gms | German Medical Science

Background: Several microscopic techniques can be used for detection of autofluorescent pigments in retinal pigment epithelial (RPE) cells. Structured illumination in fluorescence microscopy allows for high resolution imaging in a nanometer range.

Methods: The utilized structured illumination microscopy (SIM) setup is based on a common wide field microscope with different applicable excitation wavelengths (488, 568, 647 nm). The object is illuminated with a periodical pattern (i.e. structured illumination). The acquired data that consists of a superimposition of the illumination pattern and the object is processed subsequently by reconstruction software to generate high resolution images. SIM was applied to histological RPE sections and RPE cell cultures. Lipofuscin (LF) granules, melanolipofuscin granules (MLF) and melanosomes were characterized with regard to their morphology and autofluorescence distribution.

Results: SIM significantly improves the lateral resolution compared to commonly used wide field microscopes by a factor of two down to 110 nm. LF granules showed the strongest autofluorescence signal (AS) at 488 and 568 nm excitation while the melanosomes showed a red shifted fluorescence with a strong signal at 647 nm. LF granules were significantly smaller than MLF granules (976 vs. 1156 nm; p<0.001); the size of LF granules increased with decreasing distance to the bruch membrane (BM). The melanosomes were significantly further away from BM than LF and MLF granules.

Conclusion: SIM is suitable for high resolution imaging of autofluorescent LF and MLF granules and melanosomes in RPE cells. The significant improvement in lateral resolution allows for better insights in the autofluorescence distribution of RPE cells.