gms | German Medical Science

23rd Annual Meeting of the German Retina Society

German Retina Society

24.09. - 25.09.2010, Freiburg

High resolution autofluorescence microscopy of human RPE cells using structured illumination

Meeting Abstract

  • Thomas Ach - University Eye Clinic Heidelberg
  • G. Best - University of Heidelberg, Institute of Physics
  • C. Cremer - University of Heidelberg, Institute of Physics
  • F. Schütt - University Eye Clinic Heidelberg
  • S. Dithmar - University Eye Clinic Heidelberg

German Retina Society. 23rd Annual Conference of the German Retina Society. Freiburg i. Br., 24.-25.09.2010. Düsseldorf: German Medical Science GMS Publishing House; 2010. Doc10rg54

doi: 10.3205/10rg54, urn:nbn:de:0183-10rg548

This is the translated version of the article.
The original version can be found at:

Published: September 21, 2010

© 2010 Ach et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( You are free: to Share – to copy, distribute and transmit the work, provided the original author and source are credited.



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.