Photooxidation technology for correlated light and electron microscopy.
The combination of the capabilities of light microscopical techniques with the power of resolution of electron microscopy along with technical advances has led to a gradual decline of the gap between classical light and electron microscopy. Among the correlative techniques using the synergistic opportunities, photooxidation methods have been established as valuable tools for visualizing cell structures at both light and electron microscopic level. Fluorescent dyes are used to oxidize the substrate diaminobenzidine, which in its oxidized state forms fine granular precipitates. Stained with osmium, the diaminobenzidine precipitates are well discernible in the electron microscope, thus labelling and defining the cellular structures, which at light microscopy level are recorded by fluorescent probes. The underlying photooxidation reaction is based on the excitation of free oxygen radicals that form upon illumination of fluorochromes; this is a central step in the procedure, which mainly influences the success of the method. This article summarizes basic steps of the technology and progresses, shows efforts and elaborated pathways, and focuses on methodical solutions as to the applicability of different fluorochromes, as well as conditions for fine structural localizations of the reaction products.
Meisslitzer-Ruppitsch
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C
Department of Cell Biology and Ultrastructure Research, Centre for Anatomy and Cell Biology, Medical University Vienna, Vienna, Austria.
Röhrl
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Neumüller
J
J
Pavelka
M
M
Ellinger
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A
D015100:3,3'-Diaminobenzidine / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D008853:Microscopy / Q000379:methods*; D008854:Microscopy, Electron / Q000379:methods*; D009992:Osmium / Q000378:metabolism; D010084:Oxidation-Reduction; D013194:Staining and Labeling / Q000379:methods*
D005456:Fluorescent Dyes; D009992:Osmium; D015100:3,3'-Diaminobenzidine