Cryoelectron Microscopy of Fission Yeast

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.