Whole-cell Cryo-et
Whole-cell Cryo-et - Related Videos
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.
05:05
Preparation of High-Temperature Sample Grids for Cryo-EM
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Cited by 5 •
2021
This paper provides a detailed protocol for preparing sample grids at temperatures as high as 70 °C, prior to plunge freezing for cryo-EM experiments.
10:54
Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam
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Cited by 5 •
2014
Cryo Electron Microscopes, either Scanning (SEM) or Transmission (TEM), are widely used for characterization of biological samples or other materials with a high water content1. A SEM/Focused Ion Beam (FIB) is used to identify features of interest in samples and extract a thin, electron-transparent lamella for transfer to a cryo-TEM.
11:33
Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography
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Cited by 48 •
2016
We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.
10:00
Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy
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Cited by 11 •
2011
We demonstrate the fabrication of a low-cost cryogenic stage designed to fit most reflected light microscopes. This lab-built cryogenic stage enables efficient and reliable correlative imaging between cryo-light and cryo-electron microscopy.
FAQs
Rapid vitrification preserves cellular water in a glass-like state without forming ice crystals. This prevents ice-related disruption of membranes, macromolecular complexes, cytoskeletal elements, and compartments during preparation. By maintaining these structures close to their native state, the resulting images can better represent organization and interactions as they exist inside intact cells.
The microscope records projection images while the frozen-hydrated specimen is tilted through different angles. Each projection captures structural information from a distinct viewpoint. Computational reconstruction combines those views into a three-dimensional tomogram, allowing researchers to interpret the spatial arrangement of cellular components rather than relying on a single two-dimensional image.
Whole-cell cryo-ET preserves macromolecular complexes within their cellular surroundings, where neighboring structures, membranes, and compartments can influence their organization. This native context can reveal molecular interactions and spatial relationships that may be obscured when components are purified or separated from the cell. The approach therefore connects molecular architecture with cellular function.
The method can visualize macromolecular complexes, membranes, cytoskeletal elements, and cellular compartments within intact cells. Examining these features together helps researchers relate their architecture to broader cellular organization. In biochemistry, that context is valuable for investigating how molecular assemblies are positioned and associated with structures that support cellular function.
The workflow begins by rapidly vitrifying the cell sample so its water and structures remain preserved without ice crystals. The frozen-hydrated specimen is then imaged repeatedly as it tilts through a range of angles. Finally, computational reconstruction converts the projection series into a three-dimensional tomogram for structural analysis.
Researchers would choose this approach when they need structural information from intact cells rather than isolated or heavily processed material. It supports investigations of molecular interactions, structural dynamics, infection, and cellular organization. Its main value is showing these phenomena in the spatial context of membranes, compartments, and other cellular structures.
In biochemistry, the technique links the architecture of molecular assemblies to their cellular setting and potential function. It can show how macromolecular complexes relate to membranes, cytoskeletal elements, and compartments while preserving near-native organization. This makes it useful for studying cellular mechanisms that fixation, staining, or purification may obscure.