1
STRATEGIC RESEARCH DEVELOPMENT
GRANT (SRDG)
Emerging Opportunites and Challenges
INTERDISCIPLINARY CENTRE
FOR
MEDICAL PHOTONICS
Interim Report October 2005
Professor Kishan Dholakia, Professor Wilson Sibbett, Professor Andrew Riches,
Dr. Peter Bryant
University of St. Andrews
Professor Sir Alfred Cuschieri, Dr. PaulCampbell,
University of Dundee
2
Table of Contents
1
Overview of specific targets and milestones
2
Detailed scientific evaluation
3
Summary and forward look
4
Appendix of published journal papers
5
Appendix of publicity generated from the work
3
1. Progress with specific aims and objectives
(a)
2. Increased capacity and expansion of the research base
(b) New laboratory areas in Physics have been refurbished and new dedicated laser
rooms developed. A tissue culture laboratory has been included in this plan to allow
rapid and convenient access to the laser facilities. An optical tweezers facility has
been established in the Bute Medial School to facilitate the work on cell and
chromosome separation.
(c ) Research development and innovation
(d) interdisciplinary and multi-disciplinary research activities
Regular Biophotonics Tuesday teatimes have been instigated alternating between the
Physics and Medical School site and have proved a useful forum for discussion
between all staff. Regular meetings of the SRDG group have also been undertaken to
ensure integration and planning These biophotonics meeting take the form of a lunch
with two or three talks on various on-going projects. A Cancer Colloquium on
“Biophysical Approaches to Cancer Diagnosis : cancer diagnosis under a new light.”
brought International experts to St. Andrews for a highly successful meeting.
(e) Core research programmes and research teams
Thematically a number of key areas have emerged from the grant
(f) opportunities for postgraduate research
Next generation of researchers
Training, PhDs, postdocs, courses
(g)
4
3. Knowledge Transfer
(h) Engagement with users
category of other users by type
location of users
(j) Response to other users
(k) spin-outs
(l) industry interaction
4. Communication, collaboration and dissemination
(m) enhanced reputation
(n) networks/strategic partnerships
(p) publications
Peer reviewed journals
(k) Spin-out/IP
(l) Specific Targets / Milestones.
5. Sustainability and forward look
(q)
(r)
(s) Future plans
5
6. Finanical Report
Appendix One
Copies of journal papers published
Appendix two
Publicity generated
Year 1 and 2
To:

recruit staff and refurbish existing laboratories.
Action :Three excellent postdoctoral fellows have been recruited.

establish the proper environment and group dynamics between medical scientists,
physicists and clinical scientists through internal workshops and research
colloquia.
Action: Regular Biophotonics Tuesday teatimes have been instigated alternating
between the Physics and Medical School site and have proved a useful forum for
discussion. Regular meetings of the SRDG group have also been undertaken to
ensure integration and planning. A Cancer Colloquium on “Biophysical
Approaches to Cancer Diagnosis : cancer diagnosis under a new light.” brought
International experts to St. Andrews for a highly successful meeting.

establish
milestones
within
the
initial
research
themes
and
ensure
interdisciplinary effort.
Action: Initial meetings set up milestones and experimental plans. The results are
summarised below.
6

run specialist Workshops to disseminate interface science to healthcare and
scientific communities and pave the way for wider interactions
Action :
7
Scientific Summary of Work
1. Targeted gene and drug delivery using optical systems.
The cell membrane represents the outer extremity of all eukaryotic cells.
In
mammals, this is a thin (5nm) bi-layer film of lipids, embedded with various protein
molecules at interspersed locations. The membrane encloses the cell, defines its
boundaries and maintains the essential physio-chemical differences between the
cytoplasm and the extracellular environment. Under normal circumstances, the lipid
nature of the cell membrane acts as an impermeable barrier to the passage of most
water soluble molecules. Thus the selective introduction of therapeutic agents to the
inside of dysfunctional or diseased cells remains problematic. Only a handful of
useful approaches for the delivery of membrane impermeants have been devised thus
far. These include physical injection into individual cells using glass micropipettes
membrane fusion of loaded lyposomes, ballistic introduction of coated gold nanospheres (gene gun), delivery of therapeutic agents encapsulated in membrane
permeable shells (vectors), local permeabilisation of cells via the application of pulsed
electric fields, local permeabilisation of cells via the application of diagnostic
ultrasound (Sonoporation). The introduction of foreign DNA into cells (transfection)
is a key procedure in genetic analysis and recombinant protein experiments. Various
methods for puncturing the cell membrane without causing any collateral damage
have been implemented. While it is possible to introduce genes into cells by a variety
of methods, it is very difficult to do this in a targeted way so that specific cells in a
population are targeted. We have now developed a method of doing this using laser
targeting within this SRDG proposal which is protected by Intellectual Property.
