Ex Vivo Confocal Spectroscopy of Autofluorescence in Age-Related Macular Degeneration

We investigated the autofluorescence (AF) signature of the microscopic features of retina with age-related macular degeneration (AMD) using 488 nm excitation. The globes of four donors with AMD and four age-matched controls were embedded in paraffin and sectioned through the macula. Sections were excited using a 488 nm argon laser, and the AF emission was captured using a laser scanning confocal microscope (496-610 nm, 6 nm resolution). The data cubes were then analyzed to compare peak emission spectra between the AMD and the controls. Microscopic features, including individual lipofuscin and melanolipofuscin granules, Bruch's Membrane, as well macroscopic features, were considered. Overall, the AMD eyes showed a trend of blue-shifted emission peaks compared with the controls. These differences were statistically significant when considering the emission of the combined RPE/Bruch's Membrane across all the tissue cross-sections (p = 0.02). The AF signatures of ex vivo AMD RPE/BrM show blue-shifted emission spectra (488 nm excitation) compared with the control tissue. The magnitude of these differences is small (~4 nm) and highlights the potential challenges of detecting these subtle spectral differences in vivo.