Computational aberration-corrected volumetric imaging of single retinal cell dynamics in the living eye

Abstract The eye offers a unique non-invasive window for accessing single-cell level structures and functions of the central nervous system (CNS) throughout the retina. However, strong, space-varying ocular aberrations and limited volumetric imaging speed challenges large-scale single-cell imaging in living eyes. Here, we present plenoptic illumination scanning laser ophthalmoscopy (PI-SLO), a 3D fluorescence retinal imaging modality that enables high-speed, widefield, volumetric single-cell imaging with low phototoxicity. By capturing multiple angular views of fluorescence signals from the entire retinal volume, PI-SLO performs digital aberration correction and reconstructs 3D images over a >20° field of view at >23 volume per second. We apply PI-SLO to investigate three key aspects of CNS physiology through the living mouse retina: microglial process dynamics, vascular perfusion, and light-evoked calcium activity in inner retinal neurons. PI-SLO provides a versatile, non-invasive platform for studying vision, retinal disease, and CNS physiology at cellular resolution.