Cryogenic Electron Tomography
Cryogenic Electron Tomography - Related Videos
02:03
Preparation of Legionella pneumophila for Cryogenic Electron Tomography
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2025
Source: Chetrit, D., et al. Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System. J. Vis. Exp. (2020)The video describes how to prepare Legionella pneumophila samples for cryo-electron tomography by combining bacterial culture, gold nanoparticle marking, and vitrification techniques. The process preserves native cellular structures and enables high-resolution imaging of membrane-associated secretion...
08:16
Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition
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Cited by 8 •
2021
The increasing demand for large-scale data collection in cryogenic electron tomography requires high-throughput image acquisition routines. Described here is a protocol that implements the recent developments of advanced acquisition strategies aimed at maximizing the time-efficiency and throughput of tomographic data collection.
08:29
Workflow Using a Cryogenic Coincident Fluorescence, Electron, and Ion Beam Microscope for Targeted Milling of Cells
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Cited by 2 •
2025
This workflow enables lamella production targeting fluorescently labeled biological structures that are small (<1 μm in axial extent) and rare (1 copy per cell) using a cryogenic tri-coincident imaging platform. This platform integrates fluorescence microscopy, focused ion beam milling, and scanning electron microscopy at a single focal position and enables simultaneous fluorescence microscopy while milling.
09:59
Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography
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Cited by 24 •
2014
To preserve neuronal processes for ultrastructural analysis, we describe a protocol for plating of primary neurons on electron microscopy grids followed by flash freezing, yielding samples suspended in a layer of vitreous ice. These samples can be examined with a cryo-electron microscope to visualize structures at the nanometer scale.
09:53
Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows
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Cited by 29 •
2021
The goal of this protocol is to direct cell adhesion and growth to targeted areas of grids for cryo-electron microscopy. This is achieved by applying an anti-fouling layer that is ablated in user-specified patterns followed by deposition of extra-cellular matrix proteins in the patterned areas prior to cell seeding.
FAQs
Rapid vitrification prevents ice crystal formation while the specimen is frozen. This preserves cellular organization and molecular architecture in a near-native state, allowing proteins, membranes, organelles, and macromolecular complexes to be examined without losing their spatial relationships. The preservation is especially important when biological structure must be interpreted within its surrounding cellular context.
The electron microscope records a series of two-dimensional projections while the frozen specimen is tilted through a range of angles. Computational methods then combine these projections into a tomogram, a three-dimensional representation of the sample. Because the images come from multiple viewing angles, the reconstruction can show structures and their organization throughout the specimen.
The near-native frozen state retains relationships among proteins, organelles, membranes, and larger macromolecular complexes. This makes it possible to study molecular architecture together with the organization of the surrounding cell. Rather than viewing structures only as isolated components, researchers can examine how cellular structures are positioned within their biological environment.
Subtomogram averaging and correlative microscopy add complementary levels of interpretation. Subtomogram averaging can support analysis of repeated molecular structures within tomograms, while correlative microscopy helps connect molecular structure with broader cellular context. Together, these approaches expand the information obtained from cryo-ET beyond a single reconstructed volume and support links between structural and cell biology.
A typical workflow begins with a biological specimen that is rapidly vitrified to avoid ice crystals. The frozen sample is placed in an electron microscope, where it is tilted through a range of angles to collect a projection series. Computational reconstruction then combines the images into a tomogram for biological interpretation.
Researchers use cryo-ET when they need structural information together with cellular organization. The technique supports investigations in infection, cell biology, and structural biology, where proteins, membranes, organelles, and macromolecular complexes may need to be examined in place. Its results can reveal how molecular architecture relates to the larger organization of a biological specimen.