Two-photon fluorescence imaging has a number of advantages for retinal imaging. Our group was the first to demonstrate two-photon imaging in the living primate eye (Hunter et al, 2011) we are using those results as a stepping stone for the following projects:
Two-photon imaging permits the study of molecular species like NADH, FAD, retinol and others. These molecules are direct indicators of cellular function and their excitation regime is in the ultraviolet, which lies outside the transmittance spectrum of the ocular media. Two-photon absorption using infrared light is the only way to excite fluorescence from these molecules and we use this method to study cellular metabolism and the visual cycle in the living eye.
Two-photon imaging of fluorophores using infrared light prevents unwanted stimulation of photoreceptors during imaging. This property is useful for measuring the response of ganglion cells labeled with calcium indicators like G-CaMP for studying the retinal circuitry (ganglion cell function).
By combining adaptive optics with fluorescence lifetime imaging, we are able to resolve the fluorescence decay with high resolution both spatially and temporally. Each fluorophore in the retina has a unique fluorescence lifetime which can be modified by environmental factors including enzyme binding. As a result, AOFLIO may be able to help us identify the contribution from retinal fluorophores and how the retinal environment changes when the retina becomes unhealthy. We have shown that cones and rods have different fluorescence lifetimes in the living macaque retina.