title: Photooxidation technology for correlative light and electron microscopy.
abstract: Correlative microscopic approaches combine the advantages of both light and electron microscopy. Here we show a correlative approach that uses the photooxidation capacity of fluorescent 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.
authors.affiliation: Department of Cell Biology and Ultrastructure Research, Center for Anatomy and Cell Biology, Medical University of Vienna, Vienna, Austria.
authors.forename: Claudia
authors.initials: C
authors.lastname: Meisslitzer-Ruppitsch
authors.forename: Clemens
authors.initials: C
authors.lastname: Röhrl
authors.forename: Carmen
authors.initials: C
authors.lastname: Ranftler
authors.forename: Herbert
authors.initials: H
authors.lastname: Stangl
authors.forename: Josef
authors.initials: J
authors.lastname: Neumüller
authors.forename: Margit
authors.initials: M
authors.lastname: Pavelka
authors.forename: Adolf
authors.initials: A
authors.lastname: Ellinger
mesh_terms: D015100:3,3'-Diaminobenzidine / Q000737:chemistry; D001896:Boron Compounds / Q000737:chemistry; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D002518:Ceramides / Q000737:chemistry / Q000378:metabolism; D011232:Chemical Precipitation / Q000528:radiation effects; D002863:Chromogenic Compounds / Q000737:chemistry; D042783:Endothelial Cells / Q000648:ultrastructure; D005456:Fluorescent Dyes / Q000737:chemistry; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D008856:Microscopy, Fluorescence; D008867:Microtomy; D010084:Oxidation-Reduction / Q000528:radiation effects; D055668:Photochemical Processes
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D002518:Ceramides; D002863:Chromogenic Compounds; D005456:Fluorescent Dyes; D015100:3,3'-Diaminobenzidine