Correlative Super Resolution and Electron Microscopy to Detect Molecules in Their Native Cellular Context

Tyler Ogunmowo, Sumana Raychaudhuri, Grant Kusick, Shuo Li, Shigeki Watanabe

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Nano-resolution fluorescence electron microscopy (nano-fEM) provides the precise localization of biomacromolecules within electron micrographs. Classically, electron microscopy has provided the highest possible cellular detail, boasting nanometer-scale resolution. However, while cellular ultrastructure is clearly defined, molecular identity is obscured even when electron dense tags in the form of antibodies or locally polymerized moieties are used. Fluorescence microscopy complements electron microscopy by providing significant molecular specificity. Further, super-resolution techniques surpass the diffraction limit and localize labelled proteins at ~20 nm resolution. However, sparse light-emitting points do little to provide the subcellular context of labeled molecules. In nano-fEM, fluorescently tagged biological samples are first high-pressure frozen and processed via freeze substitution for fixation and to preserve fluorescence. Afterwards, samples are embedded in hydrophilic resin, cut into ultrathin sections, and visualized by, for example, direct stochastic optical reconstruction microscopy (dSTORM) followed by transmission electron microscopy. Fluorescence and electron micrographs are correlated by use of fiduciary markers and post-processing. This approach also provides 3D information similar to Array Tomography by serial sectioning of ultrathin sections followed by super-resolution microscopy and electron microscopy of each section in a sequential manner, enabling 3D reconstruction of the z axis.

Original languageEnglish (US)
Title of host publicationNeuromethods
PublisherHumana Press Inc.
Pages1-13
Number of pages13
DOIs
StatePublished - 2020

Publication series

NameNeuromethods
Volume155
ISSN (Print)0893-2336
ISSN (Electronic)1940-6045

Keywords

  • CLEM
  • Fluorescence EM
  • Localization microscopy
  • Nano-fEM (nano resolution fluorescence electron microscopy)
  • Photoactivated localization microscopy (PALM)
  • Stochastic optical reconstruction microscopy (STORM)
  • Super-CLEM

ASJC Scopus subject areas

  • Neuroscience(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Pharmacology, Toxicology and Pharmaceutics(all)
  • Psychiatry and Mental health

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