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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...

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Related Experiment Video

Updated: Jun 8, 2026

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

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

Published on: August 15, 2014

Cryonegative staining of macromolecular assemblies.

Sacha De Carlo¹, Holger Stark

¹Department of Chemistry, Institute for Macromolecular Assemblies, City University of New York, City College Campus, New York, USA.

Methods in Enzymology

|October 5, 2010

Summary

Cryonegative staining improves signal-to-noise ratio for cryoelectron microscopy (cryo-EM) by enhancing contrast during specimen preparation. This method preserves hydrated biological samples, yielding clearer images of macromolecular assemblies.

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Last Updated: Jun 8, 2026

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

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Published on: August 15, 2014

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Published on: February 6, 2018

Area of Science:

Structural Biology
Biophysics
Microscopy

Background:

Cryoelectron microscopy (cryo-EM) is vital for molecular resolution studies of biological assemblies.
Improving signal-to-noise ratio (SNR) in cryo-EM is crucial due to noisy electron micrographs.
Current SNR enhancement primarily relies on image processing techniques.

Purpose of the Study:

To introduce cryonegative staining as an alternative method for enhancing contrast at the specimen preparation stage.
To present two efficient cryonegative staining procedures for macromolecular assemblies.
To improve SNR while maintaining biological samples in a hydrated, frozen state.

Main Methods:

Cryonegative staining using ammonium molybdate, similar to conventional cryo-EM.
Cryonegative staining with uranyl formate or acetate using a carbon-sandwich method.
Observation of samples at liquid nitrogen temperature in the electron microscope.

Main Results:

Cryonegative staining increases SNR by at least a factor of three compared to conventional cryo-EM.
Both presented cryonegative staining procedures maintain sample hydration throughout the process.
Examples of attainable data using these methods are provided.

Conclusions:

Cryonegative staining offers a significant SNR improvement over conventional cryo-EM.
The presented methods provide efficient ways to prepare hydrated biological samples for cryo-EM.
Cryonegative staining expands possibilities for high-resolution structural analysis of macromolecular assemblies.