Visible-Light Optical Coherence Tomography Fibergraphy for Quantitative Imaging of Retinal Ganglion Cell Axon Bundles

Abstract

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-beta-III 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.

Longitudinal analysis of retinal ganglion cell damage at individual axon bundle level in mice using visible-light optical coherence tomography fibergraphy

“…Taking advantage of the improved axial resolution offered by vis-OCTF and associated data processing methods [34], we established an analytic tool for tracking changes in individual RGC axon bundles rather than the bulk thickness of the RNFL in vivo . Briefly, four vis-OCT volumes were acquired from the same eye with the optic nerve head (ONH) aligned to each of the four corners of the field of view.…”

“…Each vis-OCT image volume was approximately 700 μm × 700 μm × 1500 μm (x × y × z). For each area, we generated vis-OCT fibergrams from the vis-OCT volume [34]. We used an intensity-based threshold method to detect the surface of the retina and cropped the RNFL by selecting the first ~15 μm in depth, and then calculated the mean intensity projection along the axial (z) direction to generate the fibergram image composed of RGC axon bundles and surrounding vasculature.…”

“…As a result, vis-OCT provides unique anatomical and functional imaging capabilities that facilitate RGC damage evaluation [33]. Recently, we developed vis-OCT fibergraphy (vis-OCTF) to visualize and quantify changes in individual RGC axon bundles in mice [34][35][36]. In this study, we applied vis-OCTF to track the changes of axon bundles following the ONC injury by quantifying four parameters: (1) lateral bundle width, (2) axial bundle height, (3) cross-sectional area, and (4) bundle shape.…”

“…We recently applied visible-light OCT (vis-OCT), which operates from 510 nm to 610 nm and reaches an axial resolution of 1.3 μm, in the mouse retina [33, 34]. Because optical scattering contrast nearly exponentially decays with increasing wavelength, the visible-light spectral range offers a much higher contrast than the near-infrared (NIR) range [34]. As a result, vis-OCT provides unique anatomical and functional imaging capabilities that facilitate RGC damage evaluation [33].…”

The Development and Clinical Application of Innovative Optical Ophthalmic Imaging Techniques

“…Vis-OCT has continued to develop. Registration and averaging of multiple volumes have allowed the visualization of single cells, and the concept of Vis-OCT fibergraphy (Vis-OCTF) of imaging-specific retinal ganglion cell (RGC) axon bundles has been introduced in animal models (108)(109)(110). Ultrahigh resolution Vis-OCT (UHR Vis-OCT) is capable of imaging sublayers within retinal layers, such as the IPL (Figure 2), and improvements in the axial resolution have also been made using rapid spectral shaping, axial tracking, and in vivo spatially dependent numerical dispersion compensation (111)(112)(113).…”