Related Experiment Video
Updated: Jan 13, 2026
Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography
Published on: October 25, 2021
Reproducible Sample Preparation of Virus-Infected Cells for Cryo-FIB/ET Using Manual Plunge Freezing
Nathalie R Lavoie1,2, Ekaterina E Heldwein1,2
1Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA, USA.
Abstract:
Most viruses extensively remodel their host cells to establish productive infection. Visualization of virus-induced cellular remodeling by electron microscopy (EM) has been revolutionized in recent years by advances in cryo-focused ion beam (cryo-FIB) milling paired with cryo-electron tomography (cryo-ET). As cryo-FIB/ET becomes more widely available, there is a need for beginner-friendly guides to optimize the preparation of virus-infected mammalian cells on EM grids. Here, we provide an in-house protocol for new users for preparing samples of cells infected with herpes simplex virus 1 (HSV-1) for cryo-FIB/ET. This protocol guides users in how to seed infected cells onto grids, blot, and plunge-freeze grids using basic, manual equipment. It also provides tips on how to screen and prioritize grids for efficient milling and data collection. Key features • A beginner-friendly protocol for users without access to a cryo-EM core/suite at their institution that utilizes basic equipment. • This protocol focuses on optimizing cell seeding and blotting to yield grids with thin ice and evenly distributed cells. • Grids prepared using this protocol can be used for focused ion beam milling.
Insights
This study presents a beginner-friendly protocol for preparing herpes simplex virus 1-infected mammalian cells for cryo-electron tomography (cryo-ET). The method uses basic equipment to optimize sample preparation for visualizing virus-induced cellular changes.
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.
More Related Videos
09:16Manual Blot-and-Plunge Freezing of Biological Specimens for Single-Particle Cryogenic Electron Microscopy
Published on: February 7, 2022
13:35Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy
Published on: May 5, 2017
