation
at 244 nm allows the investigation of the drugs and the biological targets in
aqueous solution at biological low concentrations (a few µM). Structural
Raman marker bands are assigned for relaxed DNA (before the action of the
gyrase) and supercoiled DNA (after the action of gyrase). The detailed
assignment of the vibrational bands of the fluoroquinolones is assisted by
DFT calculations [1].
In-vivo experiments with bacteria experiencing varying drug concentration
revealed changes in the vibrational bands of the protein and DNA components
within the bacterial cell caused by the action of the drug. Due to the complexity
of the bacterial spectra advanced multivariate statistics in combination with
variable selection methods proved to be useful in the data analysis. [2, 3].
Acknowledgement
Support of the Deutsche Forschungsgemeinschaft (DFG), Germany
(Sonderforschungsbereich 630, Teilprojekt C1) is gratefully acknowledged.
References:
[1] U. Neugebauer, A. Szeghalmi, M. Schmitt, W. Kiefer, J. Popp, U. Holzgrabe,
Spectrochimica Acta Part A 2005, 1505-1517.
[2] U. Neugebauer, U. Schmid, K. Baumann, W. Ziebuhr, S. Kozitskaya, V.
Deckert, M. Schmitt, J. Popp, ChemPhysChem 2006, in print
[3] U. Neugebauer, U. Schmid, K. Baumann, U. Holzgrabe, W. Ziebuhr, S.
Kozitskaya, W. Kiefer, M. Schmitt, and J.Popp, Biopolymers 2006, 82, 306311.
6633-69, Poster Session
Raman label for DNA detection by means of
SERRS
K. K. Hering, R. Möller, J. Popp, Friedrich-Schiller-Univ. Jena (Germany)
Currently the most accepted method for the detection of DNA sequences is
fluorescence spectroscopy. Unfortunately there are some disadvantages
concerning multiplexing.
Instead of fluorescence, Surface Enhanced Resonance Raman Scattering
(SERRS) can be used as a greatly selective and sensitive detection technique
for biological assays. Additionally it can provide detection limits, which are
at least as well as those of fluorescence [1]. Labelling of single-strand DNA
with Raman-active dyes with narrow-band spectroscopic fingerprints is an
useful tool for multiplex identification of specific sequences of DNA [2,3,4].
SERRS requires molecules with chromophor systems, which have an
absorption maximum close to the frequency of the laser light. Furthermore
the label must be very close or in contact with a metal surface. In most cases
this is realised by analysing the dye in a colloidal solution of gold or silver
nanoparticles or by functionalising the label with gold nanoparticles.
This study uses short synthetic oligonucleotides on silicium oxide and glass
surfaces. Several options of linking position of the label are tested in
combination with different possibilities of bringing the gold nanoparticles in
close proximity to the dye. Furthermore alternative metal depositioning
techniques are examined.
Acknowledgement:
The Funding of the research project “Jenaer Biochip Initiative (JBCI)” within
the framework “InnoProfile - Unternehmen Region” from the Federal Ministry
of Education and Research (BMBF) Germany is gratefully acknowledged.
References:
[1] K. Faulds, R.P. Barbagallo, J.T. Keer, W.E. Smith, D. Graham, Analyst
129, 567-568 (2004)
[2] K. Faulds, W.E. Smith, D. Graham, Anal. Chem. 76, 412-417 (2004)
[3] D. Graham, B.J. Mallinder, W.E.Smith, Biopolymers (Biospectroscopy)
57, 85-91 (2000)
[4] Y.C. Cao, R. Jin, C. Mirkin, Science 297, 1536-1540 (2002)
6633-70, Poster Session
Physical limits to autofluorescence signals
recordings in the rat olfactory bulb in vivo: a
Monte Carlo study
B. L’Heureux, H. Gurden, L. Pinot, R. Mastrippolito, F. Lefebvre, P.
Lanièce, F. Pain, Univ. Paris-Sud II (France)
Intrinsic Optical Signal Imaging (IOSI) allows in vivo imaging of brain activity
in small animals with a high spatio-temporal resolution. Intrinsic signals are
mainly related to changes in the optical absorption of hemoglobin. To
understand neuro-vascular coupling mechanisms related to cerebral
activation, we record IOSI signals in the olfactory bulb following odor
presentation in anesthetized rats [1]. More precisely, we study olfactory
glomeruli, the functional modules allowing the first step of olfactory coding
in the brain. Recently a new complementary approach relying on
autofluorescence properties of Flavin Adenine Dinucleotide (FAD) or
Nicotinamide Adenine Dinucleotide (NADH) was proposed [2]. It allows the
observation of intracellular metabolic mechanisms due to changes in the
redox state of FAD/FADH and NAD/NADH.
European Conferences on Biomedical Optics 2007 •
Here, we investigate the physical limits to spatial resolution for in vivo
autofluorescence imaging in the olfactory bulb. We performed standard Monte
Carlo simulations [3] to model photons scattering and absorption at the
excitation and emission wavelengths of FAD (Exc 420-490 Em 500-560nm)
and NADH (Exc 320-380nm Em.420-490nm) fluorescence. We validate the
simulations accuracy with experimental measurements on in vitro optical
phantoms. We evaluate the influence of the following parameters on the spatial
resolution and signal intensity: i) depth of the glomerular layer ii) focusing
depth within brain tissues iii) depth of field of the optical apparatus. Finally,
we compare these results to similar simulations of the IOSI signals in the
olfactory bulb to evaluate the contribution of both signals sources at the
fluorescence wavelengths.
[1] Gurden H. et aL. (2006) Neuron. 52: 335-45.
[2] Murakami H, et al.. (2004) Eur J Neurosci. 19: 1352-60.
[3]: Prahl SA et al. (1989) SPIE Proceedings of Dosimetry of Laser Radiation
in Medicine and Biology, IS 5: 102-111
6633-71, Poster Session
Towards ultra-stable fluorescent dyes for singlemolecule spectroscopy
R. Kasper, Bielefeld Univ. (Germany)
The wide-spread use of fluorescent dyes in molecular diagnostics and
fluorescence microscopy together with new developments such as singlemolecule fluorescence spectroscopy provide researchers from various
disciplines with an ever expanding toolbox. Single-molecule fluorescence
spectroscopy relies to a large extent on extraordinary bright and photostable
organic chromophores such as rhodamine- or cyanine- derivatives. While in
the last decade single-molecule equipment and methodology have
significantly advanced and in some cases reached theoretical limits (e.g.
detectors approaching unity quantum yields), instable emission (? blinking)
and photobleaching become more and more the bottleneck of further
development and spreading of single-molecule fluorescence studies. Here,
we present a model which accounts for the most relevant photophysical
processes and describe a new strategy to influence the photophysical
pathways with the aim to prevent photobleaching and blinking. We present
stable single-molecule fluorescence transients of several minutes duration
recorded under close to physiological conditions. The applicability to different
dye classes further underlines the generality of the concept.
6633-72, Poster Session
Two photon microscopy for studies of
xenobiotics in human skin
C. Simonsson, M. Smedh, C. Jonsson, M. B. Ericson, A. Karlberg,
Göteborg Univ. (Sweden)
For successful uptake and distribution of drugs, cosmetic products and skin
treatment products from transdermal formulations it is essential to understand
the barrier functions of the skin. This is equally important when evaluating
biological effects after exposure to harmful xenobiotics, e.g. skin sensitizers.
Innovative advances in modern microscopy techniques have provided
valuable tools to study the interaction between the skin and xenobiotics.
Optical sectioning using two photon laser scanning microscopy (TPLSM)
permits non-invasive visualization of fluorescent compounds in the skin.
TPLSM offers an advantage over other confocal techniques in 3D imaging of
optically thick tissues such as the skin by increasing the maximum imaging
depth and reducing out of focus photobleaching and phototoxic effects.
We use TPLSM to study the absorption and distribution of various fluorescent
compounds, e.g. sulforhodamine B (SRB) and the action of penetration
enhancers, e.g. oleic acid, in epidermis. The experiment is performed with
human skin from breast reduction surgery. Two photon imaging is carried out
after passive diffusion of fluorophores through epidermal membranes,
separated form dermis and mounted in vertical diffusion cells. Changes in
the barrier properties after application of penetration enhancers are evaluated
by image processing and analysis followed by a mathematical interpretation
of the enhancer effects.
We have imaged different fluorophores, e.g. SRB at different depths in the
skin. Increased penetration of SRB has been observed in the presence of
oleic acid. Structural differences in SC depending on the vehicle formulation
have also been visualized. At present we are using TPLSM and diffusion
cells to further study the enhancer effects on absorption, distribution and
diffusion of fluorescent compounds in SC and epidermis.
6633-73, Poster Session
Uncovering of melanin fluorescence in human
skin tissue
M. Scholz, G. Stankovic, G. S. Seewald, D. Leupold, LTB Lasertechnik
Berlin GmbH (Germany)
Because of its extremely low fluorescence quantum yield, melanin
fluorescence is masked by several other endogenous and possibly also
exogenous fluorophores (e.g. NADH, FAD, Porphyrins) in the conventionally
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Conference 6633: Biophotonics 2007: Optics in Life Science
(one-photon) excited autofluorescence of skin tissue. A first step to enhance
the melanin contribution was realized by two-photon fs pulse excitation in
the red/near IR, based on the fact that melanin can be excited by stepwise
two-photon absorption [1,2], whereas all other fluorophores in this spectral
region allow only simultaneous two-photon excitation.
