Cryoelectron Microscopy of Fission Yeast
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Published on: November 20, 2021
Mary K Morphew1, Thomas H Giddings2, J Richard McIntosh1,2
1Laboratory for 3D Electron Microscopy, University of Colorado, Boulder, Colorado 80309-0347.
Abstract:
Fission yeast cells can be prepared for electron microscopy (EM) in the frozen-hydrated state. This eliminates the requirement for dehydration and heavy metal staining when preparing samples for EM. As with room temperature imaging, however, the yeast must be sectioned to make them thin enough for transmission of the electron beam. Cutting sections of vitreous ice with a microtome is challenging. An alternative method that uses a focused ion beam to make a thin sample by milling away much of the sample at liquid nitrogen temperatures is under development but is not yet available for routine use. Imaging frozen-hydrated samples by EM is also a challenge. The technique involves battling low image contrast, high sensitivity to the electron beam, and mechanical distortions produced during the sectioning process. When used successfully, however, the method holds promise of providing excellent molecular detail without the disruption characteristic of dehydration or isolating a structure from its cellular environment. Cryo-EM of tilted views can be used to examine small structures and macromolecular complexes in their native cellular environment. If a structure exists in multiple copies, or has a repeating unit, it can be investigated at higher resolution using subvolume averaging. This protocol focuses on the preparation of cells for cryo-EM.
Insights
Preparing fission yeast for cryo-electron microscopy (cryo-EM) involves freezing cells to preserve their native state. This method avoids damaging dehydration and staining, enabling high-resolution imaging of cellular structures.
Area of Science:
- Cell Biology
- Microscopy Techniques
- Structural Biology
Background:
- Traditional electron microscopy (EM) sample preparation for fission yeast requires dehydration and heavy metal staining, which can introduce artifacts.
- Sectioning frozen-hydrated samples for transmission electron microscopy (TEM) is technically challenging.
- Current methods struggle with low contrast and beam sensitivity in frozen samples.
Purpose of the Study:
- To outline a protocol for preparing fission yeast cells for cryo-electron microscopy (cryo-EM).
- To highlight the advantages of cryo-EM for preserving cellular structures in their native state.
- To address the challenges associated with imaging frozen-hydrated biological samples.
Main Methods:
- Cells are prepared in a frozen-hydrated state, eliminating the need for dehydration and heavy metal staining.
- Discusses the challenges of sectioning vitreous ice using a microtome.
- Mentions the development of focused ion beam milling as an alternative sectioning technique at liquid nitrogen temperatures.
- Addresses imaging challenges including low contrast, beam sensitivity, and mechanical distortions.
Main Results:
- Successful cryo-EM preparation allows for imaging with excellent molecular detail.
- Preserves cellular structures without the disruption caused by dehydration.
- Enables investigation of macromolecular complexes within their native cellular environment.
- Tilted views and subvolume averaging can be used for higher resolution analysis.
Conclusions:
- Cryo-EM offers a powerful approach to study cellular ultrastructure and macromolecular complexes in their native state.
- Despite technical challenges, cryo-EM provides superior preservation compared to traditional EM methods.
- This protocol facilitates high-resolution structural studies of fission yeast.
