Spectral profiling of autofluorescence associated with lipofuscin, Bruch's membrane, and sub-RPE deposits in normal and AMD eyes

PURPOSE. To compare the autofluorescence spectra of retinal pigment epithelium (RPE)-associated lipofuscin, Bruch's membrane, and sub-RPE deposits (drusen and basal laminar-linear deposits) in eyes of donors with age-related macular degeneration (AMD) against eyes of age-matched control donors. METHODS. Cryosections were cut from the maculae of unfixed human donor eyes with AMD or from age-matched control eyes. Tissues were excited at wavelengths of 364, 488, 568, and 633 nm. Emission spectra were collected with a confocal microscope equipped with a spectrophotometric detector at 10-nm wavelength intervals between 400 and 800 nm. RESULTS. RPE lipofuscin had strong autofluorescent emissions that were excited at all wavelengths. Bruch's membrane exhibited strong autofluorescence with an emission peak of 485 ± 5 nm when excited with 364-nm light. At 488-, 568-, and 633-nm excitations, Bruch's membrane and sub-RPE deposits in normal eyes exhibited minimal autofluorescence. In AMD eyes, however, both the 364- and 488-nm excitation wavelengths stimulated substantial blue-green emissions from sub-RPE deposits and Bruch's membrane, with average pixel intensities substantially exceeding that elicited in the yellow-orange range by RPE lipofuscin. CONCLUSIONS. These data suggest that an increase in blue-green autofluorescence of Bruch's membrane relative to the yellow-orange autofluorescence of RPE-associated lipofuscin is associated with AMD. Knowledge of these spectra will be useful in evaluating animal models of macular degenerative disease and in diagnosis of AMD, and will provide a novel signature for further analysis of the molecular entities emitting these fluorescent signatures.