Now, the next and decisive step has been realized: Using an extremely
sensitive detection system, for the first time two-photon fluorescence of skin
tissue excited with pulses in the ns range could be measured. The motivation
for this step was based on the fact that the population density of the
fluorescent level resulting from a stepwise excitation has a different
dependence of the pulse duration than that from a simultaneous excitation
(?t2 vs. ?t). Due to this strong discrimination between the fluorophores,
practically pure melanin fluorescence is obtained. Examples for in-vivo, exvivo as well as paraffin embedded skin tissue will be shown. The information
content with respect to early diagnosis of skin diseases will be discussed.
[1] K. Teuchner et al (1999). Femtosecond Two-photon excited Fluorescence
of Melanin. Photochem Photobiol Vol. 70, pp. 146-151
[2] K. Teuchner et al (2000). Fluorescence Studies of Melanin by Stepwise
Two-Photon Femtosecond Laser Excitation. J Fluorescence Vol. 10, pp. 275281
The authors gratefully thank Dr´s R. Eichhorn (Berlin), K. Hoffmann and M.
Stücker (Bochum) for cooperation. Partial financial support by BMBF (grant
13N8787) is also acknowledged.
6633-74, Poster Session
Image reconstruction of the location of macroinhomogeneity in random turbid medium by
using artificial neural networks
B. A. Veksler, Cranfield Univ. (United Kingdom); A. V. Kovaleva, Saratov
State Univ. (Russia); I. V. Meglinski, Cranfield Univ. (United Kingdom); I.
L. Maksimova, Saratov State Univ. (Russia)
Nowadays the artificial neural network (ANN), an effective powerful technique
that is able denoting complex input and output relationships, is widely used
in different biomedical applications. In present Letter the applying of ANN for
the determination of characteristics of random highly scattering medium (like
bio-tissue) is considered. Spatial distribution of the backscattered light
calculated by Monte Carlo method is used to train ANN for single and multiply
scattering regimes. The potential opportunities of use of ANN for image
reconstruction of an absorbing macro inhomogeneity located in topical layers
of random scattering medium are presented. This is especially of high priority
because of new diagnostics/treatment developing that is based on the
applying gold nano-particles for labeling cancer cells.
6633-75, Poster Session
Photoinduced electron transfer (PET)-probes for
the study of enzyme activity at the ensemble
and single-molecule level
S. Henkenjohann, S. Doose, P. Tinnefeld, M. Sauer, Bielefeld Univ.
(Germany)
The development of efficient probes for enzyme activity is of fundamental
importance for cancer and medical diagnosis. Especially, the ability to easily
and specifically determine the efficiency of inhibitors has attracted great
interest in the development of new probes. To date, different fluorescencebased assays have been developed to proof the presence of specific protease
or nucleases in a sample using labeled enzyme substrates. Commonly,
enzyme substrates, e.g. a specific peptide sequence, are doubly labeled
with a donor and an acceptor fluorophore in a way that ensures efficient
fluorescence resonance energy transfer (FRET). Upon cleavage of the peptide
substrate by a protease the spatial contact between the donor and acceptor
fluorophore gets lost. To circumvent unspecific probe enzyme interactions
and affinity problems associated with the use of two extrinsic labels we
developed a new method that takes advantage of properties of naturally
occurring amino acids and nucleic acids. The novel technique relies on the
use of singly labeled, quenched peptide probes based on photoinduced
electron transfer (PET) reactions between selected fluorophores and
tryptophan (Trp) or guanosine (G) residues. The basic idea of the experiment
is that the fluorescence of suitable fluorophores is efficiently quenched only
upon contact formation with Trp or G. the general validity of the technique is
demonstrated at the ensemble level using various fluorescently labeled
enzyme substrates. The rapid response time of the probes enables real-time
monitoring of enzyme activity and provides quantitative data including enzyme
velocity and Michaelis-Menton kinetic parameters. Furthermore, we
demonstrate that our novel probes can be used advantageously to monitor
enzyme activity at the single-molecule level, a prerequisite for the improved
understanding of enzyme mechanisms.
6633-76, Poster Session
Towards a real-time technology for the
identification of native bioaerosols
M. Krause, P. Roesch, Friedrich-Schiller-Univ. Jena (Germany); M.
Lankers, rap.ID Particle Systems GmbH (Germany); H. Thiele, KayserThrede GmbH (Germany); J. Popp, Friedrich-Schiller-Univ. Jena
(Germany) and Insitute for Physical Hightechnology (Germany)
Microorganisms are present in all kinds of matrices or surfaces. Most of them
are tolerated in human environment without any effect. Some strains are
even helpful and can be used in production of foods (cheese, beer, yoghurt)
or pharmaceutical products. But on the other hand some microorganisms
can cause harmful diseases. Or they become resistant against antibiotics
(super bacteria) and lead to a new origin of danger. Therefore a biological
monitoring for example in clean rooms of pharmaceutical production is
necessary. The total encumbrance is assessed by counting colony forming
units (CFU) but this is time consuming. Faster methods like flow cytometry
are also limited to total count enumeration. Continuative identification to strain
level is reserved to microbiology. Cultivation steps need about 2 to 3 days to
get the results. Quicker but more expensive and limited to given primers is
method called polymerase chain reaction.
To overcome all the limitations a new method was developed on basis of
micro Raman spectroscopy to identify single microbial cells without
cultivation. Within some minutes, a Raman spectrum of a bacterium is
generated and compared to a database. Before Raman measurements, the
sample can be analysed by auto fluorescence imaging to separate biotic out
of abiotic particles. The results presented give an impression on the challenge
of sample preparation when just one out of millions of particles in native
samples is biotic.
Acknowledgement
The funding of the research project FKZ 13N8369 within the framework
‘Biophotonik’ from the Federal Ministry of Education and Research, Germany
(BMBF) is gratefully acknowledged.
6633-77, Poster Session
Drug search: in situ UV Raman microscopic
localization of anti malaria active agents in plant
material
T. Frosch, L. Zedler, M. Schmitt, Friedrich-Schiller-Univ. Jena (Germany);
T. Noll, G. Bringmann, Univ. Würzburg (Germany); J. Popp, FriedrichSchiller-Univ. Jena (Germany)
Deep UV resonance Raman micro spectroscopy (lexc. = 244 and 257 nm)
was applied for a highly sensitive, selective and gentle in situ localization of
the antiplasmodials quinine and dioncophylline A in very low concentrations
in plant material of cinchona bark [1] and Triphyophyllum peltatum [2, 3]
respectively.
Malaria is a re-emerging infectious disease with tremendous impact on the
economical development primarily on sub-Saharan African countries [4, 5].
This is because of arising resistances [5] against well established drugs like
chloroquine and mefloquine [6, 7] on a global scale. The design and acquisition
of new active agents against malaria is therefore of utmost importance.
Fortunately traditional medical plants, like cinchona bark and the tropical
liana Triphyophyllum peltatum from the Ivory Coast, have been used by natives
to fight fever for centuries and are therefore a source of established as well
as new, promising active agents.
Therefore the presented results of a highly sensitive and selective in situ
localization of the active agents [1-3] are of high importance for the acquisition
of new antimalarials and for plant science in general.
6633-78, Poster Session
A parallel approach for sub-wavelength
molecular surgery using gene-specific
positioned metal nanoparticles as laser light
antennas
A. Csaki, G. Festag, F. Garwe, Institut für Physikalische
Hochtechnologie e.V. (Germany); G. Maubach, Institute of Bioengineering and Nanotechology (Singapore); K. Mrasek, Friedrich-Schiller-Univ.
Jena (Germany); I. Riemann, Fraunhofer-Institut für Biomedizinische
Technik (Germany); T. Schüler, A. Steinbrück, Institut für Physikalische
Hochtechnologie e.V. (Germany); A. Weise, Friedrich-Schiller-Univ. Jena
(Germany); K. König, Fraunhofer-Institut für Biomedizinische Technik
(Germany); W. Fritzsche, Institut für Physikalische Hochtechnologie e.V.
(Germany)
An optical technique for the parallel manipulation of nanoscale structures
with molecular resolution is presented. Bioconjugated metal nanoparticles
are thereby positioned at the location of interest, such as e.g. certain DNA
sequences along metaphase chromosomes, prior to pulsed laser light
irradiation of the whole sample. The positioned particles serve as a very
bright optical label that allows for easy visualization of chromosome
88
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substructure such as bands or regions. The nanoparticles are designed to
absorb the introduced energy highly efficiently, in that way acting as
nanoantenna. As result of the interaction, structural changes of the sample
with sub-wavelength dimensions and nanoscale precision are observed at
the location of particles. The process leading to the nanolocalized destructions
is caused by particle ablation as well as thermal damages of the surrounding
material.
6633-79, Poster Session
Investigation of biotic and abiotic soil
components by means of various spectroscopic
methods
A. Walter, P. Roesch, S. Jezewski, M. Reinicke, E. Kothe, FriedrichSchiller-Univ. Jena (Germany); J. Popp, Friedrich-Schiller-Univ. Jena
(Germany) and Institut für Physikalische Hochtechnologie, Jena
(Germany)
To develop new biological remediation strategies for contaminated areas our
research focuses on the interaction between biotic and abiotic soil
components. To establish a more fundamental understanding of the influence
of the environmental parameters on bacteria as well as the bacteria on the
inorganic components for example rocks, several spectroscopic methods
especially vibrational spectroscopy are applied.
The investigation of the anorganic components concentrates on the
distribution of minerals in rock material by means of Raman imaging methods.
