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G. Xu, J. Qu, L. Zhao, Y. Sun, Z. Ding, H. Niu; Ex vivo Study of 3-dimensional Distribution of Lipofuscin in RPE Cells of Normal Subjects Using Two-Photon Excitation Fluorescence. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1186.
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Lipofuscin is the dominant endogenous fluorophore in RPE cells. The goal of this work is to study the 3-dimensional distribution of lipofuscin granules in RPE cells of normal pig subjects using two-photon excitation fluorescence (TPEF).
Normal 8-10 month porcine eyes are harvested as the study objects (n=15). Eye balls are enucleated after death, kept in a moist and freezing chamber (0-4°C), and an RPE-choroid-sclera sample complex is trephined, without fixing, embedding and slicing. TPEF imaging is performed on a laser scanning confocal microscope, equipped with a mode-locked femtosecond Ti:sapphire laser for two-photon excitation. The excitation wavelength for this study is 860 nm.
The shape of the RPE cell is hexagon and its typical diameter is 12-15µm. Autofluorescence images of the RPE cells at different depths are obtained at a 0.5µm axial interval. 3-dimensional reconstruction of the images shows that fluorescent granules distribute around the shell of a hemisphere whose external diameter is 7-11µm, and the thickness of the shell is 2-3µm. At the bottom of the hemisphere, there are many round fluorescent granules in the cytoplasm and adjacent to the cellular membranes, with diameter of 0.6-1.0 µm. Some oval fluorescent granules, whose typical length is 2µm, locate on the top of the hemisphere. There is a peak at 600 nm in the TPEF spectrum of single round granules. The FWHM of the spectrum is about 150nm. When the detection channel is tuned to 580-630nm which is corresponding to the lipofuscin emission, the measured fluorescence intensity decreases slightly, but the image of the granules is sharp, which suggests that the round fluorescent granules are lipofuscin. Although the fluorescence of the oval granules is very weak, the anatomic location and its fluorescence characters suggest that they are oxidized melanin or melanin-lipofuscin.
TPEF enables reduced photobleaching and photodamage, enhanced penetration depth and high signal-to-noise ratio imaging of biological specimens in three dimensions at submicron resolution. It can be used to measure the 3-dimensional distribution of lipofuscin and other pigment granules in RPE cells, which makes it a potential tool for the study of the pathogenesis of some fundus diseases at subcellular level.
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