Additionally, colleagues at Dundee in collaboration with St Andrews and researchers
in the USA have performed studies of cell membrane disruption using microbubble
injection. This too is covered by Intellectual Property.
A low cost, compact, violet diode laser, of 405 nm wavelength, was used to transfect
Chinese hamster ovary (CHO) cells with plasmid DNA. The strongly focused beam,
with an optical power density of around 1200 MW/m2, creates a hole in the cell
plasma membrane allowing uptake of plasmid expression vector. This power density
is six orders of magnitude less than femtosecond, infrared lasers (around 104 TW/m2),
8
that have also been used for photoporation. Laser-assisted cell transfection techniques
offer the attraction of sterility, a high degree of selectivity, and compatibility with
standard microscopes.
Figure : An example of photoporation on four CHO cells in one sample using a 405
nm violet diode laser at a power of 0.3 mW focused to a spot of 1 m diameter. A
x100 microscope objective is used to both image the cells and focus the laser beam.
9
Figure : a) Transfected, live, CHO cells expressing DsRed-Mito, viewed under x 100
magnification, using the Rhodamine channel of a Zeiss Axioscope.
b) CHO cells expressing EGFP, viewed under x63 magnification, using the FITC
channel of a Zeiss Axioscope.
Transfection was achieved, in both cases, by
photoporation with a violet diode laser.
2. Optical micro-manipulation.
a. Chromosome tweezing and FISH probe generation
An optical tweezer system (employing an Nd:YAG 1064nm laser; see Fig. 1) has
been developed to tweeze and isolate single chromosomes from suspensions of
Chinese Hamster Ovary (CHO) cell chromosome preparations. CHO cell lines are
maintained in the laboratory and mitotic cells are harvested for chromosome
preparation.
Chromosomes are trapped in the focus of the laser beam and manipulated towards the
end of fine-bore glass capillaries which are produced using a micro-electrode puller.
The capillary is attached to a micro-syringe allowing 1µl aliquots of suspension to be
dispensed for use in Polymerase Chain Reaction (PCR) experiments to generate
probes for use in Fluorescent in-situ Hybridisation (FISH).
10
Fig.1 – Optical tweezer system
Fig.2 – Chromosome tweezed to capillary
Chromosomes have been successfully tweezed as demonstrated in Fig.2 above and are
being used in PCR. Fig.3 below shows hybridisation of a PCR-generated fluorescent
probe in CHO cell metaphase spreads.
Fig.3 – FISH experiment on CHO metaphase spreads. Arrows represent
hybridisation of probe.
Work is continuing to optimise conditions for probe production by varying PCR
parameters and conditions.
b. Optical tweezing using a femtosecond laser
c. Cell sorting using a Bessel beam
Tailored optical potential landscapes have been used to accumulate
microscopic particles and to arrange articles in pre-described arrays for example in
linear fringes (MacDonald et al. 2001) or extended lattices (Korda et al. 2002,
MacDonald et al. 2004). A mixture of microscopic particles with different optical
properties may be separated when placed on a sculpted or modulated optical potential
by exploiting differing particle responses to the pattern. Particles of different sizes,
11
shapes or refractive indices may be separated in such an optical pattern. The Bessel
beam is used as the optical potential landscape with which to sort mixtures of spheres
of differing sizes and also different cell types.
The Bessel beam is described as ‘nondiffracting’, and consists of a series of
concentric rings surrounding a central beam. Each ring of the Bessel beam acts as a
potential well within which particles can reside and undergo Brownian motion.
Random hopping can occur according to Kramers’ theory (Kramers. 1940; McCann
et al. 1999). Each ring has equal optical power therefore the intensity of the rings
increase closer to the beam centre. The rings of the Bessel beam define a series of
potential wells that increase in depth towards the centre. Particles smaller than, or
similar to, the well diameter will reside within the optical potential well and thermal
activation will lead to the particles hopping very slowly across the optical potential
landscape of the Bessel beam. Larger particles which straddle two or more potential
wells in the pattern respond to the overlying envelope of the pattern rather than
respond to each individual well. Therefore, the gradient force of the overlying
envelope draws the larger particles rapidly towards the beam centre. This allows the
particles to be sorted and separated without the need to implement microflows within
the system. The separation of erythrocytes and lymphocytes has been performed in
this manner.
An approximation to a Bessel beam was created by passing a Gaussian beam
from a Nd:YAG laser through an axicon, then telescoping the beam into a sample
chamber containing the cell mixture. The beam had a central core of 5 m diameter,
ring width of 3 m, ring spacing of 2 m and a propagation distance of 3 mm.