For the study of the biotic soil fauna we examine strains of Streptomyces
that have been isolated from the testfield in Ronneburg - a former uranium
mining area. If the bacteria suffer various heavy metal exposure during their
development they carry along different chemical information. Those we will
investigate by means of vibrational spectroscopy, especially Raman
spectroscopy. With the choice of various excitation wavelengths different
information can be retrieved from the fingerprint region of the Raman spectra.
If the excitation wavelength is in the range of the visible light, the identification
of bacteria is based on phenotypic characteristics. On the other hand by
applying UV-resonance Raman spectroscopy the direct investigation of DNA
becomes possible and genotypic information are used for the classification.
To identify microbes on strain level chemometrical methods are applied on
the Raman and IR spectra1,2.
Acknowledgement:
We gratefully acknowledge financial support from the Deutsche
Forschungsgemeinschaft (Graduiertenkolleg “Alteration and element mobility
at the microbe-mineral interface”).
References:
[1] P. Rösch, M. Harz, M. Schmitt, K.D. Peschke, O. Ronneberger, H.
Burkhardt, H.W. Motzkus, M. Lankers, S. Hofer, H. Thiele, J. Popp, Appl.
Environm. Microbiol. 2005, 71, (3), 1626-1637.
[2] M. Harz, P. Rösch, K.-D. Peschke, O. Ronneberger, H. Burkhardt and J.
Popp,
Analyst, 2005, 130, 1543-1550.
6633-80, Poster Session
Characterization of human plasma by means of
vibrational spectroscopy
M. K. Harz, R. Claus, P. Roesch, C. Bockmeyer, K. Kentouche,
Friedrich-Schiller-Univ. Jena (Germany); H. Deigner, Univ. of East Anglia
Norwich (United Kingdom); J. Popp, Friedrich-Schiller-Univ. Jena
(Germany)
Various diseases shift the composition of human plasma; hence, the relative
quantification of plasma constituents offers the opportunity, to gain
information on novel diagnostic and prognostic factors. By thrombotic
microangiopathy the protein composition is changed; in patients with severe
infections a hugh amount of von Willebrand factor (VWF) multimers with a
heightened molecular weight could be observed [1]. Conventional analysis
of blood plasma is based on electrophoretic methods that may require up to
several days. Hence these methods are incapacitative for therapeutically
monitoring. Furthermore comparison and reproducibility of these methods
are deficient. For rapid diagnosis in order to expedite an earlier initiation of a
therapeutically plasma exchange, a fast and reliable analytical technique is
necessary. As a promising alternative method vibrational spectroscopic
techniques such as Fourier-Transform-Infrared spectroscopy (FT-IR) [2] and
Raman spectroscopy can be applied due to sensitivity for protein vibrations.
In this study cryoprecipitated human plasma samples of healthy donors and
patients with thrombotic microangiopathy are investigated by means of FTIR and UV-resonance Raman spectroscopy (UVRR). UVRR-spectroscopy is
the more beneficial method for analyzing plasma compared to FT-IR
spectroscopy since with excitation in the UV region an increase of intensity
and a selective enhancement of protein vibrations occur. For characterization
spectra of cryoprecipitated plasma were compared with spectra of plasma
components such as proteins, aromatic amino acids, proteolyzed von
Willebrand factor (VWF) fragments, as well as different pigments and whole
blood. In addition, the application of different chemometric approaches such
European Conferences on Biomedical Optics 2007 •
as hierarchical cluster analysis enables a differentiation between spectra of
plasma samples of patients and healthy controls. [3]
Acknowledgement
We gratefully acknowledge support from the Deutsche
Forschungsgemeinschaft (PO 563/7-1).
References
[1] R. A. Reiter, K. Varadi, P. L. Turecek, B. Jilma, P. Knoebl, Thrombosis and
Haemostasis 2005, 93, 554-558.
[2] C. Petibois, G. Cazorla, A. Cassaigne, G. Deleris, Applied Spectroscopy
2002, 56, 1259-1267.
[3] M. Harz, R. A. Claus, C. L. Bockmeyer, M. Baum, P. Rösch, K. Kentouche,
H. P. Deigner, J. Popp, Biopolymers 2006, 82, 317-324.
6633-81, Poster Session
Retinal image quality with the different types of
intraocular lenses including new idea of the
hybrid IOLs
D. Siedlecki, M. Zajac, J. Nowak, Politechnika Wroclawska (Poland)
Cataract is one of the most frequent reasons of blindness all around the
world. Its treatment relies on removing the pathologically altered crystalline
lens and replacing it with an artificial intraocular lens (IOL). There exists plenty
of types of such implants, which differ in the optical materials and designs
(shapes). But one of the important features, which is rather overlooked in the
development of the intraocular implants is the chromatic aberration and its
influence on the retinal image quality.
In this study authors try to estimate the influence of the design and optical
material of the implant on the image quality in the polychromatic light, taking
into consideration several exemplary types of IOLs which are commercially
available. Authors also propose the partially achromatized hybrid IOLs, the
longitudinal chromatic aberration (LCA)of which reduces the total LCA of the
phakic eye to the level of a healthy eye’s LCA. Several image characteristics,
as the polychromatic Point Spread Function (PSF) and the Modulation Transfer
Function (MTF) and the polychromatic encircled energy are estimated.
The results of the simulations show the significance of the partial chromatic
aberration correction.
6633-82, Poster Session
Peptide-based optical contrast agents for
targeting of intestinal malignancies
A. Frey, N. Röckendorf, N. Fujimoto, K. Wehry, Research Ctr. Borstel
(Germany); M. Bürger, Gesellschaft für Silizium Mikrosysteme mbH
(Germany); J. Helfmann, Laser- und Medizin-Technologie GmbH Berlin
(Germany)
Intestinal tumors exhibit cell surface properties that differ from neighboring
healthy epithelia and thus allow tumor-specific molecular targeting.
Ganglioside GM1 is such a discriminatory target. Although expressed in the
apical membrane of all intestinal epithelial cells it is accessible for
nanoparticulate ligands only on tumor cells.
To exploit this phenomenon we want to develop a nanoparticulate optical
contrast agent equipped with a peptidic GM1 binding ligand. For identification
of potential binders a novel screening platform was designed where putative
ganglioside GM1 binding peptides are synthesized on glass capillary plates
using microfluidic non-contact arraying techniques. These three-dimensional
supports are easy to handle and show better sensitivity than either flat glass
or cellulose membrane supports because of their large inner surface and low
interference with readout systems. Binding of fluorescently-labeled GM1 to
the capillary plate-immobilized peptides is screened with a fluorescence
reader that was designed to comply with the specific optical behaviour of
this array type. The reader uses a small numerical aperture for excitation and
a large numeric aperture for detection in epifluorescence-mode. Background
noise from fluorescence and Raman scattering is reduced by time gated
photon counting.
Using this platform the peptides are improved by several rounds of an
evolutionary procedure which in each generation creates peptides with
increased affinity. The final peptides with high affinity to ganglioside GM1 will
be fluorescently labelled with a red/near infrared dye and conjugated to a
nanoparticulate carrier. The resulting optical contrast agent shall be used for
fluorescence endoscopic intestinal tumor screening.
6633-83, Poster Session
Objective evaluation of linear feature orientation
in a two-dimensional image: applications on skin
imaging
G. N. Stamatas, A. Nkengne, A. Lopes, C. Bertin, A. Rossi, Johnson &
Johnson Consumer France S.A.S. (France)
A rotationally invariant algorithm was developed to evaluate the orientation
direction and orientation coherence of features in a two-dimensional image.
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Conference 6633: Biophotonics 2007: Optics in Life Science
The algorithm was validated on test images. It was then applied on in vivo
confocal microscopy images for the evaluation of collagen fiber orientation
and on skin microrelief images for the calculation of the primary direction of
microglyphics. The results show that the proposed algorithm can be applied
on biomedical imaging to extract useful information concerning linear features.
6633-84, Poster Session
Development of microfluidic structures for high
throughput flow cytometric characterization of
blood cells
A. Kummrow, H. Yildirim, Physikalisch-Technische Bundesanstalt
(Germany); J. Theisen, Technische Univ. Berlin (Germany); K. Brattke,
Physikalisch-Technische Bundesanstalt (Germany); C. Sprenger, M.
Schmidt, Technische Univ. Berlin (Germany); J. Neukammer,
Physikalisch-Technische Bundesanstalt (Germany)
Flow cytometry is a high throughput method for cell characterization and is,
therefore, widely used to support medical diagnosis. Microfabricated devices
integrating all elements required for dedicated flow cytometric analysis are
highly interesting since they could be designed for one way use taking
advantage of cost-efficient production. In addition, microstructures can be
designed to combine functionalities, such as various measuring quantities
and integrated sample preparation, not easily available in conventional flow
cytometry. We have designed different microstructures for optical and
impedance analysis of single particles. Mold inserts were fabricated by ultra
precision milling to produce three dimensional structures. Such fluidic
structures are needed to achieve two dimensional hydrodynamic focusing,
which is a prerequisite for accurate sample characterization in high throughput
measurements. Following hot embossing in polycarbonate or PMMA and
mounting optical and electrical interconnects upper and lower part of the
devices were assembled using laser welding. Microfabricated flow cytometers
featuring single stage or cascaded hydrodynamic focusing were used for
concurrent optical and impedance counting demonstrating coefficients of
variation down to about 7% and 5% for optical impedance counting
respectively. By simultaneously detecting forward light scatter at 633 nm
and 488 nm we succeeded to differentiate red blood cells and platelets in a
diluted whole blood sample.
