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cryoEM pp 3–23Cite as

Getting Started with In Situ Cryo-Electron Tomography

Part of the Methods in Molecular Biology book series (MIMB,volume 2215)

Abstract

Cryo-electron tomography (cryo-ET) is an extremely powerful tool which is used to image cellular features in their close-to-native environment at a resolution where both protein structure and membrane morphology can be revealed. Compared to conventional electron microscopy methods for biology, cryo-ET does not include the use of potentially artifact generating agents for sample fixation or visualization. Despite its obvious advantages, cryo-ET has not been widely adopted by cell biologists. This might originate from the overwhelming and constantly growing number of complex ways to record and process data as well as the numerous methods available for sample preparation. In this chapter, we will take one step back and guide the reader through the essential steps of sample preparation using mammalian cells, as well as the basic steps involved in data recording and processing. The described protocol will allow the reader to obtain data that can be used for morphological analysis and precise measurements of biological structures in their cellular environment. Furthermore, this data can be used for more elaborate structural analysis by applying further image processing steps like subtomogram averaging, which is required to determine the structure of proteins.

Key words

  • Cell biology
  • Cellular morphology
  • Cryo-electron tomography
  • Mammalian cells
  • Segmentation
  • Structural cell biology
  • Tilt series
  • Vitrification

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  • DOI: 10.1007/978-1-0716-0966-8_1
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Acknowledgments

This work was supported in part by the Office of Science of the US Department of Energy DE-AC02-O5CH11231 (K.M.D.), the Human Frontier Science Program fellowship LT000234/2018-L (D.S.) and UCB Start-up funds (K.M.D.).

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Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA

    Daniel Serwas & Karen M. Davies

  2. Molecular Biophysics and Integrative Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Karen M. Davies

Authors
  1. Daniel Serwas
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  2. Karen M. Davies
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Corresponding authors

Correspondence to Daniel Serwas or Karen M. Davies .

Editor information

Editors and Affiliations

  1. Howard Hughes Medical Institute Departments of Biological Chemistry and Physiology, University of California, Los Angeles, Los Angeles, CA, USA

    Tamir Gonen

  2. Chemical Engineering, School for Engineering of Matter, Transport and Energy; Center for Applied Structural Discovery Biodesign Institute, Arizona State University, Tempe, AZ, USA

    Brent L. Nannenga

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Serwas, D., Davies, K.M. (2021). Getting Started with In Situ Cryo-Electron Tomography. In: Gonen, T., Nannenga, B.L. (eds) cryoEM. Methods in Molecular Biology, vol 2215. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0966-8_1

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  • DOI: https://doi.org/10.1007/978-1-0716-0966-8_1

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