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Methods Mol Biol. 2014;1117:503-26. doi: 10.1007/978-1-62703-776-1_22.

Nanometer-resolution fluorescence electron microscopy (nano-EM) in cultured cells.

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1
Department of Biology, University of Utah, Salt Lake City, UT, USA.

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.

PMID:
24357377
DOI:
10.1007/978-1-62703-776-1_22
[Indexed for MEDLINE]
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