6633-85, Poster Session
Highly sensitive detection of target molecules
using a new fluorescence-based bead assay
S. Scheffler, D. Strauss, M. Sauer, Univ. Bielefeld (Germany)
Development of fluorescence based assays with improved sensitivity,
specificity and reliability is of major interest in modern bioanalytical research.
We describe the development of a new fluorescence-based bead assay on
the basis of specific antigen-antibody-interactions and accumulation of the
signal on 2-8 µm beads in combination with the use of highly fluorescent
extrinsic labels. The basic principle comprises immobilization of capture
molecules on the bead surface and fluorescence labeling of secondary
detection antibodies. Upon incubation with the sample and binding of target
molecules the fluorescence signal accumulates on the bead surface and
therefore allows readout of the fluorescence intensity without any washing
step by conventional fluorescence microscopy. The new assay can be easily
modified by rearranging the order of coatings and assay conditions.
Depending on the target molecule, antibodies (ABs), holoproteins or small
protein epitopes can be chosen as capture peptides. We compared our novel
assay with alternative assays based on (i) fluorescence correlation
spectroscopy (FCS) in solution and (ii) total internal reflection (TIR)
fluorescence microscopy on capture molecules immobilized on glass
surfaces. Our method is characterized by a high sensitivity and a large dynamic
range. The limit of detection for monoclonal ABs was determined to 10-15 10-10 mol/L, depending on bead coating and assay conditions. Furthermore,
the assay enables the detection of polyclonal AB from undiluted blood sera
without limits in sample volume. Here we used a 12mere epitope of the centre
region of p53 to capture auto ABs in blood sera of patients with various
types of cancer. The newly developed bead-based assay is easy to perform
with superior sensitivity to the current available ELISA.
6633-86, Poster Session
Protein chip analysis by probing time-resolved
UV-fluorescence
P. M. Schellenberg, Institut für Physikalische Hochtechnologie e.V.
(Germany); R. Dietrich, Schott Jenaer Glas GmbH (Germany); W.
Fritzsche, Institut für Physikalische Hochtechnologie e.V. (Germany); K.
O. Greulich, P. Grigaravicius, Fritz Lipmann Institute (Germany); U. Horn,
Hans-Knöll-Institute (Germany); D. Knoll, Schott Jenaer Glas GmbH
(Germany); S. Peters, Institut für Physikalische Hochtechnologie e.V.
(Germany)
Fluorescence detection techniques are very sensitive, but their usability for
protein interaction studies is hampered by the necessity of attaching
90
European Conferences on Biomedical Optics 2007 •
fluorophors to the proteins, which may perturb their structure and functionality.
Therefore, several optical methods to probe protein interactions without the
need for labeling have been put forward, such as imaging ellipsometry and
surface plasmon resonance.
These techniques are favourably complemented by a new approach based
on the decay pattern analysis of the proteins’ intrinsic UV -fluorescence. By
this method the aromatic amino acids TRP and TYR serve as internal probes
to detect alterations of their environment upon coupling to a binding partner
such as another protein. The method can also be exploited for probing binding
events of small ligands to proteins, that may evade detection by other
diagnosis techniques such as surface plasmon resonance or ellipsometry,
since the sensitivity of these methods usually scales with the size of the
binding partner.
We also automated the protein chip analysis by spacially scanning the
microarray, thereby getting a fluorescence lifetime image by employing time
correlated single photon counting.
We acknowledge financial support by the BMBF, project no. 13N8028 and
by the Federal State of Thuringia through a cooperative research project,
which is conducted together with the University of Jena, Dept. of Nutritional
Science and with Schott -Nexterion.
6633-87, Poster Session
Useful sun strategy based on light-converting
materials
R. N. Khramov, Institute for Theoretical and Experimental Physics
(Russia); G. Cheremisin, B. M. Sinelnikov, V. A. Vorobiev, IQlink Ky
(Finland)
We propose a new way: a “useful sun” strategy with application of non-toxic
red-light luminescence compounds (RLLC) that absorb the UV component
of sunlight and transform it into red light (600-640 nm). It is based on use of
light-converting materials and allows “loving sun instead of to be afraid of
it”, to use its beneficial possibilities without injuring skin and organism in
whole, due to the earlier harmful UV light becomes useful. Experiments in
noncontact use of light-converting films and glasses and agricultural
engineering resulted in
Life sciences:
- accelerate by 14-30% the period of healing of trophic skin ulcers, longhealing and burnt wounds in man, using solar or UV radiarions,
- enhance (up to 100%) of the EEG effects of hypothalamus in rats to a
dopamine agonist - priming effect, using UV radiation. The light induction of
dopaminergic priming opens a new direction in study of regulatory
mechanisms of motor activity and its correction at various diseases, in
particular, Parkinsonism,
- enhance (up to 60%) of physical endurance of mice’s (swimming test),
using solar simulation (xenon light) irradiation;
Agricultural engineering:
- increases the harvest of tomatoes, cucumbers, cabbage and radish up
100%,
- accelerates the ripening of fruits and improves the quality of fruits (higher
contents of sugar, vitamin C, carotene, and lower nitrate contents);
These materials may have an enormous commercial potential in production
of: polymer materials, sunscreens, sunglasses, varnishes; new type of
textiles; glasses for car windscreens and buildings, cell growing reactors for
biotechnology.
6633-88, Poster Session
Diffractometry analysis of human and rat
erythrocytes deformability under ischemia
A. E. Lugovtsov, A. V. Priezzhev, S. Y. Nikitin, V. B. Koshelev, M.V.
Lomonosov Moscow State Univ. (Russia)
Ischemic diseases of people and animals are accompanied with deterioration
of microrheologic properties of their blood, in particular, with impairing
erythrocyte deformability. In this work, the analysis of human and rat
erythrocytes deformability in norm and ischemia was performed by means
of the laser diffractometry. The essence of the method is in obtaining and
processing the diffraction images from a cell suspension moving in a shear
flow in a Couette chamber. The measurements result in the calculation of the
mean index of deformability of erythrocytes (IDE) as a function of shear stress.
In order to obtain the diffraction images, a 1-mm thick layer of the suspension
was illuminated with a collimated beam of a He-Ne laser (633 nm, 0.5 W/
cm2). In the experiments with human erythrocytes, we investigated blood
samples from 16 volunteers, 8 ones being patients suffering from ischemic
diseases and other 8 volunteers - practically healthy individuals. It was shown,
that IDE of ischemic patients was in average 12 - 14 % lower than of the
healthy people.
Experimentally induced ischemia (EII) in rats is an animal model frequently
used for studying the response of an organism to ischemia. Semax is a
medication lowering the effects of ischemia. We studied the effect of Semax
on IDE totally on 32 rats, from which 16 ones were in the experimental group
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Conference 6633: Biophotonics 2007: Optics in Life Science
(rats with EII), and the rest 16 rats were in the control group (rats without EII).
We show that the administration of semax positively influences the
microrheologic properties of erythrocytes of ischemi? rats.
Basing on these results one can conclude that the positive effect of semax
when used to treat the human patients with ischemic diseases is also related
to the improvement of the microrheologic properties of erythrocytes.
We can conclude that the laser diffractometry technique can be used for
investigation of deformability properties of erythrocytes and responses of
the organism to different influences.
6633-89, Poster Session
Ultraweak delayed luminescence of dry seeds
R. Neurohr, G. A. Stanciu, Univ. Politehnica Bucuresti (Romania)
Since the first publications of Veselova et al. more than 30 years ago, it is
known that dry seeds are emitting a very weak and slowly decaying light
signal after exposure to external light stimulus. These early reports
demonstrated, that the intensity of delayed photon emission is correlated
with parameters such as seed age, germination capacity and / or seed water
content, thus providing a non-invasive tool for seed testing and fundamental
investigation in general seed research.
Here we are presenting some new results, pointing out to a more complex
relationship between delayed luminescence (DL) and seed physiology,
especially in respect to the biological clock phenomenon. The intensity of
seed DL is obviously correlated to seasonal and circaseptan fluctuations in
water uptake during early imbibition, which according to Spruyt et al. is related
to the biological clock phenomenon.
These experimental results will be completed by some of our most recent
investigations related to the development of non-invasive seed testing in
practice, which are including confocal or two photon excitation imaging on
artificially aged seeds.
We used an Ar- ion laser and a Ti:sapphire femtosecond laserin a Leica
spectral confocal laser microscope. Excitations were made using 488 nm
from Ar - ion laser and 780 nm wavelength in the two photon excitation case.
We examined emission spectra of the seeds.
6633-90, Poster Session
Preparation and optical characterization of coreshell bi-metal nanoparticles
A. Steinbrueck, A. Csáki, G. Festag, T. Schüler, W. Fritzsche, Institut für
Physikalische Hochtechnologie e.V. (Germany)
Chemical approaches allow for the synthesis of highly defined metal
heterostructures, such as core-shell nanospheres. Because the material of
metal nanoparticles determines the plasmon resonance-induced absorption
band, the control of particle composition results in control of the absorption
band.
Metal deposition on gold or silver nanoparticles yielded core-shell particles
with modified optical properties. UV-VIS spectroscopy on solution-grown
and immobilized particles was conducted as ensemble measurements,
complemented by single particle spectroscopy of selected structures.