Erythrocytes (bi-concave disc-shaped, approximately 8 m diameter, 2 m wide) are
well known to re-orient or align in optical traps (Grover et al.) with their longest axis
in the direction of beam propagation. When erythrocytes were placed in a low power
Bessel beam (150 mW) many of them were transported towards the beam centre
where they re-oriented and were then guided upwards within the central core or they
re-oriented in the first or second rings. At higher beam power, the rings in which the
erythrocytes aligned were further away from the beam centre, for example 550 mW of
total beam power, the erythrocytes re-orient and were guided within the third, fourth
and fifth rings. Lymphocytes (spherically shaped, 8 m diameter) straddled two of
the Bessel beam rings and were always transported to the beam centre where they
12
were guided upwards. As the lymphocytes moved closer to the beam centre, their
velocity increased due to the gradient force of the overlying envelope of the pattern.
As power increased, the velocity of lymphocytes traveling to the beam centre also
increased due to enhanced convection (figure 1). Separation of lymphocytes and
erythrocytes as performed at 550mW as shown in figure 2. The erythrocytes aligned
and were guided in the outer rings, whereas lymphocytes were transported to the
beam centre and were then guided within the central core. Frame (e) of figure 2
shows how lymphocytes may be extracted from the central core using a
microcapillary.
Subpopulations of cells were also be labeled, via antibodies to cell surface markers,
with silica spheres. Due to the increased refraction and scattering of the laser light by
silica spheres, these cell-sphere conjugates were transported to the beam centre and
were guided upwards more rapidly than unlabelled cells. This enhanced separation
can be seen in figure 3, which shows a mixture of lymphocytes containing Tlymphocytes labeled - via the CD2 cell-surface marker - with 5 m diameter, silica
microspheres. The sphere-labeled cells were separated from the unlabelled cells due
to greater refraction of the beam through the spheres than through the cells resulting
in a stronger gradient force on the spheres. Therefore they traveled more rapidly than
unlabeled cells across the Bessel beam optical potential landscape to the central core,
where they were guided upward by radiation pressure. The final frame shows a stack
of two sphere-T-cell conjugates, viewed from above, at the top of the sample chamber
in the central core of the Bessel beam, separated from the unlabelled cells.
In
summary, cell sorting was achieved in the optical potential landscape of a Bessel
beam. Exploiting the differing interactions of different cell types with the beam leads
to some cell types residing within the individual wells, and some cell types
accumulating in the beam centre. This mechanism of sorting is static and requires no
fluid flow as is easy to implement using simple optics.
Figures
13
Figure 1: Velocity of lymphocytes (white blood cells) across the Bessel beam rings
for three power regimes.
Figure 2: Separation of lymphocytes and erythrocytes in a Bessel beam of 550 mW.
Figure 3: Separation of sphere-labeled T-lymphocytes from unlabeled lymphocytes in
the Bessel beam.
14
References
MacDonald, M.P., L. Paterson, W. Sibbett, K. Dholakia and P.E. Bryant.. Trapping
and manipulation of low-index particles in a two- dimensional interferometric optical
trap. Optics Letters: 26, 863-865 (2001).
Korda, P. T., M. B. Taylor and D. G. Grier. Kinetically locked-in colloidal transport
in an array or optical tweezers. Physical Review Letters: 89, art. no.128301 (2002).
MacDonald, M.P., G.C. Spalding and K. Dholakia. Microfluidic sorting in an optical
lattice. Nature: 426, 421-424, (2003).
Kramers, H.A. Brownian motion in a field of force and the diffusion model of
chemical reactions. Physica: 7, 284 (1940).
McCann, L.I., M. Dykman and B. Golding. Thermally activated transitions in
bistable three-dimensional optical trap. Nature: 402, 785-787 (1999).
Grover, S.C., R.C. Gauthier and A.G. Skirtach. Analysis of the behaviour of
erythrocytes in an optical trapping system. Optics Express: 7, 533-539 (2000).
15
Published Journal Papers
Femtosecond optical tweezers for in-situ control of two-photon fluorescence
B. Agate, C. T. A. Brown, W. Sibbett and K. Dholakia, Optics Express 12, 3011
(2004).
Optical guiding of microscopic particles in femtosecond and continuous wave
Bessel light beams
H. Little, C.T.A. Brown, V.Garcés-Chávez, W. Sibbett and K. Dholakia, Opt. Express
12, 2560 (2004)
Imaging in optical micromanipulation using two-photon excitation
K Dholakia, H Little, C T A Brown, B Agate, D McGloin, L Paterson and W Sibbett,
New J. Phys. 6 136 (2004)
Photoporation and cell transfection using a violet diode laser
L. Paterson, B. Agate, M. Comrie, R. Ferguson, T. K. Lake, J. E. Morris, A. E.
Carruthers, C. T. A. Brown, W. Sibbett, P. E. Bryant, F. Gunn-Moore, A. C. Riches,
K. Dholakia, Opt. Express (2005)
Membrane disruption by optically controlled microbubble cavitation,
Paul Prentice, Alfred Cuschieri, Kishan Dholakia, Mark Prausnitz and Paul Campbell,
accepted for Nature Physics