Human pluripotent stem cell (hPSC)-derived retinal cell culture holds great promise for treatment of retinal diseases that cause blindness. However, high-throughput non-invasive functional assays are needed for rapid optimization and quality control of these stem cell culture systems. Here we use simultaneous two- and three-photon excited fluorescence lifetime imaging microscopy (FLIM) to characterize the autofluorescent signature of light response in photoreceptor cells that originates from the fluorescent vitamin A compounds (i.e., retinoids) of the visual cycle. This multiphoton microscopy technique resolves the dynamics of visual pigment photobleaching and recycling following light exposure, including conversion of different retinoids to one another.
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