title: Nanometer-resolution fluorescence electron microscopy (nano-EM) in cultured cells.
abstract: Nano-resolution fluorescence electron microscopy (nano-fEM) pinpoints the location of individual proteins in electron micrographs. Plastic sections are first imaged using a super-resolution fluorescence microscope and then imaged on an electron microscope. The two images are superimposed to correlate the position of labeled proteins relative to subcellular structures. Here, we describe the method in detail and present five technical advancements: the use of uranyl acetate during the freeze-substitution to enhance the contrast of tissues and reduce the loss of fluorescence, the use of ground-state depletion instead of photoactivation for temporal control of fluorescence, the use of organic fluorophores instead of fluorescent proteins to obtain brighter fluorescence signals, the use of tissue culture cells to broaden the utility of the method, and the use of a transmission electron microscope to achieve sharper images of ultrastructure.
authors.affiliation: Department of Biology, University of Utah, Salt Lake City, UT, USA.
authors.forename: Shigeki
authors.initials: S
authors.lastname: Watanabe
authors.forename: Martin
authors.initials: M
authors.lastname: Lehmann
authors.forename: Edward
authors.initials: E
authors.lastname: Hujber
authors.forename: Richard D
authors.initials: RD
authors.lastname: Fetter
authors.forename: Jackson
authors.initials: J
authors.lastname: Richards
authors.forename: Berit
authors.initials: B
authors.lastname: Söhl-Kielczynski
authors.forename: Annegret
authors.initials: A
authors.lastname: Felies
authors.forename: Christian
authors.initials: C
authors.lastname: Rosenmund
authors.forename: Jan
authors.initials: J
authors.lastname: Schmoranzer
authors.forename: Erik M
authors.initials: EM
authors.lastname: Jorgensen
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans; D002478:Cells, Cultured; D016591:Histocytological Preparation Techniques; D007091:Image Processing, Computer-Assisted; D051379:Mice; D008854:Microscopy, Electron / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*