1. Molecular Imaging True Color Spectroscopic (METRiCS) OCT Francisco E. Robles 1,2, Christy Wilson 3, Gerald Grant 3 and Adam Wax 1,2 Nature Photonics DOI: 10.1038/NPHOTON.2011.257 Department of Biomedical Engineering 1, Medical Physics Program 2, and Pediatric Neurosurgery 3 Duke University, Durham, North Carolina 27708, USA

2. 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

3. 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

4. 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

5. 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 (SO 2 )

6. 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).

7. 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

8. Robles et al. Nature Photonics DOI: 10.1038/NPHOTON.2011.257

9. 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 SO 2 levels x-y scale bars are 100 µm

10. 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

11. Robles et al. Nature Photonics DOI: 10.1038/NPHOTON.2011.257

12. 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 = NaFS max abs /Hb max abs

13. 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

14. Acknowledgements Duke University BIOS Lab: Francisco E Robles, PhD Adam Wax, PhD Pediatric Neurosurgery: Christy Wilson, PhD Gerald 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