Increasing layers of a second metal lead to a shift in the absorption band,
and shell diameter in the scale of the original particle diameter lead to a
predominant influence of the core material. The extent of shell growth could
be controlled by reaction time or the concentration of either the metal salt or
the reducing agent. Besides the optical characterization, the utilization of
AFM, SEM and TEM yielded important information about the ultrastructure
of the nanoparticle complexes.
6633-91, Poster Session
Luminescent nanoparticles for molecular
medicine
and thermal induced denaturation events of DNA strands read out by FRET
processes as well as immuno assays are presented.
6633-92, Poster Session
Studying sigma54-dependent transcription at
the single-molecule level using alternating-laser
excitation (ALEX) specroscopy
M. Heilemann, K. Lymperopoulos, Univ. of Oxford (United Kingdom); S.
Wigneshweraraj, M. M. Buck, Imperial College London (United
Kingdom); A. N. Kapanidis, Univ. of Oxford (United Kingdom)
Gene transcription, the vital biological process of copying genetic information
from DNA to RNA, is orchestrated by RNA polymerase (RNAP). In bacteria,
RNAP directs transcription after forming a functional complex (“holoenzyme”)
with transcription-initiation proteins known as sigma factors. Most of the
published work has focused on sigma70-dependent transcription; here, we
study sigma54-dependent transcription which operates distinctly from its
sigma70 counterpart. Our goal is to understand how RNAP is remodeled by
sigma54 to function in a different way; studying sigma54-dependent
transcription may also explain elements of eukaryotic transcription, since
both mechanisms require ATP hydrolysis, specific DNA sequences known
as enhancers, and specific activator proteins.
Here, we present single-molecule studies of sigma54-dependent transcription
using single-molecule fluorescence resonance energy transfer (smFRET) and
alternating-laser excitation (ALEX) spectroscopy. The ability to study one
biomolecular machine at the time allowed us to resolve and analyse sample
heterogeneities and extract structural information on subpopulations and
transient intermediates of transcription; such information is hidden in bulk
experiments.
Using site-specifically labelled sigma54 proteins and site-specifically labelled
promoter-DNA fragments, we demonstrate that we can observe single
diffusing transcription-initiation RNAP-sigma54-DNA complexes, and that
we can measure distances and distance changes within such complexes;
the identity of the complexes has also been confirmed using electrophoreticmobility-shift assays. Our studies pave the way for understanding the
mechanism of abortive initiation and promoter escape in sigma54-dependent
transcription.
6633-93, Poster Session
The luminescent manifestation of the DNA:
tribetamid interaction
A. O. Dudko, National Taras Shevchenko Univ. of Kyiv (Ukraine)
The complete understanding of the therapy mechanism action of drugs is
impossible without studies of the interaction of these compounds with
biological objects on the molecular level. In our work some results of the
investigations the DNA - amitozine (plant origin - Chelidonium majus L. drug with anticancer and immune modulation properties) are presented.
The absorbtion, fluorescence and phosphorescence of amitozine were studied
in water solution without and in presence of the DNA. The fluorescence
maximum amitozine without DNA depends on excitation wavelength but
fluorescence maximum amitozine in presence DNA doesn’t depend.
Simultaneously the fluorescence intensities increase approximately 10 times
(see Fig.1). This phenomenon is connected, to our opinion with the adsorption
one of the one amitozine’s alcaloid on the DNA macromolecules (amitozine
molecule consists from several alkaloids).
According to our investigations the triplet excitations in DNA are localized
mainly on amitozine’s alkaloid - berberine (the phosphorescence spectra
DNA+berberin are very close to berberine water solution spectra). It was
obtained from studies of the phosphorescence dependence of
DNA+berberine solution on berberine concentration, that average value of
the triplet excitation displacement at least reaches the 20 base sequence
length (7 nm.)
H. Hummel, V. Weiler, Philips Research Labs. (Germany); W. Hoheisel,
Bayer Technology Services GmbH (Germany); C. Walter, M. Haase,
Univ. Osnabrück (Germany)
6633-28, Session 7
Optical technologies play an important role in the rapid developing fields of
molecular medicine such as molecular diagnostics and molecular imaging.
To detect processes on a molecular level target-specific labels are needed.
Luminescent nanoparticles like so called quantum dots or nanophosphors
are promising as optical tags due to their emission characteristics and
efficiencies, high photostability and their capability for multiplexing. Possible
applications for such luminescent nanomaterials range from in-vivo optical
contrasting via in-vitro labeling, to components of therapeutic agents.
Here we present novel optical labels based on nanophosphor materials. Core
particles can be synthesised smaller than 10nm and stabilized in aqueous
media. Polymer or silica coatings of individual nanoparticles increase long
term stability and introduce functional groups of interest for bioconjugation
chemistry. Strategies for bioconjugation of these materials will be discussed.
In feasibility studies for in-vitro diagnostic applications nanophosphors feature
their advantage over organic dyes. For example, the detection of hybridization
J. A. Käs, Univ. Leipzig (Germany)
European Conferences on Biomedical Optics 2007 •
Optical deformability as a new cell marker
The cytoskeleton a compound of highly dynamic polymers and active nanoelements inside biological cells is responsible for a cell’s stability and
organization. Light has been used to observe cells since Leeuwenhoek’s
times; however, we use the forces caused by light described by Maxwell’s
surface tensor to feel the cytoskeleton. The optical stretcher exploits the
nonlinear, thus amplified response of a cell’s mechanical strength to small
changes between different cytoskeletal proteomic compositions as a high
precision cell marker that uniquely characterizes different cell types.
Consequentially, the optical stretcher detects tumors and their stages with
accuracy unparalleled by molecular biology approaches. This precision allows
us to isolate adult stem cells for regenerative medicine without contamination
through molecular markers.
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Conference 6633: Biophotonics 2007: Optics in Life Science
6633-29, Session 7
Live cell opto-perforation by femtosecond laser
pulses
J. Baumgart, Laser Zentrum Hannover e.V. (Germany); W. Bintig, A.
Ngezahayo, W. A. Ertmer, Univ. Hannover (Germany); H. Lubatschowski,
A. Heisterkamp, Laser Zentrum Hannover e.V. (Germany)
Transfection of foreign DNA into cells by the use of chemical carriers and
electroporation is limited by the efficiency in some cells such as primary
cells or by the viability of the cells after electroporation. [1] Another method
for introduction of large molecules into cells is the perforation of the membrane
realized by femtosecond (fs) laser pulses. [2, 3]
Transient pores are created by focusing the laser beam on the membrane for
some milliseconds. Through this pore, the proteins can enter into the cell.
This was demonstrated in a proof of principle experiment for a few cells, but
it is essential to develop an opto-perforation system for large numbers of
cells in order to obtain statistically significant samples for biological
experiments. The relationship between pulse energy, irradiation time, repetition
rate and efficiency of the transfer of a chromophor into the cells as well as
the viability of the cells were analysed. The cell viability was observed up to
90 minutes after manipulation. Additionally the membrane potential of the
treated cells was studied. This allows the determination of possible changes
in the concentration of the ions inside the cell volume, which changes if the
outer cellular media diffuses through the laser created pore into the cytoplasm.
[1] E. Tekle, R. D. Astumian, P. B. Chock, Proc. Nadl. Acad. Sci. USA, Vol. 88,
pp. 4230-4234, 1991
[2] U.K. Tirlapur, K. König, Nature, Vol. 418, 290-291, 2002
[3] D. Stevenson, B. Agate, X. Tsampoula, P. Fischer, C.T.A. Brown, W. Sibbett,
A. Riches, F. Gunn-Moore, and K. Dholakia, Opt. Express, 14(16), 7125-7133,
2006
6633-30, Session 7
able to calculate the whole curve of the optical force as a function of radial
position, including the effects of spherical aberration due to the refractive
index mismatch interface, generally oil-water. We present an analytical
treatment to determine axial optical forces for arbitrary sized scatterers (Mie
regime), polarization, beam profile and position of the beam with respect to
the dielectric microsphere including the aberration. The axial force is shown
to be independent of the incident field’s polarization. As for the sharp
fluctuations of the absolute electric field in the focal region, a ripple structure
appears on the axial optical force curve, these could be shown to be a natural
ruler to calibrate the axial distances for beam movement as compared to
other recent approaches, such as photoluminescence by two photon
absorption or interference of the forward scattered light.
6633-32, Session 7
Vascular end-to-side soldering using a dyeenhanced albumin solder
S. Bogni, A. Alfieri, M. Reinert, M. A. Constantinescu, E. Knall, A. Bregy,
M. Frenz, Univ. Bern (Switzerland)
The sucess of revascularization procedures or bypass techniques is limited
by factors such as size of the anastomosed vessels and secondary intimal
hyperplasia, leading to secondary occlusion of the bypass. Therefore research
for improving anastomosisis techniques is focused on sutureless techniques
such as laser tissue soldering. In this talk a new intraluminal method to solder
end-to-side anastomoses is presented. Porcine vessels with an inner diameter
of 0.6 mm to 0.8 mm were used to perform the anastomoses. As a solder
material a 25 % albumin solution enhanced with indocyanine green was used.
A quartz fiber with a melted focussing lens at one fiber end was inserted into
one vessel to irradiate the albumin solder, which was placed arround the
vessel. For irradiation a diode laser at a wavelength of 808 nm in a continuous
wave regime was used. With this method tensile strengths of more than 1 N
were obtained. This new method allows to perform very precise side-to-end
anastomoses without suturing and in a shorter time.
