Protein localization in electron micrographs using fluorescence nanoscopy View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2011-01

AUTHORS

Shigeki Watanabe, Annedore Punge, Gunther Hollopeter, Katrin I Willig, Robert John Hobson, M Wayne Davis, Stefan W Hell, Erik M Jorgensen

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. More... »

PAGES

80

Identifiers

URI

http://scigraph.springernature.com/pub.10.1038/nmeth.1537

DOI

http://dx.doi.org/10.1038/nmeth.1537

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1036618702

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/21102453


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43 schema:description 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.
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