Identification and location of the pigment granules in the retinal pigment epithelium cells using fluorescence technology

Detection of autofluorescence is employed for measuring early dysfunctions of the fundus. Laser scanning confocal ophthalmoscope has greatly facilitated the study of fundus autofluorescence, but the complexity of fundus endogenous fluorophores makes it hard to interpret the results. To better understand the autofluorescence characteristics of fluorophores at fundus, such as spectra, intensity, distribution and lifetime, we take advantage of multiphoton microscope to investigate the autofluorescence of retinal pigment epithelium cells which is abundant in endogenous fluorophores, such as pigment granules (lipofuscin, melanin), structural protein (collagen, elastin), coenzyme (FAD), and so on. In this paper, we use a modified laser scanning confocal microscope coupled with a time-correlated single photon count module to identify and locate the pigment granules in retinal pigment epithelium cells. According to the results of two-photon excited autofluorescence (spectrum peak at ~600 nm) and fluorescence lifetimes (tau₁=0.6~0.7 ns, tau₂=1.4~2.2 ns), we conclude that there are many fluorophores inside the RPE cell, and the mainly contributing component to autofluorescence is lipofuscin. Both lipofuscin and melanin granules co-locate around cellular membranes and produce higher autofluorescence intensity around the membranes than other areas inside the cells. The coenzyme FAD and oxidized melanin granules contribute to the wide (>100 nm) fluorescence spectrum.