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Related Concept Videos

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...
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Preparation of Samples for Electron Microscopy01:20

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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: Jul 24, 2025

Optimizing Sample Preparation for Cryogenic Electron Microscopy
06:32

Optimizing Sample Preparation for Cryogenic Electron Microscopy

Published on: April 11, 2025

499

Challenges in making ideal cryo-EM samples.

Bong-Gyoon Han1, Agustin Avila-Sakar1, Jonathan Remis2

  • 1Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720, USA.

Current Opinion in Structural Biology
|July 1, 2023
PubMed
Summary
This summary is machine-generated.

Cryo-electron microscopy (cryo-EM) faces challenges at the air-water interface (AWI). Immobilizing samples on grids and controlling thickness are key strategies to improve cryo-EM and cryo-electron tomography (cryo-ET) data quality.

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Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction
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Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction
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Manual Blot-and-Plunge Freezing of Biological Specimens for Single-Particle Cryogenic Electron Microscopy
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Area of Science:

  • Structural Biology
  • Biophysics
  • Electron Microscopy

Background:

  • The air-water interface (AWI) presents a significant challenge in cryo-electron microscopy (cryo-EM) sample preparation.
  • Uncontrolled interaction with the AWI can lead to particle denaturation and aggregation, compromising structural integrity.
  • Both single-particle cryo-EM and cryo-electron tomography (cryo-ET) are negatively impacted by AWI effects.

Purpose of the Study:

  • To review current methodologies for mitigating air-water interface (AWI) issues in cryo-EM.
  • To highlight promising techniques for sample preparation, focusing on particle immobilization and thickness control.
  • To explore future applications of prepared samples in time-resolved biochemical assays.

Main Methods:

  • Review of existing cryo-EM sample preparation techniques.
  • Focus on particle immobilization strategies, particularly using affinity grids.
  • Analysis of methods for achieving precise control over sample thickness to prevent AWI contact.

Main Results:

  • Particle immobilization on affinity grids is identified as a highly promising approach to avoid AWI.
  • Effective control of sample thickness is crucial for preventing immobilized particles from contacting residual buffer AWI.
  • The importance of AWI avoidance is underscored for both single-particle cryo-EM and cryo-ET.

Conclusions:

  • Avoiding the air-water interface is critical for successful cryo-EM and cryo-ET.
  • Immobilization techniques and precise thickness control are key strategies for high-quality cryo-EM data.
  • Future directions include using immobilized samples for in-situ time-resolved biochemical experiments on EM grids.