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:

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