Reproducible Sample Preparation of Virus-Infected Cells for Cryo-FIB/ET Using Manual Plunge Freezing.

Area of Science:

• Virology
• Cell Biology
• Microscopy

Background:

• Viruses extensively remodel host cells for infection.
• Electron microscopy (EM) techniques like cryo-focused ion beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET) have advanced virus visualization.
• There is a need for accessible protocols for preparing virus-infected cells for cryo-FIB/ET.

Purpose of the Study:

• To provide an in-house, beginner-friendly protocol for preparing herpes simplex virus 1 (HSV-1)-infected mammalian cells for cryo-FIB/ET.
• To guide new users through sample preparation using basic, manual equipment.
• To optimize cell seeding, blotting, and plunge-freezing for efficient data collection.

Main Methods:

• Developed a protocol for seeding HSV-1 infected cells onto EM grids.
• Detailed manual blotting and plunge-freezing techniques.
• Included tips for screening and prioritizing grids for milling and data acquisition.

Main Results:

• The protocol yields grids with thin ice and evenly distributed cells.
• Successfully prepared samples suitable for focused ion beam milling.
• The method is designed for users without access to advanced cryo-EM facilities.

Conclusions:

• This protocol enables new users to prepare virus-infected cells for cryo-FIB/ET using basic equipment.
• Optimized sample preparation is crucial for successful visualization of virus-induced cellular remodeling.
• The developed method facilitates the study of viral infections at a high resolution.