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Cryo-electron Microscopy of Vitreous Sections

Part of the Methods in Molecular Biology book series (MIMB,volume 1117)

Abstract

More than 30 years ago two groups independently reported the vitrification of pure water, which was until then regarded as impossible without a cryoprotectant [1, 2]. This opened the opportunity to cryo-electron microscopy (cryo-EM) to observe biological samples at nanometer scale, close to their native state. However, poor electron penetration through biological samples sets the limit for sample thickness to less than the average size of the mammalian cell. In order to image bulky specimens at the cell or tissue level in transmission electron microscopy (TEM), a sample has to be either thinned by focused ion beam or mechanically sectioned. The latter technique, Cryo-Electron Microscopy of Vitreous Section (CEMOVIS), employs cryo-ultramicrotomy to produce sections with thicknesses of 40–100 μm of vitreous biological material suitable for cryo-EM. CEMOVIS consists of trimming and sectioning a sample with a diamond knife, placing and attaching the section onto an electron microscopy grid, transferring the grid to the cryo-electron microscope and imaging. All steps must be carried on below devitrification temperature to obtain successful results. In this chapter we provide a step-by-step guide to produce and image vitreous sections of a biological sample.

Key words

  • Cryo-ultramicrotomy
  • Cryo-transfer
  • Cryo-transmission electron microscopy
  • Trimming
  • Sectioning
  • Vitrification

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Acknowledgements

We are grateful to Gérard Pehau-Arnaudet for taking photographs for Figs. 1, 2, 3, 4, 5, 6, 7, and 8 and to Gregory M. Becker (RMC Products) and Mark A. Kukucka (Leica Microsystems Inc.) for providing photographs for Figs. 1a, b and 8a, b, respectively.

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Chlanda, P., Sachse, M. (2014). Cryo-electron Microscopy of Vitreous Sections. In: Kuo, J. (eds) Electron Microscopy. Methods in Molecular Biology, vol 1117. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-776-1_10

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  • DOI: https://doi.org/10.1007/978-1-62703-776-1_10

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