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Cryo-electron Microscopy01:28

Cryo-electron Microscopy

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Conventional electron microscopy (EM) involves dehydration, fixation, and staining of biological samples, which distorts the native state of biological molecules and results in several artifacts. Also, the high-energy electron beam damages the sample and makes it difficult to obtain high-resolution images. These issues can be addressed using cryo-EM, which uses frozen samples and gentler electron beams. The technique was developed by Jacques Dubochet, Joachim Frank, and Richard Henderson, for...
3.9K
Preparation of Samples for Electron Microscopy01:20

Preparation of Samples for Electron Microscopy

6.3K
To be visualized by an electron microscope, either transmission or scanning, biological samples need to be fixed (stabilized) so the electron beam does not destroy them and dried thoroughly (desiccated/dehydrated) so the vacuum does not affect them. Fixation needs to be done as quickly as possible because the sample properties will start changing as soon as it is removed from its natural environment. For example, in a tissue sample, the oxygen levels begin decreasing, causing an altered...
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Related Experiment Video

Updated: Nov 24, 2025

Single Particle Cryo-Electron Microscopy: From Sample to Structure
11:52

Single Particle Cryo-Electron Microscopy: From Sample to Structure

Published on: May 29, 2021

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Progress Towards CryoEM: Negative-Stain Procedures for Biological Samples.

Shane Gonen1

  • 1Department of Molecular Biology and Biochemistry, University of California Irvine, Irvine, CA, USA. gonens@uci.edu.

Methods in Molecular Biology (Clifton, N.J.)
|December 28, 2020
PubMed
Summary
This summary is machine-generated.

Negative-stain electron microscopy (EM) is a valuable screening tool that can optimize sample preparation and conditions before high-resolution cryo-electron microscopy (CryoEM) studies of biological macromolecules.

Keywords:
AveragingCryoEMCrystalsElectron crystallographyElectron microscopyNegative-stainSingle-particleUranyl acetateUranyl formate

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Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy
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Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Published on: August 15, 2014

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Area of Science:

  • Structural Biology
  • Biophysics
  • Electron Microscopy

Background:

  • Electron cryo-microscopy (CryoEM) is a key technique for high-resolution structural studies of biological macromolecules.
  • CryoEM experiments can be lengthy and resource-intensive, necessitating efficient sample screening methods.
  • Negative-stain EM offers a rapid preliminary assessment of samples prior to CryoEM.

Purpose of the Study:

  • To detail the advantages and disadvantages of using negative-stain EM for screening biological samples.
  • To emphasize the utility of Uranyl stains as a preparatory step for CryoEM.
  • To provide practical guidance, including protocols and material lists, for negative-stain EM applications.

Main Methods:

  • Review of negative-stain electron microscopy principles and applications.
  • Specific focus on the use of Uranyl stains for biological sample preparation.
  • Development of a detailed protocol and materials list for negative-stain EM screening.

Main Results:

  • Negative-stain EM effectively screens biochemical conditions, sample integrity, binding, and composition.
  • It provides an estimation of sample grid concentration, crucial for CryoEM success.
  • Map resolutions from negative-stain EM can be biologically informative, comparable to some CryoEM applications.

Conclusions:

  • Negative-stain EM is an indispensable tool for optimizing sample quality and experimental design before undertaking CryoEM.
  • Uranyl stains are particularly effective for screening biological samples for imaging and diffraction-based EM methods.
  • The described protocol facilitates the efficient application of negative-stain EM, enhancing the success rate of subsequent high-resolution studies.