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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: Aug 31, 2025

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging
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Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

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A versatile enhanced freeze-substitution protocol for volume electron microscopy.

Sébastien Bélanger1, Heather Berensmann2,3, Valentina Baena2,3

  • 1Donald Danforth Plant Science Center, Saint Louis, MO, United States.

Frontiers in Cell and Developmental Biology
|August 25, 2022
PubMed
Summary

This study introduces a new method to improve heavy metal staining for volume electron microscopy. This technique enhances contrast in diverse biological samples, aiding 3D imaging of cells and tissues.

Keywords:
C. elegansOTOSaccharomyces cerevisiaefreeze-substitution fixationhigh-pressure freezing (HPF)plant specimensquick freeze-substitution (QFS)volume electron microscopy (vEM)

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Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy
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Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy
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Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

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Area of Science:

  • Electron Microscopy
  • Cell Biology
  • Biotechnology

Background:

  • Volume electron microscopy (VEM) requires robust contrast for 3D imaging.
  • Conventional fixation improves VEM contrast, but freeze-substitution methods lag.
  • Organic solvent-based freeze-substitution lacks sufficient heavy metal staining.

Purpose of the Study:

  • To develop an improved freeze-substitution protocol for enhanced heavy metal staining in VEM.
  • To enable high-contrast 3D imaging of diverse and challenging biological specimens.

Main Methods:

  • A novel approach using osmium tetroxide, acetone, and water substitution fluid for freeze-substitution.
  • Gradual transition from organic solvent to aqueous osmium tetroxide.
  • Post-fixation staining with aqueous potassium ferrocyanide, thiocarbohydrazide, osmium tetroxide, uranyl acetate, and lead acetate.

Main Results:

  • Substantial and consistent increase in heavy metal staining observed.
  • Successful application across diverse organisms: plants (Hordeum vulgare), nematodes (Caenorhabditis elegans), and yeast (Saccharomyces cerevisiae).
  • Improved contrast in difficult-to-fix tissues.

Conclusions:

  • The developed method significantly enhances heavy metal staining for VEM.
  • This protocol combines cryo-preservation benefits with superior contrast for diverse biological samples.
  • Opens new avenues for high-resolution 3D structural analysis in biology.