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Y. Sun, J. Qu, G. Xu, L. Zhao, H. Niu; Autofluorescence Lifetime Imaging of Lipofuscin and Melanin in Retinal Pigment Epithelium Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2965.
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© ARVO (1962-2015); The Authors (2016-present)
The goal of this work is to investigate the spatial distribution and their morphologic characteristics of two dominant endogenous fluorophores in the retinal pigment epithelium (RPE) cell, lipofuscin and oxidized melanin, using two-photon excitation fluorescence lifetime imaging (TPE-FLIM).
Normal porcine eyeballs are obtained from a local slaughterhouse. After asepsis treatment of the eyeball, a 6 mm diameter RPE/choroid/sclera tissue sample within 5~15 deg of the macular field is cut off from eyecup with a surgical trephine. Two-photon excitation (of wavelength 860nm) of the sample is provided by a mode-locked femtosecond Ti:sapphire laser (Mira 900F, Coherent Inc.). Fluorescence microscopy of fluorophores at differentin depths and of different shapes is performed with an inverted laser scanning confocal microscope system (TCS SP2, Leica Microsystems). Fluorescence lifetime imaging of the sample is obtained with a time-correlated single photon counting (TCSPC) imaging module (SPC-730, Becker & Hickl GmbH) that is coupled to the optional exit port of the microscope.
The RPE cell contains two classes of pigment, lipofuscin and melanin. At the bottom of the RPE cells, the dominant fluorophore is lipofuscin which is largely located adjacent to the cell membranes and around nucleus. Melanin is the amorphous pigment which is mainly present at the top of the RPE cells. Although melanin granules usually do not emit autofluorescence, their oxidized forms exhibit fluorescence emission smilar to that of lipofuscin, and the fluorescence intensity can be increased by increasing light exposure. By analyzing the histogram of the fluorescence lifetime image and the fluorescence decay curves using two-exponential component fitting model, the results show that the lifetime of lipofuscin varies from 500ps to 650ps and the lifetime of oxidized melanin is ~738ps. In addition, the results reveal the presence of a fluorephore with longer lifetime (1.4ns) in the most part of the cytoplasm in RPE cells, which is believed to be FAD bound protein.
Primary results show that TPE-FLIM reveals the spatial distribution of the fluorophores in different layers of the RPE cells and provides complementary functional information for investigating the metabolic state of the RPE cells.
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