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Updated: Jul 28, 2026
13:35
Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy
Published on: May 5, 2017
Electron microscopy of frozen biological objects: a study using cryosectioning and cryosubstitution
Journal of Microscopy
|May 20, 1998
Summary
High-pressure freezing improves structural preservation of biological samples by forming smaller ice crystals, enabling detailed cryomicroscopy and revealing novel cellular structures. This method enhances cryo-electron microscopy applications.
Area of Science:
- Cryo-electron microscopy
- Structural biology
- Biophysics
Background:
- Traditional freezing methods at atmospheric pressure often result in large ice crystals, compromising the structural integrity of biological specimens.
- Cryoprotectants are typically required to prevent ice crystal formation, but some biological samples possess natural cryoprotectants or low water content, facilitating vitrification.
Purpose of the Study:
- To investigate the effects of different freezing methods on biological sample preservation.
- To explore the potential of high-pressure freezing for achieving vitrification and improving structural analysis.
- To compare structural preservation and identify artefacts across various biological samples.
Main Methods:
- X-ray cryodiffraction was used to analyze ice crystal formation under different freezing conditions.
- High-pressure freezing and atmospheric pressure freezing were compared.
- Cryo-sectioning and electron cryomicroscopy were employed for vitrified samples.
- Cryosubstitution and resin embedding were used for samples with crystalline ice.
Main Results:
- High-pressure freezing produces smaller ice crystals (hexagonal, cubic, high-pressure forms) compared to atmospheric pressure freezing, leading to better structural preservation.
- Vitrification is achievable, especially with high-pressure freezing, allowing for cryo-sectioning and high-resolution imaging (<2 nm) and diffraction (<0.2 nm).
- Novel cellular compartment organizations were observed in bacteria, yeast, and marine worm elytrum, not discernible with classical fixation techniques. A high-pressure artefact in Paramecium trichocysts was identified.
Conclusions:
- High-pressure freezing is a superior method for preserving biological structures, enabling advanced cryo-electron microscopy.
- Vitrification via high-pressure freezing allows for detailed structural analysis of cellular components.
- This technique reveals previously undescribed cellular organizations and aids in identifying potential artefacts.
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