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Cryo-electron Microscopy

Cryo-electron Microscopy

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...

Preparation of Samples for Electron Microscopy

Preparation of Samples for Electron Microscopy

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 4, 2025

Optimizing Sample Preparation for Cryogenic Electron Microscopy

Optimizing Sample Preparation for Cryogenic Electron Microscopy

Published on: April 11, 2025

Improving particle quality in cryo-EM analysis using a PEGylation method.

Zhikuan Zhang¹, Hideki Shigematsu², Toshiyuki Shimizu¹

¹Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

Structure (London, England : 1993)

|May 28, 2021

Summary

This summary is machine-generated.

PEGylation of proteins prevents aggregation during cryo-electron microscopy (cryo-EM) sample preparation. This new method simplifies vitrification, enabling structural biology studies of previously unusable samples.

Keywords:

PEGylation aggregation cryo-EM denaturation single-particle analysis vitrification

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Last Updated: Nov 4, 2025

Optimizing Sample Preparation for Cryogenic Electron Microscopy

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Single Particle Cryo-Electron Microscopy: From Sample to Structure

Single Particle Cryo-Electron Microscopy: From Sample to Structure

Published on: May 29, 2021

Area of Science:

Structural biology
Biophysics
Biochemistry

Background:

Cryo-electron microscopy (cryo-EM) is a powerful technique for determining the three-dimensional structures of biomolecules.
A critical step in cryo-EM is sample vitrification, which involves rapid freezing to preserve native structures.
However, the air-water interface during vitrification often leads to particle aggregation and denaturation, limiting its applicability.

Purpose of the Study:

To develop a method to mitigate particle aggregation and denaturation during cryo-EM sample vitrification.
To improve the success rate of cryo-EM studies for challenging protein samples.

Main Methods:

Protein PEGylation: Covalently attaching polyethylene glycol (PEG) chains to protein surfaces.
Vitrification: Applying the PEGylated protein solution to a grid and rapidly freezing it in liquid ethane.
Cryo-EM data collection and analysis: Imaging the vitrified samples to assess particle quality and structure.

Main Results:

PEGylation effectively prevented protein aggregation and denaturation at the air-water interface during vitrification.
The method simplified the optimization of vitrification conditions, reducing experimental effort.
Previously problematic samples, prone to aggregation, became amenable to cryo-EM analysis.

Conclusions:

Protein PEGylation is a robust strategy to overcome sample preparation limitations in cryo-EM.
This technique enhances the scope of cryo-EM by enabling the structural analysis of a wider range of biomolecules.
The simplified workflow facilitates routine cryo-EM structure determination.