Molecular Imaging True Color Spectroscopic
(METRiCS) OCT
Francisco E. Robles1,2, Christy Wilson3, Gerald Grant3 and Adam Wax1,2
Nature Photonics DOI: 10.1038/NPHOTON.2011.257
Department of Biomedical Engineering1, Medical Physics Program2, and Pediatric Neurosurgery3
Duke University, Durham, North Carolina 27708, USA
Introduction
OCT provides high resolution cross-sectional imaging
A novel form of spectroscopic OCT (SOCT) is introduced to provide molecular imaging with
high spatial and spectral resolution
Spectroscopy provides insight into the molecular composition of samples
• 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 trade-off 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*
*Robles et al. Opt. Express 17, 6799-6812 (2009)
High spatial and spectral resolution from the DW
method allows for a quantitative treatment of the depth resolved
spectra
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 y-dimension to acquire the three-dimensional (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
Robles et al. Nature Photonics DOI: 10.1038/NPHOTON.2011.257
– 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)
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– 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).
FDA approved sodium fluorescein
*Huang, Q. et al. Nature Biotechnol. 17, (1999).
METRiCS OCT using Endogenous Contrast
• 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., vessels >100 µm in diameter) produce ‘shadow’ effect
Robles et al. Nature Photonics DOI: 10.1038/NPHOTON.2011.257
Conventional OCT image
METRiCS OCT image
x-z scale bars are 100 µm
Robles et al. Nature Photonics DOI: 10.1038/NPHOTON.2011.257
METRiCS OCT using Endogenous Contrast
Robles et al. Nature Photonics DOI: 10.1038/NPHOTON.2011.257
• 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 spatially-resolved 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
METRiCS OCT using Exogenous Contrast
Conventional OCT image
METRiCS OCT image
Robles et al. Nature Photonics DOI: 10.1038/NPHOTON.2011.257
x-z scale bars are 100 µm
• To date, conventional OCT has shown limited success in using exogenous contrast agents
• METRiCS OCT provides clear molecular contrast form the exogenous agent
- 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
Robles et al. Nature Photonics DOI: 10.1038/NPHOTON.2011.257
x-y scale bars are 100 µm
• 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
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 BIOS Lab:
Francisco E Robles, PhD
Adam Wax, PhD Pediatric Neurosurgery:
Christy Wilson, PhDGerald Grant, MD
A.W. is the founder and chairman of Oncoscope, which licenses the rights to intellectual property underlying this work.
Funding NIH (NCI R01 CA 138594-01)
Robles et al. Nature Photonics DOI: 10.1038/NPHOTON.2011.257