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

Shigeki Watanabe, Martin Lehmann, Edward Hujber, Richard D. Fetter, Jackson Richards, Berit Söhl-Kielczynski, Annegret Felies, Christian Rosenmund, Jan Schmoranzer, Erik M. Jorgensen

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

Original languageEnglish (US)
Title of host publicationElectron Microscopy
Subtitle of host publicationMethods and Protocols
PublisherHumana Press Inc.
Pages503-526
Number of pages24
ISBN (Print)9781627037754
DOIs
StatePublished - 2014

Publication series

NameMethods in Molecular Biology
Volume1117
ISSN (Print)1064-3745

Keywords

  • Correlative light and electron microscopy
  • Direct stochastic optical reconstruction microscopy (dSTORM)
  • Fluorescence electron microscopy
  • Fluorescence nanoscopy
  • Ground-state depletion and single-molecule return (GSDIM)
  • Nano-fEM
  • Photoactivated localization microscopy (PALM)
  • Protein localization
  • Stochastic optical reconstruction microscopy (STORM)
  • Super-resolution fluorescence microscopy

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Fingerprint Dive into the research topics of 'Nanometer-resolution fluorescence electron microscopy (Nano-EM) in cultured cells'. Together they form a unique fingerprint.

  • Cite this

    Watanabe, S., Lehmann, M., Hujber, E., Fetter, R. D., Richards, J., Söhl-Kielczynski, B., Felies, A., Rosenmund, C., Schmoranzer, J., & Jorgensen, E. M. (2014). Nanometer-resolution fluorescence electron microscopy (Nano-EM) in cultured cells. In Electron Microscopy: Methods and Protocols (pp. 503-526). (Methods in Molecular Biology; Vol. 1117). Humana Press Inc.. https://doi.org/10.1007/978-1-62703-776-1_22