Visible-light optical coherence tomography fibergraphy for quantitative imaging of retinal ganglion cell axon bundles

Purpose: To develop a practical technique for visualizing and quantifying retinal ganglion cell (RGC) axon bundles in vivo. Methods: We applied visible-light optical coherence tomography (vis-OCT) to image the RGC axon bundles, referred to as vis-OCT fibergraphy, of healthy wild-type C57BL/6 mice. After vis-OCT imaging, retinas were flat-mounted, immunostained with anti-betaIII tubulin (Tuj1) antibody for RGC axons, and imaged with confocal microscopy. We quantitatively compared the RGC axon bundle networks imaged by in vivo vis-OCT and ex vivo confocal microscopy using semi-log Sholl analysis. Results: Side-by-side comparison of ex vivo confocal microscopy and in vivo vis-OCT confirmed that vis-OCT fibergraphy captures true RGC axon bundle networks. The semilog Sholl regression coefficients extracted from vis-OCT fibergrams (3.7 ± 0.8 mm^–1)and confocal microscopy (3.6 ± 0.3 mm^–1) images also showed good agreement with each other (n = 6). Conclusions: We demonstrated the feasibility of using vis-OCT fibergraphy to visualize RGC axon bundles. Further applying Sholl analysis has the potential to identify biomarkers for non-invasively assessing RGC health. Translational Relevance: Our novel technique for visualizing and quantifying RGC axon bundles in vivo provides a potential measurement tool for diagnosing and tracking the progression of optic neuropathies.