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Sabrina Rossberger, Thomas Ach, Gerrit Best, Christoph Cremer, Stefan Dithmar; High Resolution Analysis of autofluorescent Granules within Drusen using Structured Illumination Microscopy. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4784.
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Drusen play a key role in AMD, but very little is known about composition and the autofluorescent (AF) material sometimes found within drusen. Here we present a detailed analysis of drusen containing autofluorescent material using Structured Illumination Microscopy (SIM), which provides a lateral resolution twice as high as conventional fluorescence microscopy.
Ten histological RPE sections obtained from ten human donor eyes (range 57 to 95 years) were examined by SIM using laser light of different wavelengths (488, 568, and 647 nm). Drusen were studied regarding size and shape. AF material within drusen was analyzed in terms of size, shape, AF behaviour and distribution across drusen.
A total of 400 drusen were found, of which 101 contained AF material. 90 % of these drusen were smaller than 63 µm (mean: 35.63 µm ± 0.25 µm). AF material within drusen were identified as lipofuscin (LF, n=183) and melanolipofuscin (MLF, n=36) granules. Up to 11 LF and/or 3 MLF granules were found within one single druse. Clusters of LF granules could be observed. Nearly all granules were located in the lower 2/3 of the drusen (LF: 83.8 %; MLF: 97.4 %).
Recently we have shown that Structured Illumination Microscopy (SIM) can be used to image the autofluorescence emitted by LF and MLF granules generally found in RPE cells. This study shows that SIM is able to detect much more autofluorescent granules within drusen than reported until now. Shape and AF behaviour of the material embedded in drusen suggest that the AF material emerges from the overlaying RPE cells.
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