PS-OCT imaging, processing and birefringence calculation

The technical details of the PS-OCT system and methods used in this study to generate PS-OCT images have been previously described in detail (14,22,32-35,44-46). Briefly, we used a custom-built fiber-based swept-source OCT system with a center wavelength of 1310 nm, a bandwidth of 110 nm, and an A-line acquisition rate of 66 kHz. A benchtop microscope, with a telecentric lens with a spot size approximately 20 μm, was used to image the tissue samples. The axial resolution of the OCT system was approximately 7 μm in tissue. Lateral and transverse scanning were achieved using galvonometric mirrors.

Conventional structural OCT and PS-OCT images were obtained simultaneously, and images were generated from the raw datasets off-line. Structural OCT images were processed and displayed by use of an inverse gray-scale lookup table. Polarization sensitivity was achieved by modulating the polarization state of the source between perpendicular polarization states in successive axial depth profiles (A-lines), then combining A-line pairs by Stokes vector analysis (22,32-35,46). PS-OCT images were processed using a spectral binning method to mitigate polarization mode dispersion, which has previously been described in detail (46). Differential (local) retardation was calculated from spectrally binned data using a depth offset of approximately 24 μm (in tissue).

Out of frame averaging was performed on OCT images over 30 frames, or approximately 60 μm, to reduce speckle noise. Structural OCT images were thresholded to remove signal < 30 dB in order to remove noise. PS-OCT birefringence was thresholded to remove signal < 20 degrees/100 microns, based on birefringence values from previously published data (22). The thresholded structural image was then used to mask the corresponding thresholded PS-OCT image to isolate birefringence signal arising from tissue. The PS-OCT region of interest (ROI) was defined as the distance between the two registration ink marks (ranging 2.6–8.8 mm) by 0.25 mm depth. The depth of 0.25 mm was chosen to mitigate signal roll off within tissue. The percent fibrosis in the PS-OCT ROI was calculated as the number of pixels with birefringence measurements ≥ 20 degrees/100 microns divided by the total number of pixels in the ROI. All data processing was performed using Matlab (Natick, MA).