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J. Yu, M. Han, F. Holz, J. Bille; Ultrastructural Imaging of Human Rpe Cells and Single Lipofuscin Granules With 4–pi Fluorescence Microscopy . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4064.
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© ARVO (1962-2015); The Authors (2016-present)
Age–related macular degeneration(AMD) is the leading cause of blindness and visual disability in patients older than 60 years. One potential pathogenesis of AMD is the excessive accumulation of lipofuscin (LF) granules in retinal pigment epithelial(RPE) cells, which leads to the degeneration of RPE cells. The major source of LF granules is the incompletely digested photoreceptor distal segments. LF granule are comprised of a group of autofluorescent(AF) lipid–proteins including a photoactive material A2–E. Compared with other conventional methods, two–photon 4Pi–confocal fluorescence microscopy enhances the depth resolution of fluorescence microscope by a factor of 3 to 7 times, resulting in a axial resolution of 100 nm.
The human retinas were obtained from postmortem donor eyes from the eye hospital, University of Bonn, Germany. A RPE cell monolayer is prepared either manually or ablated with a 308 XeCl excimer laser. The Leica TCS–4Pi microscope provides a stable and user–friendly platform for fluorescence imaging with a resolution of 100nm in 3D inside of RPE cell and LP granules. It consists of a 4Pi–unit and a standard confocal scanning microscope(TCS SP2, Leica). The 4Pi–unit is mounted to the microscope turret to maintain the scanning and spectral detection capabilities of the confocal system. As the excitation source for two–photon fluorescence imaging, a mode–locked Ti:Sapphire laser is coupled into the scanner.
4–Pi microscope enhanced axial resolution by utilizing two opposing objective lenses which are illuminated coherently and the fluorescent signal is almost collected in all directions. Compared with TPEF imaging with a typical axial resolution of 1um, 4–Pi microscope can reach an axial resolution of 100 nm. With 4–Pi microscopy, the 3D spatial distribution of LF granules in RPE cells and the ultrastructures of single LP granules can be imaged with super–resolution compared to confocal microscopes. .
Although many questions regarding AMD pathogenesis remain open, the 3D–imaging of RPE cell and LP granule with 4–Pi fluorescence microscopy provide a novel method to investigate the pathogenesis of RPE cell degeneration. We can resolve the ultrastructure of RPE cell and lipofuscin granule with unmatched depth resolution, which is invaluable to reveal the sub–cellular structural disorder of RPE cells from ageing patient and may lead to better understanding of the pathology of AMD.
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