Molecular Imaging True
Color Spectroscopic
(METRiCS) OCT
Francisco E. Robles1,2, Christy Wilson3, Gerald
Grant3 and Adam Wax1,2
Department of Biomedical Engineering1, Medical Physics Program2, and
Pediatric Neurosurgery3
Duke University, Durham, North Carolina 27708, USA
Nature Photonics DOI: 10.1038/NPHOTON.2011.257
Introduction
OCT provides high
resolution crosssectional imaging
Spectroscopy provides
insight into the molecular
composition of samples
A novel form of spectroscopic OCT (SOCT) is
introduced to provide molecular imaging with
high spatial and spectral resolution
• A dual window processing methods is used to achieve high
spatial and spectral resolution
• A laser light source with a bandwidth (BW) that spans the
visible region of the spectrum is used
• Quantitative molecular imaging in true color is achieved
Dual Window (DW) Method
• Typical processing methods for SOCT, including short time Fourier
transforms and wavelet transforms, suffer from an inherent tradeoff between the spatial and spectral resolution
• The DW method avoids this resolution trade-off by using two
orthogonal windows that independently tune the resolution in
each dimension*
High spatial and spectral
resolution from the DW
method allows for a
quantitative treatment
of the depth resolved
spectra
*Robles et al. Opt. Express 17, 6799-6812 (2009)
Parallel Frequency Domain OCT
System
• The light source consists of a super continuum laser
– Detection centered in the visible spectrum (450-700 nm)
– Large BW yields an experimental axial resolution of 1.2 µm
• A cylindrical lens is used to deliver a line of illumination
onto the sample
• Lateral resolution is 6 µm
• Detection is achieved by using
an imaging spectrograph which
detects 400 interferograms
simultaneously
• Sample is translated along the ydimension to acquire the threedimensional (3D) data set
Robles et al. Nature Photonics DOI: 10.1038/NPHOTON.2011.257
Processing for METRiCS OCT
• Each interferogram is processed using the DW method
• The spectra at all points in the sample is divided into red,
greed, and blue channels to provide a hue map with the
sample’s true colors
– This provides an intuitive form
of display of the spectral data
• The spectra may also be
analyzed quantitatively to
obtain parameters of
interest such as hemoglobin
oxygen levels (SO2)
Robles et al. Nature Photonics DOI: 10.1038/NPHOTON.2011.257
Animal model
• An in-vivo CD1 nu/nu normal mouse
dorsal skinfold window chamber
model was used*
• Mice were anaesthetized and the
window chambers were removed
before imaging
• Endogenous (Hb) and exogenous
(sodium fluorescein; NaFS) molecular
contrast were utilized
FDA approved sodium fluorescein
– NaFS has an extinction maxima at ~494 nm,
thus transmitted light appears red (bottom
left). It fluoresces with a peak wavelength
of ~521 nm thus appearing green at low
concentrations (bottom right).
*Huang, Q. et al. Nature Biotechnol. 17, (1999).
METRiCS OCT using Endogenous
Contrast
Conventional OCT image
• Conventional OCT imaging
revealed tissue structures
- E.g., muscle layer layer at the
surface, lumen of blood vessels
and the subcutaneous layer
• METRiCS OCT reveals the same
structures with the addition of
true-color molecular contrast
- Muscle layer appears relatively
colorless due to low Hb
concentrations
- Once light traverses through the
vasculature network, a red shift is
clearly observed due to the higher
concentrations of Hb
- Highly attenuating regions (e.g.,
x-z scale bars are 100 µm
vessels >100 µm in diameter)
produce ‘shadow’ effect
Robles et al. Nature Photonics DOI: 10.1038/NPHOTON.2011.257
METRiCS OCT image
Robles et al. Nature Photonics DOI: 10.1038/NPHOTON.2011.257
METRiCS OCT using Endogenous
Contrast
• An en-face view of the volumetric data provides a global prospective of the
vasculature network
- The major vessel on the left is an artery, while the one of the right is a vain
• An important capability
of METRiCS OCT is the
ability to provide a
quantitative analysis
from the spatiallyresolved spectra
- Spectra from points (b)(e) are measured and
plotted on the right
- The figure also shows the
computed Hb SO2 levels
x-y scale bars are 100 µm
Robles et al. Nature Photonics DOI: 10.1038/NPHOTON.2011.257
METRiCS OCT using Exogenous
Contrast
Conventional OCT image
METRiCS OCT image
• To date, conventional OCT
has shown limited success
in using exogenous
contrast agents
• METRiCS OCT provides
clear molecular contrast
form the exogenous agent
-
-
x-z scale bars are 100 µm
Robles et al. Nature Photonics DOI: 10.1038/NPHOTON.2011.257
The presence of NaFS is
evident by a severe red
shift in hue
NaFS also shows an
increase in scattering from
within the vessels
Robles et al. Nature Photonics DOI: 10.1038/NPHOTON.2011.257
METRiCS OCT using Exogenous
Contrast
• Vessels in the en-face METRiCS OCT image are now characterized by
the red hue of NaFS
• Large vessels still exhibit a ‘shadow’
• Spectra of four points
are quantitatively
analyzed
-
-
-
The spectra now exhibits
contributions form three
absorbing species: oxy-Hb,
deoxy-Hb and NaFS
NaFS only absorbes in the
lower wavelength regions,
thus SO2 levels may still be
computed
ε = NaFSmax abs/Hbmax abs
x-y scale bars are 100 µm
Robles et al. Nature Photonics DOI: 10.1038/NPHOTON.2011.257
Conclusions
• METRiCS OCT provides molecular contrast using
endogenous and/or exogenous agents
• The DW method allows for a thorough quantitative
analysis
• Use of the large visible spectral bandwidth provides
1.2 µm resolution and allows for a true color
representation of samples
• Introduces possibility of using readily available
contrast agents for molecular contrast (e.g., using
FDA approved NaFS)
Robles et al. Nature Photonics DOI: 10.1038/NPHOTON.2011.257
Acknowledgements
Duke University
Funding
BIOS Lab:
Francisco E Robles, PhD
Adam Wax, PhD
Pediatric Neurosurgery:
Christy Wilson, PhD
Gerald Grant, MD
NIH (NCI R01 CA 138594-01)
A.W. is the founder and chairman of
Oncoscope, which licenses the rights
to intellectual property underlying
this work.
Robles et al. Nature Photonics DOI: 10.1038/NPHOTON.2011.257