Photooxidation technology for correlative light and electron microscopy

Claudia Meisslitzer-Ruppitsch, Clemens Röhrl, Carmen Ranftler, Herbert Stangl, Josef Neumüller, Margit Pavelka, Adolf Ellinger

Research output: Chapter in Book/Report/Conference proceedingsChapterpeer-review

2 Citations (Scopus)

Abstract

Correlative microscopic approaches combine the advantages of both light and electron microscopy. Here we show a correlative approach that uses the photooxidation capacity offluorescent dyes. Through illumination with high energetic light, the chromogen diaminobenzidine is oxidized and stable deposits are formed at the sites of the former fluorescent signals, which after osmification are then visible in the electron microscope. The potential of the method is illustrated by tracing the endocytic pathway of three different ligands: the lipid ceramide, high density lipoproteins, and the lectin wheat germ agglutinin. The ligands were labeled either with BODIPY or Alexa dyes. Following cell surface binding, uptake, and time-dependent intracellular progression, the route taken by these molecules together with the organelles that have been visited is characterized. Correlative microscopic data are recorded at various levels. First, by fluorescence and phase contrast illumination with the light microscope, followed by the analysis of semithin sections after photooxidation, and finally of thin sections at the ultrastructural level

Original languageEnglish
Title of host publicationCell Imaging Techniques
Subtitle of host publicationMethods and Protocols
PublisherHumana Press Inc.
Pages423-436
Number of pages14
Volume931
ISBN (Print)9781627030557
DOIs
Publication statusPublished - 2012
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume931
ISSN (Print)1064-3745

Keywords

  • Alexa Fluor dyes
  • BODIPY
  • Correlative microscopy
  • DAB-photooxidation
  • Humans
  • Cells, Cultured
  • Chromogenic Compounds/chemistry
  • Fluorescent Dyes/chemistry
  • Microscopy, Electron, Transmission/methods
  • Ceramides/chemistry
  • Photochemical Processes
  • Boron Compounds/chemistry
  • Oxidation-Reduction/radiation effects
  • Chemical Precipitation/radiation effects
  • 3,3'-Diaminobenzidine/chemistry
  • Golgi Apparatus/metabolism
  • Cell Culture Techniques
  • Endothelial Cells/ultrastructure
  • Microscopy, Fluorescence
  • Microtomy

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