Autofluorescence imaging of human RPE cell granules using structured illumination microscopy
- Thomas Ach1,
- Gerrit Best1,2,
- Sabrina Rossberger1,2,
- Rainer Heintzmann3,4,
- Christoph Cremer2,5,
- Stefan Dithmar1
- 1Department of Ophthalmology, University of Heidelberg, Germany
- 2Kirchhoff Institute for Physics, University of Heidelberg, Germany
- 3Institute of Photonic Technology, Jena, Germany
- 4Randall Division, King's College London, London, UK
- 5Institute of Molecular Biology, Mainz, Germany
- Correspondence to Professor Stefan Dithmar, Department of Ophthalmology, University Hospital Heidelberg, Im Neuenheimer Feld 400, Heidelberg 69120, Germany;
Contributors All authors have made substantive contributions to this study. T Ach: conception and design, acquisition of data, analysis and interpretation of data, drafting the article, final approval of the version to be published. G Best: acquisition of data, drafting the article, final approval of the version to be published. S Rossberger: analysis and interpretation of data, critically revising the article, final approval of the version to be published. R Heintzmann: substantial contributions to conception, critically revising the article, final approval of the version to be published. C Cremer: substantial contributions to conception, critically revising the article, final approval of the version to be published. S Dithmar: analysis and interpretation of data, critically revising the article, final approval of the version to be published.
- Accepted 21 May 2012
- Published Online First 3 July 2012
Background/aims To characterise single autofluorescent (AF) granules in human retinal pigment epithelium (RPE) cells using structured illumination microscopy (SIM).
Methods Morphological characteristics and autofluorescence behaviour of lipofuscin (LF) and melanolipofuscin (MLF) granules of macular RPE cells (66-year-old donor) were examined with SIM using three different laser light excitation wavelengths (488, 568 and 647 nm). High-resolution images were reconstructed and exported to Matlab R2009a (The Mathworks Inc, Natick, MA, USA) to determine accurate size and emission intensities of LF and MLF granules.
Results SIM doubles lateral resolution compared with conventionally used wide-field microscopy and allows visualisation of intracellular structures down to 110 nm lateral resolution. AF patterns were examined in 133 LF and 27 MLF granules. LF granules (968±220 nm) were significantly smaller in diameter than MLF granules (1097±110 nm; p<0.001). LF granules showed an inhomogeneous intragranular pattern, and the average intensity negatively correlated with the size of these granules when excited at 647 nm. The autofluorescence of MLF granules was more homogeneous, but shifted towards higher excitation wavelengths in the centre of the granules.
Conclusion SIM is a useful tool for examining AF signals within single LF and MLF granules in RPE cells. This allows new insights into RPE autofluorescence patterns.
Funding This study was supported by Ernst and Berta Grimmke Stiftung. The funding source had no involvement in the study design, the collection, analysis and interpretation of data, in the writing of the report or in the decision to submit the paper for publication.
Competing interests None.
Ethics approval Ethics Committee of University Heidelberg.
Provenance and peer review Not commissioned; externally peer reviewed.