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Fluorescence lifetime imaging microscopy (FLIM) is a technique that analyzes the metabolic state of tissues based on the spatial distribution of fluorescence lifetimes of certain interacting molecules. We used multiphoton FLIM to study the metabolic state of developing C57BL6/J and rd10 retinas based on the fluorescence lifetimes of free versus bound nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate (NAD(P)H), with free NAD(P)H percentages suggesting increased glycolysis and bound NAD(P)H percentages indicating oxidative phosphorylation. The mice were sacrificed and enucleated at various time points throughout their first 3 months of life. The isolated eyecups were fixed, sectioned using a polyacrylamide gel embedding technique, and then analyzed with FLIM. The results suggested that in both C57BL6/J mice and rd10 mice, oxidative phosphorylation initially decreased and then increased, plateauing over time. This trend, however, was accelerated in rd10 mice, with its turning point occurring at p10 versus the p30 turning point in C57BL6/J mice. There was also a noticeable difference in oxidative phosphorylation rates between the outer and inner retinas in both strains, with greater oxidative phosphorylation present in the latter. A greater understanding of rd10 and WT metabolic changes during retinal development may provide deeper insights into retinal degeneration and facilitate the development of future treatments.

Fluorescence lifetime imaging microscopy (FLIM) evaluates the metabolic state of tissue based on reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD). Fluorescence lifetime imaging ophthalmoscopy (FLIO) can image the fundus of the eyes, but cannot detect NAD(P)H. We used multiphoton FLIM to study the metabolic state of the retina in fixed eyes of wild-type mice C57BL6/J. We sectioned the eye using a polyacrylamide gel-embedding technique and estimated the percentage of bound NAD(P)H. We found that oxidative phosphorylation was the predominant metabolic state, particularly in the inner retina, when a fixed retina was used. We also demonstrated the feasibility of FAD imaging of the retina. In addition, we demonstrated that autofluorescence and various FLIM channels, such as hemoglobin, melanin and collagen, can be used to evaluate the structure of the retina and other parts of the eye without any special staining.