Automatic segmentation of cell nuclei in bladder
tissue for karyometric analysis
6633-33, Session 8
V. R. Korde, College of Optical Sciences/The Univ. of Arizona (USA); H.
G. Bartels, J. Ranger-Moore, J. K. Barton, The Univ. of Arizona (USA)
Photodynamic therapy: state-of-the-art and
further perspectives
Objective: To automatically segment cell nuclei in bladder tissue for
karyometric analysis of chemopreventive agents.
Materials/Methods: This robust segmentation technique used image
properties to optimally process the image and segment cell nuclei. Image
processing parameters and the segmentation threshold were automatically
selected based on the image histogram. The initial thresholding created a
number of closed four-way chain code nuclei segmentations. Statistical
properties of the segmentations and cusp locations were evaluated and then
stored with the segmentation. Segmentation property deviations by more
than one standard deviation from the group mean were also stored with the
segmentation. A nucleus segmentation was placed in the salvageable
category based on cusps and measures indicative of an unsmooth
segmentation. Erosion dilation and rethresholding was performed on
salvageable nuclei that fit the appropriate criteria. Most salvageable nuclei
were fixed using these two methods. Properties of any resulting
segmentations were evaluated and statistically analyzed.
Results: 10 bladder tissue images from 5 different patients were segmented
by hand and automatically segmented using this program. The automatic
segmentation resulted in a sensitivity of 87%. The average difference between
hand and automatic segmentations of 52 nuclei were calculated for each of
the 95 features used in karyometric analysis. Average differences ranged
between 0 and 18.1%, with an average of 3.1%. A 1.3% difference in nuclear
area and a 2.1% difference in optical density were found.
Conclusion: The close agreement in karyometric features show that
automated segmentation can be used for karyometric analysis.
H. Berlien, Elisabeth Klinik (Germany)
6633-31, Session 7
6633-34, Session 8
Axial optical trapping and position detection
through a dielectric interface for an arbitrary
beam
Skin cancer imaging and evaluation by
multidimensional non-linear microscopy
A. A. R. Neves, A. Fontes, L. C. Barbosa, Univ. Estadual de Campinas
(Brazil); A. Camposeo, R. Cingolani, Univ. degli Studi di Lecce (Italy); D.
Pisignano, Istituto per la Microelettronica e Microsistemi (Italy); C. L.
Cesar, Univ. Estadual de Campinas (Brazil)
The dual optical trapping is the preferred setup to apply the force due to
ease of manipulating particles, coupling to spherical microcavity resonance
modes, spectroscopy, and noise reduction. The generalized Lorenz-Mie theory
is the most adequate for optical trapping of particles of arbitrary size, valid
for both, Rayleigh and Geometrical Optics, regimes, the main difficulty was
the partial wave decomposition of a highly focused non-paraxial beam
fundamental to establish a true trapping in all three dimensions, where a full
vectorial description of the incident beam is required. We developed an exact
partial wave expansion of highly focused beams from which we have been
92
European Conferences on Biomedical Optics 2007 •
Photodynamic Therapy: State of the art and further perspectives
H.-Peter Berlien, Carsten. M. Philipp
Abt. Lasermedizin, Elisabeth Klinik, Luetzowstr. 24-26, D-10785 Berlin
Phone: +49 30 2506902
Fax: +49 30 2506923
e-mail: lasermed\@elisabeth-klinik-berlin.de
Photodynamic Therapy (PDT) has emerged from an experimental treatment
into a commonly used clinical therapy option during the last decade. The
topical photosensibilization with 5-ALA has become a safe and effective
procedure for the treatment of BCC and other skin tumours and received
clearance by the FDA and within the EC. The same substance is used in
diagnostics not only topical but also, employing systemically administration,
guiding the surgeon towards the tumours. Other common substances as
hematoporphyrin-derivatives (HPD) have a longer history in systemic PDT
but were accompanied by a number of side effects as prolonged
photosensibilization of skin that may be responsible for the lack of acceptance
in the medical community. More recently developed drugs as m-THPC
showing a higher quantum yields but still suffer from the need for prolonged
light protection. The next generation of photosensitizers is currently under
investigation and promising. Short elimination times, discrete treatment
windows, an optimized light dosimetry with lasers and non-laser light sources
and the better understanding of cellular and molecular interactions (e.g. the
employment of type I or type II reactions) will provide us with new options
not only for the fight against cancer.
R. Cicchi, S. Sestini, V. De Giorgi, D. Stambouli, P. Carli, D. Massi, F. S.
Pavone, Univ. degli Studi di Firenze (Italy)
We performed a morpho-functional analysis of human skin by combining
multiple non-linear laser scanning imaging multiphoton techniques, including
two photon microscopy, second harmonic generation microscopy,
fluorescence lifetime imaging microscopy, and multispectral two photon
imaging. Basal cell carcinoma ex-vivo samples, excised during dermatological
surgery, were layer-by-layer optically sectioned, characterized, and compared
to corresponding healthy skin ex-vivo samples using all the microscopy
techniques described above. Morphological and spectroscopic differences
were found between malignant skin and corresponding healthy skin tissues.
In comparison with normal healthy skin, cancer tissue showed a different
morphology, a blue-shifted fluorescence emission, a high fluorescence
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Conference 6633: Biophotonics 2007: Optics in Life Science
response at 800 nm excitation wavelength, and a mean fluorescence lifetime
distribution slightly shifted towards higher values. Topical application of deltaaminolevulinic acid to the skin lesion three-four hours before excision
enhanced the fluorescence signal arising from malignant tissue, helping the
morphological discrimination of the tumor. Contrast enhancement was
observed at tumor borders by both two photon fluorescence microscopy
and fluorescence lifetime imaging. Non-linear detected images showed a
good correlation with conventional histological images arising from the same
sample, confirming the diagnostic accuracy of our method. Multidimensional
imaging enabled the discrimination between benign and malignant tissues
in ex-vivo human skin samples, thus offering a non-invasive tool for the invivo skin diagnostic.
6633-35, Session 8
In vivo micro-lesion of single dendrite with
femtosecond laser pulses
L. Sacconi, Univ. degli Studi di Firenze (Italy); R. Panteri, Univ. Campus
Bio-Medico (Italy); A. Masi, Univ. degli Studi di Firenze (Italy); G. Diana,
Istituto Superiore di Sanità (Italy); M. Buffelli, Univ. degli Studi di Verona
(Italy); F. Keller, Univ. Campus Bio-Medico (Italy); F. S. Pavone, Univ.
degli Studi di Firenze (Italy)
Recently, two-photon microscopy has been used for high spatial resolution
imaging of the intact neocortex in living rodents. In this work we used nearIR femtosecond laser pulses for a combination of two-photon microscopy
and microdissection on fluorescently-labeled neuronal structures in living
mice. Three-dimensional reconstructions of dendrites expressing the green
fluorescence protein were made in the cortex of GFP-M transgenic mice.
Afterwards, single dendrites were laser-dissected irradiating the structure
with a high femtosecond laser energy dose. We report that laser dissection
can be performed with micrometric precision and without any visible collateral
damage of the surrounding neuronal structures. After laser irradiation, one
part of the severed dendrite underwent degeneration and disappeared within
5 hours.
Using a chronically implanted glass window, we performed long-term imaging
in the area of the dissected dendrite. Images of the long-term morphological
changes in the neuronal network after dendritic lesioning will be provided.
Laser microdissection of selected structures of the neuronal branching in
vivo represents a promising tool for neurobiological research.
6633-38, Session 9
Miniaturized pulse oximeter sensor for
continuous vital parameter monitoring
J. Fiala, S. Reichelt, Albert-Ludwigs-Univ. Freiburg (Germany); P.
Bingger, Albert-Ludwigs-Univ. Freiburg (Germany) and Univ. Freibu
(Germany); A. Werber, H. Zappe, Albert-Ludwigs-Univ. Freiburg
(Germany); K. Förster, R. Klemm, C. Heilmann, F. Beyersdorf, Univ.
Hospital Freiburg (Germany)
6633-36, Session 8
Online-visualization in cartilage tissue
engineering by two-photon microscopy
K. Liefeith, R. Schade, S. Grohmann, Institut fur Bioprozess- und
Analysenmesstechnik e.V. (Germany); J. Martini, K. Tönsing, D.
Anselmetti, Bielefeld Univ. (Germany)
The therapy of cartilage defects increasingly depend on tissue engineering
approaches. The clinical successful employed method of matrix-induced
autologous chondrocyte implantation (MACI, registered trademark) uses the
preimplantative cultivation of chondrocytes on collagen type I/III scaffolds
and the subsequent implantation of these constructs into the cartilage defect
area. The mechanical cell stimulation during the preimplantative cultivation
is suitable to sustain the differentiation capability of chondrocytes. Todate,
however, the non-invasive online control with respect to cell growth, cell
number and cell distribution is limited. Due to the principal advantages of
the two-photon laser scanning microscopy (2PLSM) in microscopic analysis
of native cell cultures and tissues this method is qualified for online analysis
of tissue engineering constructs.
We introduce biophotonic approaches using 2TPLSM (LaVision BioTec GmbH,
Bielefeld, Germany) to selectively visualize chondrocytes and collagen
scaffolds during their cultivation under hydrostatic cell stimulation as basis
for the quantitative analysis. Two in principle different techniques can be
used: a) spectral resolved detection of native autofluorescence using prisms
or appropriate filter sets of up to three filters with subsequent spectral
unmixing and b) parallel detection of broad band autofluorescence and second
harmonic generation (SHG) signals of collagen scaffolds with subsequent
image processing. Both techniques provide a fast online analysis of 3D cellscaffold networks to control the effects of mechanical cell stimulation during
the cultivation.
