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M. Han, A. Bindewald–Wittich, F.G. Holz, S. Snyder, G. Giese, M. Niemz, J.F. Bille; Two–Photon Excited Autofuorescence Imaging of Human Retinal Pigment Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4065.
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© ARVO (1962-2015); The Authors (2016-present)
Degeneration of retinal pigment epithelial (RPE) cells severely impairs the visual function of retina photoreceptors. However, little is known about the events trigger the death of RPE cells or aged–related macular degeneration (AMD). Previous studies proposed that lipofuscin (LF) granules are harmful when present in sufficient amount in the RPE cells. Due to the unique advantages of nonlinear fluorescence imaging (large sensing depth, sub–micron resolution and reduced photodamage), we employed two–photon excited autofuorescence (TPEF) imaging to study the morphological and spectral properties of single human RPE cells.
The human retinas were obtained from two postmortem donor eyes (55 and 80 years old, with normal vision). TPEF imaging was performed with an upright Zeiss multiphoton microscope (LSM 510) equipped with a mode–locked femtosecond Ti:Sapphire laser ( = 720 – 980 nm) . The AF spectra of individual RPE cells was measured by a confocal laser scanning microscope (Leica TCS SP2) coupled to a UV argon laser ( = 364 nm).
The dominant fuorophores of AF derive from LF granules that accumulate in the RPE cells. As age increases, the cytoplasms of RPE cells can be completely occupied by LF granules. Multiple cell nuclei were observed in certain RPE cells (Fig.A). The typical diameters of LF granules are less than 1 µm, but a few RPE cells (< 1 %) contain larger LF granules. For the first time, we have observed abnormal AF from these enlarged LF granules, whose AF peak is blue–shifted from 556 nm to 520 nm (Fig.B).
Instead of utilizing multi–line Argon lasers for confocal imaging, TPEF imaging with a tunable femtosecond Ti:Sapphire laser is well suited to investigate both the morphological and spectral characteristics of LF granules in human RPE cells, which may provide fresh insights into the pathological cause of RPE cell death or AMD at the subcellular level.
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