Project Summary To understand visual function and cellular metabolism in the living eye, we need a means to visualize individual cells and assess molecular dynamics non-invasively. Some of the key molecules involved in the visual cycle and cellular energy production are intrinsically fluorescent, but are inaccessible in the living eye through single-photon excitation because the needed wavelength range is not transmitted by the optics of the eye. Two-photon excited fluorescence ophthalmoscopy (TPEFO) can excite these otherwise inaccessible fluorophores with near-infrared light. In our previous experiments in the living macaque eye, we used an adaptive optics scanning light ophthalmoscope (AOSLO) to non-invasively image many different cell classes throughout the retina, including ganglion cells (Sharma, Williams et al., 2016). Additionally, we measured visual cycle function by characterizing the time course of two-photon excited fluorescence from photoreceptors (Sharma, Schwarz et al., 2016). We now propose to expand the capabilities of TPEFO in the living rodent and macaque eye to measure several important fluorescence properties, such as emission spectrum, fluorescence lifetime, and redox ratio, which may be indicators of cell health and function. This research has the potential to provide insight into normal and altered biochemical processes and improve our understanding of diseases that impact retinal metabolism and function such as glaucoma, macular degeneration and Leber hereditary optic neuropathy.