Recent studies of the detection of synthesized extracellular matrix substances
(ECM) by fluorescence lifetime imaging microscopy (FLIM) with time correlated
single photon counting (TCSPC) and high photon count rate will be presented.
6633-37, Session 8
Raman spectroscopic investigations of cellular
components in liquor cerebrospinalis
M. K. Harz, M. Kiehntopf, P. Roesch, E. Straube, T. Deufel, J. Popp,
Friedrich-Schiller-Univ. Jena (Germany)
Cerebrospinal fluid diagnostics bases on microscopic analysis of cells and
particles. Cell counting as well as a fast and specific differentiation after
European Conferences on Biomedical Optics 2007 •
enrichment of living single cell populations and staining is accomplished.
Here in general automated haematological systems or flow cytometer are
applied [1]. However samples with small volume and a limited cell number
have hampered the analysis in routine clinical practise since it is imprecise,
has wide inter-observer variability and is labor-intensive and time-consuming.
In case of bacterial detection Gram staining and cultivation is performed that
involves more than 48 hours. Thus it presents major drawbacks for analysis
since the application of antimicrobial drugs is required immediately.
Previous research has shown that micro-Raman spectroscopy provides a
facile method for analyzing single cells such as blood cells and due to the
high spatial resolution even individual bacteria in their native state may be
rapidly investigated without culturing. [2]
The aim of our research is focused on the analysis of cellular components of
cerebrospinal fluid samples by means of Raman spectroscopy for medical
diagnosis. For this purpose in order to guarantee fast analysis without the
necessity for bacterial cultivation single bacterial cells were investigated. Our
intension comprises the cell characterization and differentiation of single
bacteria and blood cells in liquor cerebrospinalis of bacterial induced
meningitis. The combination of Raman spectroscopy with chemometrical
methods allows the identification of bacteria by means of their specific
vibrational fingerprint signature. Furthermore several cell constituents were
investigated to clarify the molecular origin for characteristic bands in the
Raman spectrum of bacteria and body cells in order to elucidate doubtless
cell identification.
Acknowledgement
We gratefully acknowledge support from the Deutsche
Forschungsgemeinschaft (PO 563/7-1).
References
[1] B. Brando, D. Barnett, G. Janossy, F. Mandy, B. Autran, G. Rothe, B.
Scarpati, G. D’Avanzo, J. L. D’Hautcourt, R. Lenkei, G. Schmitz, A. Kunkl, R.
Chianese, S. Papa, J. W. Gratama, Cytometry 42, 327-346 (2000).
[2] P. Rösch, M. Harz, K.-D. Peschke, O. Ronneberger, H. Burkhardt, H.-W.
Motzkus, M. Lankers, S. Hofer, H. Thiele, J. Popp, Appl. Environm. Microbiol.
71, 1626-1637 (2004).
A miniaturized photoplethysmographic sensor system is presented which
utilizes the principle of pulse oximetry. It is designed to be implantable and
will permit continuous monitoring of important human vital parameters such
as arterial blood oxygen saturation as well as pulse rate and shape over a
long-term period in vivo. The system employs light emitting diodes and a
photo transistor embedded in a transparent elastic cuff which is directly
wrapped around an arterial vessel. This paper highlights the specific
challenges in design, instrumentation, and electronics associated with that
exceptional sensor location. In vitro measurements were performed using
an artificial circulation system which allows regulation of the oxygen saturation
and pulsatile pumping of whole blood through a section of a domestic pig’s
arterial vessel. We discuss our experimental results compared to reference
CO-oximeter measurements and determine the empirical calibration curve.
These results prove the ability of the pulse oximeter implant in a wide range
of oxygen saturation levels and pave the way for a continuous and mobile
monitoring at high-risk cardiovascular patients.
6633-39, Session 9
Examination of in vivo tear film stability after eye
blink and the eye drying
D. H. Szczesna, H. T. Kasprzak, Z. M. Kulas, Politechnika Wroclawska
(Poland); U. Stenevi, Sahlgren’s Univ. Hospital (Sweden)
The purpose of this study is to investigate the kinetics of precorneal tear film
stabilisation process after eye blink and the process of creating the tear film
break-up. The tear films of patients were examined in vivo by use of the
lateral shearing interferometer. The information about the distribution and
stability of the tear film over the cornea is carried by the wave front reflected
from the surface of tears and coded in interference fringes. The 20 sec video
sequences are registered with frequency of 25Hz. Smooth and regular fringes
indicate the smooth surface of tears over the cornea. Immediately after eye
blink the observed interference fringes are visible on a background of bright
and dark areas. The contrast of this background structure fades with time
slowly, and after about 1-3 sec the background structure of interference fringes
becomes uniform. The vertical orientation and instability of this structure
suggests its correlation with eyelid movement and the spread of tears. If the
eye is kept open for a long time, bright lines appear in the background of
fringes after a dozen seconds. The slowly appearing structure might signify
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Conference 6633: Biophotonics 2007: Optics in Life Science
the tear film dryness and formation of break-up. However, in case of eyes
after a corneal surgery the form of the background structure has different
nature and might be stable in time. This suggests existing of the stable
irregularities on the corneal surface.
Characterization of reperfusion dynamics
following long-term renal ischemia in a rat
model using tissue autofluorescence
R. N. Raman, Univ. of California/Davis (USA); C. D. Pivetti, Univ. of
California/Davis Medical Ctr. (USA); D. L. Mat-thews, Univ. of California/
Davis (USA) and Lawrence Livermore National Lab. (USA); C.
Troppmann, Univ. of California/Davis Medical Ctr. (USA); S. G. Demos,
Lawrence Livermore National Lab. (USA) and Univ. of California/Davis
(USA)
The autofluorescence under dual-UV excitation is used to assess in situ kidney
tissue response in a rat model during long-term ischemia (up to 150 minutes)
and reperfusion. During both phases, autofluorescence images of the
exposed surfaces of both ischemic and normal (control) kidneys were acquired
alternately under 355 nm and 266 nm excitation wavelengths, and the
respective average emission intensities were determined. The emission
intensity under 355 nm excitation (predominantly arising from NADH) was
then normalized to that under 266 nm (arising mostly from tryptophan). This
ratio is calculated as a means to account for changes in the optical properties
of the tissue not associated with metabolism (and thus ischemia) as well as
the illumination/collection parameters in future implementation of this
technique in a clinical setting using a hand-held probe. The temporal profile
of this signal ratio during the reperfusion phase extending up to 60 minutes
from the onset of reperfusion was fit to a relaxation model, and characteristic
time constants were extracted. The results demonstrate increasing mean
values of these time constants with increasing injury time. These time
constants were subsequently compared with the outcome of a chronic study
of rat survival rate for the different durations of ischemia. Furthermore, rat
strains with varying degree of susceptibility to ischemia were evaluated. We
postulate that this method may be able to assess tissue viability and predict
its ability to recover in the initial period following transplantation or
resuscitation.
6633-41, Session 9
In vivo study of contrasting properties of gold
nanoparticles for optical coherence tomography
E. V. Zagaynova, Nizhny Novgorod State Medical Academy (Russia); M.
V. Shirmanova, Nizhny Novgorod State Univ. (Russia); A. G. Orlova, V. A.
Kamensky, Institute of Applied Physics (Russia); M. Y. Kirillin, Oulun
Yliopisto (Finland); I. V. Balalaeva, Nizhny Novgorod State Univ. (Russia)
We have investigated the effect of gold nanoparticles with a diameter of 50
nm and nanoshells with a 150 nm silica core size and 25 nm thick gold shell
on optical properties of biotissues during skin application. We have analyzed
the possibility of using these particles as a contrast agent for optical coherence
tomography (OCT).
As the first step in our experiments, optical effects of gold nanoparticles
after one skin application were studied using OCT. Then we evaluated the
effects of multiple applications of 50 nm gold nanoparticles on skin in 30minute intervals. Finally, we compared optical properties of propylene glycol,
the standard clearing agent, and the gold nanoparticles. Biopsy of relevant
skin areas was performed under local anaesthesia and samples for light and
electron microscopy were prepared. Identification of skin layers on OCT
images was made by comparing with histology.
Application of gold-silica nanoshells to improvements in intensity of useful
signal, brightness of the superficial part of the dermis and contrast between
the superficial and deep parts of the dermis 30 minutes after application on
skin. After 24 hours the changes in OCT images became more pronounced
as the brightness of the superficial part of the dermis and the contrast between
the superficial and deep parts of the dermis further increased. In addition,
the border between the superficial and deep parts of the dermis became
more distinct, continuous and well discernible, permitting to accurately
differentiate these layers.
European Conferences on Biomedical Optics 2007 •
Optical sensor based system to monitor caries
activity in children
A. Shrestha, R. Tahir, A. Kishen, National Univ. of Singapore (Singapore)
6633-40, Session 9
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6633-42, Session 9
An optical sensor is utilized in this study to monitor mutans streptococcal
activity in human saliva. This visible light based sensor system monitored
spectral changes due to bacterial mediated biochemical reaction in saliva.
Experiments were conducted in two stages. In stage-1 characterization
experiments were conducted to standardize the optical sensor. In stage-2,
clinical experiments were carried out on stimulated saliva from patients’ of
age group less than 6 years. The bacterial mediated reaction with sucrose
was monitored using a photosensitive indicator for a period of 180 min.
