Label-free full-thickness imaging of porcine vagus nerve fascicular anatomy by polarization-sensitive optical coherence tomography

Improving the efficacy of vagus nerve stimulation (VNS) therapy requires a detailed understanding of the anatomical and functional organization of nerve fiber bundles and their fascicles. Various ex-vivo imaging platforms have been optimized for this purpose. However, all existing tools with micrometer resolution require labeling to enhance the fascicle contrast, and this labeling is resource-intensive and time-consuming. Polarization-sensitive optical coherence tomography (PS-OCT) was previously used to perform high-speed, label-free small animal (rat) sciatic nerve imaging but has not been applied for imaging the full-thickness large animal vagus nerves (>1 mm diameter thick) due to tissue-limited imaging depth. We developed a PS-OCT platform that circumvents this problem and demonstrate high-speed label-free imaging of full-depth, multiple centimeters-long mammalian vagus nerves (VN) for the first time. We employed a custom-built PS-OCT system with a dual-surface scanning microscope to capture opposite sides of the sample in a single frame. A tailored post-processing algorithm maximized fascicle contrast and merged the two surfaces together. Multi-centimeter-long porcine VNs were imaged. Our approach reconstructed fascicle information throughout the full-thickness of the VN when compressed to a 650 µm thickness. Moreover, we cross-validated PS-OCT measurements of fascicular organization and retardance to assess myelination against pair histology from the same specimens, showing Spearman's rank correlation coefficient value of 0.69 (p-value < 0.001). We demonstrated a label-free optical imaging method for large-volume VN imaging. The time to image a 6.8-centimeter nerve was 680 seconds with 0.1 mm/s longitudinal sample translation speed, which is more than two orders of magnitude faster than existing modalities that require labeling. With this gain in speed and the possibility of label-free quantification of a fascicle's myelination level, important studies on inter-sample variability in fascicle organization become feasible.