Two-color fluorescence scanning laser ophthalmoscopy for <i>in vivo</i> mouse retinal ganglion cell imaging at the single-cell level

Fluorescence scanning laser ophthalmoscopy (SLO) provides high-resolution <i>en face</i> 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 <i>in vivo</i> 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 <i>in vivo</i> 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 1.2×1.2<i>m</i><i>m</i>² 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.