Spectroscopic analysis showed that the absorption intensity at 540nm
decreases with time. A positive correlation was observed between the rate
of decrease in the absorption intensity recorded by the optical sensor and
the decrease in pH measured using the pH-meter. There was also a positive
correlation between the saliva samples with higher numbers of mutans
streptococci and lactobacilli determined using Dentocult SM and Dentocult
LB, respectively, and the spectral response monitored using optical sensor.
The findings from this study highlight the potential advantage of using an
optical sensor to monitor mutans streptococcal activity in human saliva.
6633-43, Session 9
Advanced non invasive light activated therapy
for root canal disinfection
A. Kishen, S. George, National Univ. of Singapore (Singapore)
Conventional endodontic treatment utilizes a chemico-mechanical approach
for the disinfection of root canals. However, there are different factors that
limit complete disinfection by chemico-mechanical approach. Antimicrobial
treatment based on light activation is gaining interest in treating localized
bacterial infections. The aim of this study is to test the effectiveness of a LAT
based treatment strategy to kill bacteria for root-canal disinfection. Methyelene
blue (MB) dissolved in different formulations such as Water, Glycerol, Poly
Ethylene Glycol, and a Mixture of Glycerol: Ethanol: Water (MIX), was analyzed
for photophysical, photochemical and photobiological characteristics.
Photophysical properties of MB showed formulation dependent variations.
Aggregation of MB molecules as evident from monomer to dimer ratio
depended on the molar concentrations of MB and was more in water
compared to other formulations at lower concentrations. MIX based
formulation significantly enhanced the model substrate photooxidation and
singlet oxygen generation compared to MB dissolved in other formulations.
MIX based MB formulation effectively penetrated the dentinal tubules. The
affinity of MB for Enterococcus faecalis (gram positive) and Actinomycetes
actinomycetemcomitans (gram negative) was found to be high in water based
formulation followed by MIX. Finally photoactivated killing was performed on
biofilms produced by both organisms under in vitro and ex vivo conditions. A
dual staged approach was utilized for optimum photosensitization and
irradiation was utilized in this study. This method highlights potential
application for root canal disinfection.
6633-44, Session 10
Optical sensors in water monitoring
G. Gauglitz, Univ. Tübingen (Germany)
No abstract available
6633-45, Session 10
Fast and reliable identification of
microorganisms by means of Raman
spectroscopy
P. Roesch, M. K. Harz, M. Krause, U. Neugebauer, Friedrich-SchillerUniv. Jena (Germany); J. Popp, Friedrich-Schiller-Univ. Jena (Germany)
and Institut für Physikalische Hochtechnologie e. V. (Germany)
The identification of bacteria is necessary as fast as possible e.g. to provide
an appropriate therapy for patients. Here the cultivation time should be kept
to a minimum. Beside microbiological identification methods Raman
spectroscopy is a valuable tool for bacteria identification. UV-resonance
Raman spectroscopy enables selective monitoring of the cellular DNA/RNA
content and allows for a genotaxonomic classification of the bacteria. [1]
Since UV excitation may lead to sample destruction the measurements are
performed on rotated bacterial films.
For a faster identification avoiding the cultivation step single bacteria analysis
is necessary. Using micro-Raman spectroscopy a spatial resolution in the
size range of bacteria can be achieved. With this Raman excitation the
chemical components of the whole cell are measured which leads to a
phenotypical classification. [2]
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Conference 6633: Biophotonics 2007: Optics in Life Science
In order to improve the characterization of bacteria the cellular compounds,
which are highly connected with strain and species differentiation, need to
be identified. For this purpose tip enhanced Raman spectroscopy (TERS)
was applied to single bacterial cells. [3] This enables the molecular
characterization of the cell surface with a spatial resolution of approximately
50 nm.
Acknowledgement
The funding of the research project FKZ 13N8369 within the framework
‘Biophotonik’ from the Federal Ministry of Education and Research, Germany
(BMBF) is gratefully acknowledged.
References:
1) K. Gaus, P. Rösch, R. Petry, K.-D. Peschke, O. Ronneberger, H. Burkhardt
and J. Popp, Biopolymers 2006, 82, 286-290.
2) P. Rösch, M. Harz, K.-D. Peschke, O. Ronneberger, H. Burkhardt, A. Schüle,
G. Schmautz, M. Lankers, S. Hofer, H. Thiele, H.-W. Motzkus and J. Popp,
Anal. Chem. 2006, 78, 2163-2170.
3) U. Neugebauer, P. Rösch, M. Schmitt, J. Popp, C. Julien, A. Rasmussen,
C. Budich and V. Deckert, ChemPhysChem 2006, 7, 1428-1430.
6633-46, Session 10
A reproducible surface-enhanced Raman
spectroscopy approach: online SERS
measurements in a segmented microfluidic
system
6633-48, Session 10
Biosensing with T-ray spectroscopy
B. M. Fischer, D. Abbott, The Univ. of Adelaide (Australia)
In the recent years, it has been shown that terahertz (or T-ray spectroscopy)
is a versatile tool for biosensing and safety applications. This is due to the
fact that the THz-spectra of many biomolecules show very characteristic,
distinct spectroscopic features. Furthermore, most non-metallic packaging
materials are nearly transparent in this frequency range; so that it is possible
to non-invasively identify even sealed substances such as pharmaceuticals,
illicit drugs or explosives by their spectroscopic signatures. This opens up
significant potential for a wide range of applications from safety applications
and quality control through to biomedical applications.
The individual spectroscopic features below approximately 5 THz, which
spurred the increased world-wide interest in T-ray spectroscopy, are mainly
due to intermolecular rather than intermolecular vibrations in the
polycrystalline samples. The spectra of more complex biomolecules, such
as proteins and nucleotides, typically show fewer or even no sharp features,
due to the lack of long-range intermolecular order. Furthermore, due to the
typically small sample amount, the signal to noise ratio is strongly increased.
Water shows strong absorption in this frequency range, which makes real
biomedical applications of T-ray spectroscopy rather difficult. Yet, by
combining careful sample preparation, novel experimental techniques, and
advanced signal processing of the experimental data, we can still clearly
distinguish between even complex biomolecules and therefore demonstrate
the potential the technique holds for biomedical applications.
K. R. Strehle, D. Cialla, Friedrich-Schiller-Univ. Jena (Germany); T.
Henkel, G. Mayer, Institut für Physikalische Hochtechnologie e.V.
(Germany); J. Popp, Friedrich-Schiller-Univ. Jena (Germany) and Institut
für Physikalische Hochtechnologie e.V. (Germany)
Surface enhanced Raman spectroscopy is a promising tool for the detection
of very low concentrations. Even single molecule detection is reported in the
literature.1 However a quantitative assessment is difficult as the conditions
like mixing and activation time have to be kept constant. In combination with
micro fluidic devices, where all these parameters are kept constant,
fluctuations in the concentration of special analytes can be quantitatively
monitored online. This is very interesting when investigating the concentration
of water pollutants in drinking water or the drug concentration in blood
serum.2, 3
In the following contribution we present the SERS detection of aqueous
analytes like drugs in water droplets embedded in a stream of oil. Via injection
ports the aqueous analyte solution as well as the colloidal solution is directed
into a stream of tetradecane. The advantage of this so called segmented
flow is that the adhesion of nanoparticle aggregates to the channel walls is
prevented by a thin film of oil which surrounds the water droplets.4
The flow velocity of the oil and the injected aqueous droplets can be regulated
with an external syringe pump system. For the detection the laser of a micro
Raman setup is focused directly into the channel and the signal is detected
in backscattering geometry.
To read out only the analyte containing droplets the spectrometer is triggered
externally. With this setup a defined amount of droplets can be read out
consecutively and the signal intensity can be accumulated without
accumulating the Raman signal of the separation medium tetradecane.
Acknowledgement:
Financial support of the Deutsche Forschungsgemeinschaft (DFG; grant
number Po 563/4-1) is gratefully acknowledged.
References:
1. Nie, S.; Emory, S. R., Probing single molecules and single nanoparticles
by surface-enhanced Raman scattering. Science (Washington, D. C.) 1997,
275, (5303), 1102-1106.
2. Lee, D.; Lee, S.; Seong, G. H.; Choo, J.; Lee, E. K.; Gweon, D.-G.; Lee, S.,
Quantitative analysis of methyl parathion pesticide in a polydimethylsiloxane
microfluidic channel using confocal surface-enhanced Raman spectroscopy.
Applied Spectroscopy 2006, 60, (4), 373-377.
3. McLaughlin, C.; MacMillan, D.; McCardle, C.; Smith, W. E., Quantitative
Analysis of Mitoxantrone by Surface-Enhanced Resonance Raman Scattering.
Analytical Chemistry 2002, 74, (13), 3160-3167.
4. Strehle, K. R.; Cialla, D.; Rösch, P.; Henkel, T.; Köhler, M.; Popp, J., A
Reproducible Surface-Enhanced Raman Spectroscopy Approach. Online
SERS Measurements in a Segmented Microfluidic System . Analytical
Chemistry 2007, in print.
6633-47, Session 10
A passive terahertz camera
H. Meyer, T. May, V. Zakosarenko, S. Anders, Institut für Physikalische
Hochtechnologie e.V. (Germany); G. Thorwirth, Jena-Optronik GmbH
(Germany); E. Kreysa, N. Jethava, Max-Planck-Institut für
Radioastronomie (Germany)
No abstract available
European Conferences on Biomedical Optics 2007 •
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