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Thomas Ach, Sabrina Rossberger, Gerrit Best, Christoph Cremer, Stefan Dithmar; Three-dimensional High Resolution Autofluorescent Imaging of Retinal Pigment Epithelium Cells by Structured Illumination Microscopy. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3096.
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© ARVO (1962-2015); The Authors (2016-present)
To use structured illumination microscopy (SIM) as a novel light microscopic technique for three dimensional high resolution imaging of autofluorescent granules within human retinal pigment epithelium (RPE) cells.
RPE cells from the macular region of a 68 year old human donor were examined using SIM with laser light (excitation: 488, 568, and 647 nm). Excitation signals were recorded from top to bottom through the cells in 200 nm steps (z-direction). Self written software allowed three dimensional reconstruction coalescing these sectional images.Intracellular granules were analyzed regarding number, appearance and distribution within RPE cells.
Structured illumination offers a significantly improved lateral resolution compared to commonly used microscopic methods down to an 100 nm range. Single granules within RPE cells can be classified as lipofuscin (LF) and melanolipofuscin (MLF) granules, and clearly be distinguished. Number of granules differs between RPE cells (range: 34 to 87 LF, 3 to 12 MLF). While LF granules are mainly located at RPE cell borders, MLF granules are found centrally. Furthermore, the reconstructed high resolution images allow measurement of autofluorescence patterns within single granules.
Structured illumination is a microscopic method for high resolution imaging of autofluorescent granules. The significant resolution improvement allows better insights in RPE cells and even in single granules, including detection of AF patterns within these granules. Therefore, SIM is a useful new tool for studies on LF and MLF pathogenesis.
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