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Preparation of Samples for Electron Microscopy

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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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Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

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Published on: November 20, 2021

Preparing Fission Yeast for Electron Microscopy.

Thomas H Giddings¹, Mary K Morphew¹, J Richard McIntosh^1,2

¹Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347.

Cold Spring Harbor Protocols

|January 5, 2017

Summary

This summary is machine-generated.

High-pressure freezing (HPF) and plunge freezing are effective methods for cryofixing fission yeast. Freeze-substitution is then used for sample preparation for electron microscopy, preserving cellular structure.

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Area of Science:

Cell Biology
Microscopy Techniques
Cryogenics

Background:

Cryofixation is crucial for preserving cellular ultrastructure.
Ice crystal formation during freezing can damage biological samples.
Fission yeast is a model organism for cell biology studies.

Purpose of the Study:

To describe reliable methods for cryofixing fission yeast.
To provide protocols for high-pressure freezing (HPF) and plunge freezing.
To detail the subsequent freeze-substitution process for electron microscopy.

Main Methods:

High-pressure freezing (HPF) involves rapid pressure increase to inhibit ice crystal formation.
Plunge freezing requires dispersing cells in a thin layer (<20 µm) and immersing in liquid cryogen (e.g., ethane).
Freeze-substitution removes frozen water with a solvent at low temperatures, followed by fixation and dehydration.

Main Results:

Both HPF and plunge freezing effectively cryofix fission yeast.
Freeze-substitution successfully preserves cellular structure for transmission electron microscopy.
Protocols for HPF, plunge freezing, and freeze-substitution are detailed.

Conclusions:

HPF and plunge freezing are reliable cryofixation techniques for fission yeast.
Freeze-substitution is essential for preparing vitrified samples for electron microscopy.
These methods enable detailed ultrastructural studies of fission yeast.