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Cell Biology JoVE (Open Access) Citable · DOI

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

DOI: 10.3791/52311-v
What you'll learn
  • ✓ Prepare and maintain cryo-holder properly
  • ✓ Prepare cryo-grids for vitrification
  • ✓ Plunge-freeze samples to preserve native state
  • ✓ Collect low-dose data for 3D reconstruction
Protocol

Biopharma Insights This paper describes the cryo-electron microscopy methodology, which is used to obtain high quality microscopic images of macromolecules in their near-native state. The method yields images suitable for further computerized processing using the single particle approach, devised to generate the 3D reconstruction of a macromolecule.

Difficulty
intermediate
Total time
~1-2 days per sample

Steps

1
Prepare and maintain cryo-holder

Clean and cool the cryo-holder to maintain optimal conditions for sample transfer and imaging.

▶ 01:21
2
Prepare cryo-grids

Apply the sample solution to the grid and remove excess liquid to form a thin film for vitrification.

▶ 02:32
3
Plunge-freeze the grid

Rapidly freeze the grid in liquid ethane to vitrify the sample, preserving its near-native structure.

▶ 03:48
4
Transfer cryo-grid to electron microscope

Mount the frozen grid onto the cryo-holder and insert it into the transmission electron microscope while maintaining cryogenic temperatures.

▶ 04:50
5
Collect low-dose electron micrographs

Use low-dose imaging conditions to acquire high-quality images minimizing radiation damage to the sample.

▶ 06:15
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