Two-color fluorescence scanning laser ophthalmoscopy for in vivo mouse retinal ganglion cell imaging at the single-cell level.

Fluorescence scanning laser ophthalmoscopy (SLO) provides high-resolution en face imaging of the retina to investigate retinal ganglion cells (RGCs) in both small animals and humans. Multicolor fluorescence SLO can further extend the anatomical and functional imaging capabilities; however, its in vivo implementation remains challenging due to optical aberrations, limited labeling strategies, and the need for multichannel calibration. In this study, we present a compact two-color fluorescence SLO system designed with registered field-of-views (FOVs) between the two color channels for in vivo imaging of RGCs at single-cell resolution in mice. Using an Eno2-YFP transgenic mouse model with dextran labeling of the RGCs, we demonstrate high-quality two-color imaging across a 1.2×1.2mm2 FOV. Furthermore, by employing adeno-associated virus (AAV)-mediated co-labeling of RGCs and mitochondria, we achieved high-resolution visualization and image overlay with precise two-channel registration. The two-color fluorescence SLO system holds promise for longitudinal studies of RGCs, with applications in neurodegeneration, glaucoma, and therapeutic development.