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Microbiology JoVE (Open Access) Citable · DOI

Electron Cryotomography of Bacterial Cells

DOI: 10.3791/1943-v
What you'll learn
  • ✓ Prepare bacterial cells for electron cryotomography via plunge freezing or high-pressure freezing
  • ✓ Collect tilt series data using cryo-transmission electron microscopy
  • ✓ Reconstruct and segment 3D tomograms to resolve macromolecular ultrastructure
  • ✓ Interpret near-native bacterial cell architecture at ~4 nm resolution
Protocol

Biopharma Insights We illustrate here how to use electron cryotomography (ECT) to study the ultrastructure of bacterial cells in near-native states, to "macromolecular" (~4 nm) resolution.

Difficulty
advanced
Total time
~3–5 days per sample (including freezing, imaging, and computational reconstruction)

Steps

1
Understand electron cryotomography principles

Review the fundamental concepts of ECT for imaging bacterial ultrastructure in near-native states at macromolecular resolution. Covers the rationale and technical overview of the entire workflow.

▶ 00:39
2
Plunge freeze thin bacterial cells

Rapidly cool thin bacterial samples by plunge freezing into cryogenic liquid to preserve native cellular ultrastructure without ice crystal damage.

▶ 02:11
3
High-pressure freeze and cryo-section thicker cells

Apply high-pressure freezing to thicker bacterial cells, then perform cryo-sectioning to prepare electron-transparent specimens for imaging.

▶ 03:41
4
Examine cells using cryo light microscope

Screen frozen specimens under cryo light microscopy to identify suitable regions and verify cell viability before transmission electron microscopy analysis.

▶ 06:43
5
Collect tilt series using cryo transmission electron microscopy

Acquire high-resolution transmission electron microscopy images across a range of tilt angles to generate a complete 3D dataset of bacterial cellular architecture.

▶ 07:49
6
Reconstruct tomogram and organize database

Process collected tilt series data through computational reconstruction algorithms to generate 3D tomograms and organize results in a searchable database.

▶ 09:08
7
Segment and annotate tomogram features

Manually or semi-automatically identify and label distinct cellular structures and macromolecular complexes within the reconstructed 3D volume.

▶ 10:24
8
Generate movies and 3D animations

Create publication-quality 3D renderings, virtual slices, and animations from segmented tomograms to visualize and communicate bacterial ultrastructural findings.

▶ 10:51
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