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

  • Tyler Ogunmowo
  • Sumana Raychaudhuri
  • Grant Kusick
  • Shuo Li
  • Shigeki WatanabeEmail author
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Part of the Neuromethods book series (NM, volume 155)

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.

Key words

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

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Tyler Ogunmowo
    • 1
    • 2
  • Sumana Raychaudhuri
    • 1
  • Grant Kusick
    • 1
    • 2
  • Shuo Li
    • 1
    • 3
  • Shigeki Watanabe
    • 1
    • 4
    Email author
  1. 1.Department of Cell Biology, School of MedicineJohns Hopkins UniversityBaltimoreUSA
  2. 2.Biochemistry, Cellular and Molecular Biology Program, School of MedicineJohns Hopkins UniversityBaltimoreUSA
  3. 3.Department of Biochemistry and Molecular Biology, Bloomberg School of Public HealthJohns Hopkins UniversityBaltimoreUSA
  4. 4.Solomon H. Snyder Department of Neuroscience, School of MedicineJohns Hopkins UniversityBaltimoreUSA

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