Cryo-electron tomography (cryoET) is a label-free cryogenic imaging technique that provides 3D snapshots of the cellular context at nanometer resolution. This is done by opening literal windows into the cell with focused ion beam (FIB) milling of a cryogenically frozen (vitrified) cell. A series of 2D images is taken of this thinned cellular sample (a cryo-lamella) that are then reconstructed into a 3D structure.
Background
Light microscopy, particularly fluorescence microscopy, is a cornerstone of modern cell biology, and advances in optics, as well as computational tools, have made it possible to view systems in a range of relevant scales. Nevertheless, there are also drawbacks to this technique.
While it allows for the identification of proteins and molecules based on bound fluorescent tags, it cannot visualize other structures that do not have fluorescent properties. This means that only that which is tagged can be seen; the surrounding cellular context is lost. Moreover, the resolution is often limited, and the presence of certain chemical agents needed for some fluorescence modalities to work (e.g. super-resolution light microscopy techniques) can also affect the native structure. This is where cryo-ET can play to its strengths, as it does not require any dehydration, staining or labeling of the sample.
Cryo tomography can provide 3D snapshots of proteins at work within their functional cellular environments, allowing us to see and understand how they, and other molecules, work together to carry out major processes in a cell. This is because cryo-ET delivers both structural information about individual proteins as well as their spatial arrangements within the cell, making it a truly unique technique. Cryo tomography has enormous potential for cell biology as it bridges the gap between light microscopy and near-atomic-resolution techniques like single particle analysis (SPA). (Note that cryo-electron tomography data can be collected with the same transmission electron microscopes as single-particle analysis data.)
This video shows the 3D visualization of a Golgi apparatus from the green alga Chlamydomonas reinhardtii. The unicellular alga was flash-frozen without any artificial stains or fixatives.
Cryo electron tomography visualization of the secretion mediated Salmonella-host cell interface, showing a plausible pathway of effector translocation. Courtesy of D. Park and J. Liu, Yale University.
Visualization of the nuclear periphery of a HeLa cell revealed by cryo-electron tomography. Courtesy of Dr. J. Mahamid, EMBL.
Cryo-electron tomography allows you to visualize macromolecular structures in situ, inside the cell. Vitreous frozen cells are first thinned with a focused ion beam and then imaged in three dimensions using a transmission electron microscope.
This video shows the 3D visualization of a Golgi apparatus from the green alga Chlamydomonas reinhardtii. The unicellular alga was flash-frozen without any artificial stains or fixatives.
Cryo electron tomography visualization of the secretion mediated Salmonella-host cell interface, showing a plausible pathway of effector translocation. Courtesy of D. Park and J. Liu, Yale University.
Visualization of the nuclear periphery of a HeLa cell revealed by cryo-electron tomography. Courtesy of Dr. J. Mahamid, EMBL.
Cryo-electron tomography allows you to visualize macromolecular structures in situ, inside the cell. Vitreous frozen cells are first thinned with a focused ion beam and then imaged in three dimensions using a transmission electron microscope.
Infectious Disease Research
Cryo-EM techniques enable multiscale observations of 3D biological structures in their near-native states, informing faster, more efficient development of therapeutics.
Structural Biology Research
Cryo-electron microscopy enables the structural analysis of challenging biological targets such as large complexes, flexible species and membrane protein.
Plant Biology Research
Fundamental plant biology research is enabled by cryo electron microscopy, which provides information on proteins (with single particle analysis), to their cellular context (with tomography), all the way up to the overall structure of the plant (large volume analysis).
Proteins Analysis
Cryo-electron microscopy provides near-atomic resolution 3D protein structure. It can determine structural information for complexes and crystallization-resistant samples, as well as vital cellular context.
Virus Analysis
Cryo-EM enables the 3D structural visualization of virus particles, and the antigen-antibody interface, at near-atomic resolutions. A virus’s inherent structural symmetry makes it the ideal target for cryo-EM analysis.
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