1. Protein localization in electron micrographs using fluorescence nanoscopy
- Author
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Shigeki Watanabe, Katrin I. Willig, M. Wayne Davis, Stefan W. Hell, Robert J. Hobson, Annedore Punge, Gunther Hollopeter, and Erik M. Jorgensen
- Subjects
Electrons ,02 engineering and technology ,Biology ,Biochemistry ,Article ,law.invention ,Histones ,Mitochondrial Proteins ,03 medical and health sciences ,law ,Organelle ,Microscopy ,Animals ,Nanotechnology ,Photoactivated localization microscopy ,Caenorhabditis elegans ,Molecular Biology ,030304 developmental biology ,Luminescent Proteins ,0303 health sciences ,STED microscopy ,Cell Biology ,021001 nanoscience & nanotechnology ,Fluorescence ,Protein subcellular localization prediction ,Cell biology ,Microscopy, Electron ,Microscopy, Fluorescence ,Electron microscope ,0210 nano-technology ,Biotechnology - 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.
- Published
- 2010