Project Summary Non-invasive methodologies to visualize individual cells and assess molecular dynamics are needed to improve our understanding of visual function and cellular metabolism in the living eye. Some of the key molecules involved in the visual cycle and cellular energy production are intrinsically fluorescent. However, they are inaccessible in the living eye using single-photon excitation because the excitation wavelength range is not transmitted by the optics of the eye. We have developed two-photon excited fluorescence ophthalmoscopy (TPEFO) to excite these otherwise inaccessible fluorophores with near- infrared light in conjunction with an adaptive optics scanning light ophthalmoscope that enables non- invasive high-resolution imaging of many different cell classes throughout the in vivo retina. With TPEFO, visual cycle kinetics can be characterized through the intensity changes of emitted two-photon excited fluorescence from photoreceptors. During this funding period, the unique capabilities of TPEFO will be enhanced to measure additional meaningful fluorescence properties in pre-clinical models. As a window into the molecular dynamics of the visual cycle, we will track, in conjunction with the kinetics of emitted fluorescence in response to visual stimulation, changes in the fluorescence lifetime of photoreceptors. Our second aim is to non-invasively measure neuronal function by tracking emitted two-photon excited fluorescence kinetics and lifetime from the inner retina. Finally, we will compare the TPEFO intensity emitted in different spectral bands to calculate the optical redox ratio, a measure of a cell's balance between aerobic and anaerobic metabolism. All of these measures may be indicators of cell health and function. In addition to characterizing the TPEFO properties in healthy eyes, we will use systemic hypoxia as a model of altered cellular respiration to represent the potential changes that occur early in disease. 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 visual function such as glaucoma, macular degeneration and Leber hereditary optic neuropathy.