A protocol for isolation and visualization of yeast nuclei by scanning electron microscopy (SEM)

Area of Science:

• Cell Biology
• Microscopy Techniques

Background:

• Studying yeast nuclear structure requires robust isolation and high-resolution imaging methods.
• Existing techniques may not adequately preserve delicate nuclear components for detailed visualization.

Purpose of the Study:

• To establish a detailed protocol for isolating yeast nuclei.
• To enable immuno-gold labeling and visualization of nuclear structures using FESEM.
• To optimize sample preparation for long-term storage and image acquisition.

Main Methods:

• Isolation of nuclei from Saccharomyces cerevisiae and Schizosaccharomyces pombe.
• Two nuclear isolation methods: enzymatic spheroplast formation or whole-cell freezing and mechanical disruption.
• Immuno-gold labeling of target proteins within isolated nuclei.
• Visualization using field emission scanning electron microscopy (FESEM).

Main Results:

• Successful isolation of yeast nuclei from both budding and fission yeast strains.
• High-resolution imaging of the yeast nuclear envelope (NE), nuclear pore complexes (NPCs), and associated cytoskeletal elements.
• Demonstration of sample stability under vacuum for extended periods, facilitating image acquisition.

Conclusions:

• The developed protocol provides a reliable method for yeast nuclear isolation and high-resolution imaging.
• This technique is valuable for studying the structural organization of the yeast nucleus, including the NE and NPCs.
• The protocol's sample preservation capabilities enhance the efficiency of FESEM-based investigations.