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T. Ach, G. Best, M. Ruppenstein, C. Cremer, S. Dithmar; Structured Illumination Microscopy for High Resolution in vivo and in vitro Imaging of Human RPE Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1198.
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© ARVO (1962-2015); The Authors (2016-present)
In aging retinal pigment epithelial (RPE) cells, accumulating autofluorescence (AF) pigments and fluorophores can be visualized with several AF microscopy techniques. Structured illumination microscopy (SIM), a form of high resolution light microscopy, allows imaging of fluorescent molecules in a nanometer range.
For in vitro imaging, deparaffined histological slides of human donor eyes, for in vivo imaging, cultured human RPE cells were used. All samples were examined using SIM technique with laser light (488, 568 and 647 nm). (Composite) SIM images of RPE cells were evaluated in terms of AF pattern and different AF granules.
SIM doubles the lateral resolution compared to conventionally used widefield microscopy and enables differentiation down to 130 nm. In in vitro RPE cells, an improved lateral resolution shows lipofuscin rings around melanin in melanolipofuscin granules. Furthermore, single hyperfluorescent clusters in lipofuscin rings are clearly detectable. Different intralysosomal fluorophores can be distinguished using varying excitation wavelength. SIM can also be used for in vivo imaging providing similar similar findings and allowing visualization of dynamic processes.
SIM can be used for high resolution AF imaging of in vitro and in vivo RPE cells. With SIM, AF structures in RPE cells can be detected with a lateral resolution of 130 nm. Wavelength-dependent AF patterns in single lysosomes and melanolipofuscin granules can be observed. Above all, this high resolution imaging technique of in vivo cells provides new sights in RPE pathogenesis.
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