Visualizing Proteins in Electron Micrographs at Nanometer Resolution

Shigeki Watanabe, Erik M. Jorgensen

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

To understand protein function, we need a detailed description of the molecular topography of the cell. The subcellular localization of proteins can be revealed using genetically encoded fluorescent proteins or immunofluorescence. However, the precise localization of proteins cannot be resolved due to the diffraction limit of light. Recently, the diffraction barrier has been overcome by employing several microscopy techniques. Using super-resolution fluorescence microscopy, one can pinpoint the location of proteins at a resolution of 20. nm or even less. However, the cellular context is often absent in these images. Recently, we developed a method for visualizing the subcellular structures in super-resolution images. Here we describe the method with two technical improvements. First, we optimize the method to preserve more fluorescence without compromising the morphology. Second, we implement ground-state depletion and single-molecule return (GSDIM) imaging, which does not rely on photoactivatable fluorescent proteins. These improvements extend the utility of nano-resolution fluorescence electron microscopy (nano-fEM).

Original languageEnglish (US)
Title of host publicationMethods in Cell Biology
PublisherAcademic Press Inc.
Pages283-306
Number of pages24
DOIs
StatePublished - 2012
Externally publishedYes

Publication series

NameMethods in Cell Biology
Volume111
ISSN (Print)0091-679X

Keywords

  • Correlative light and electron microscopy
  • Fluorescence electron microscopy
  • Fluorescence nanoscopy
  • Ground-state depletion and single-molecule return
  • GSDIM
  • Nano-fEM
  • PALM
  • Photoactivated localization microscopy
  • Protein localization
  • Super-resolution fluorescence microscopy

ASJC Scopus subject areas

  • Cell Biology

Fingerprint Dive into the research topics of 'Visualizing Proteins in Electron Micrographs at Nanometer Resolution'. Together they form a unique fingerprint.

Cite this