title: Protein localization in electron micrographs using fluorescence nanoscopy.
abstract: A complete portrait of a cell requires a detailed description of its molecular topography: proteins must be linked to particular organelles. Immunocytochemical electron microscopy can reveal locations of proteins with nanometer resolution but is limited by the quality of fixation, the paucity of antibodies and the inaccessibility of antigens. Here we describe correlative fluorescence electron microscopy for the nanoscopic localization of proteins in electron micrographs. We tagged proteins with the fluorescent proteins Citrine or tdEos and expressed them in Caenorhabditis elegans, fixed the worms and embedded them in plastic. We imaged the tagged proteins from ultrathin sections using stimulated emission depletion (STED) microscopy or photoactivated localization microscopy (PALM). Fluorescence correlated with organelles imaged in electron micrographs from the same sections. We used these methods to localize histones, a mitochondrial protein and a presynaptic dense projection protein in electron micrographs.
authors.affiliation: Department of Biology and Howard Hughes Medical Institute, University of Utah, Salt Lake City, Utah, USA.
authors.forename: Shigeki
authors.initials: S
authors.lastname: Watanabe
authors.forename: Annedore
authors.initials: A
authors.lastname: Punge
authors.forename: Gunther
authors.initials: G
authors.lastname: Hollopeter
authors.forename: Katrin I
authors.initials: KI
authors.lastname: Willig
authors.forename: Robert John
authors.initials: RJ
authors.lastname: Hobson
authors.forename: M Wayne
authors.initials: MW
authors.lastname: Davis
authors.forename: Stefan W
authors.initials: SW
authors.lastname: Hell
authors.forename: Erik M
authors.initials: EM
authors.lastname: Jorgensen
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans; D004583:Electrons; D006657:Histones / Q000032:analysis / Q000648:ultrastructure; D008164:Luminescent Proteins / Q000032:analysis* / Q000648:ultrastructure; D008854:Microscopy, Electron / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D024101:Mitochondrial Proteins / Q000032:analysis / Q000648:ultrastructure; D036103:Nanotechnology / Q000379:methods*
chemical_list: D006657:Histones; D008164:Luminescent Proteins; D024101:Mitochondrial Proteins