Integrated visible-light optical coherence tomography and fluorescence scanning laser ophthalmoscopy to image retinal ganglion cell axons.

Retinal ganglion cell (RGC) soma and axonal damage is a hallmark of optic neuropathies. Visible-light OCT fibergraphy (vis-OCTF) enables non-invasive imaging and quantitative assessment of individual RGC axon bundles; however, validating vis-OCTF using confocal fluorescence imaging of flat-mounted postmortem retina is less accurate due to structural alterations caused by flat-mount preparation and cannot be performed longitudinally. For in vivo vis-OCTF validation, we developed an integrated visible-light optical coherence tomography (vis-OCT) and fluorescence scanning laser ophthalmoscopy (SLO) system. The vis-OCT had a 100 nm bandwidth with a center wavelength of 560 nm, offering an axial resolution of 1.3 µm in the retina. The lateral resolutions of vis-OCT and SLO were 4 µm and 3.5 µm, respectively. In the transgenic Eno2-YFP mice, we showed that vis-OCTF and SLO provide consistent RGC axon bundle imaging results. Measuring 30 axon bundle widths from six mice yielded a Pearson correlation coefficient of 0.991 between SLO and vis-OCTF. Thus, the combined SLO and vis-OCT can potentially achieve multimodal longitudinal in vivo studies of RGC pathologies.