What is cryo-electron tomography
Cellular cryo-electron tomography (cryo-ET) is a label-free cryogenic imaging technique that provides 3D datasets of organelles and protein complexes at nanometer resolution. This is done by opening 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 (cryo-lamella) then reconstructed into a 3D dataset.
Correlative light electron microscopy
Light microscopy, particularly fluorescence microscopy, is a cornerstone of modern cell biology. Advances in optics, as well as computational tools, have made it possible to view systems in a range of relevant scales. It allows for the identification of proteins and molecules based on bound fluorescent tags. Structures that are not fluorescently labeled, however, cannot be visualized, meaning the context surrounding the fluorescent label is lost. Moreover, the presence of certain chemical agents needed for some fluorescence modalities to work can also affect the native structure.
Cryo-TEM sample preparation
Cryo-ET however does not require any dehydration, staining, or labeling of the sample. When used with light microscopy, which provides the location of a labeled protein of interest, cryo-ET brings together the best of both worlds: localization, specificity, and nanometer resolution. A new integrated cryo-ET workflow from Thermo Fisher Scientific and Leica Microsystems illustrates how these two techniques can be combined to achieve results.
Cryo-EM vs cryo-tomography
Cryo-electron tomography provides 3D snapshots of proteins at work within their functional cellular environments, allowing users 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-ET has enormous potential for cell biology as it bridges the gap between light microscopy and near-atomic resolution techniques like single-paticle analysis cryo-electron microscopy (cryo-EM). (Note that cryo-electron tomography data can be collected with the same transmission electron microscopes as single-particle analysis data.)
With Thermo Fisher, get to cryo-electron tomography datasets faster through automation and reliability. The Thermo Scientific Aquilos 2 Cryo-FIB allows users to take a revolutionary step forward by automating steps in the workflow to produce multiple lamellas from specific programmed locations. The best image quality and contrast for high-resolution 3D reconstruction can be achieved using the powerful combination of the Thermo Scientific Krios G4 Cryo-TEM, the Thermo Scientific Selectris Imaging Filter, and the Thermo Scientific Falcon 4 Direct Electron Detector.
Cell preparation
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.
The Thermo Fisher cryo-electron tomography workflow covers flash freezing cells to final 3D visualization. Interior cellular regions are imaged at nanometer-scale resolution and from cryo-lamellae precisely prepared with Aquilos 2 cryo-FIB.
Accelerate and Advance service packages for Cryo-tomography will support your success from the moment your purchase your Thermo Scientific Aquilos 2 Cryo-FIB system. From applications support to system remote monitoring to comprehensive maintenance, our unique blend of data-driven and hand-on services will help you quickly achieve your desired outcomes.
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.
The Thermo Fisher cryo-electron tomography workflow covers flash freezing cells to final 3D visualization. Interior cellular regions are imaged at nanometer-scale resolution and from cryo-lamellae precisely prepared with Aquilos 2 cryo-FIB.
Accelerate and Advance service packages for Cryo-tomography will support your success from the moment your purchase your Thermo Scientific Aquilos 2 Cryo-FIB system. From applications support to system remote monitoring to comprehensive maintenance, our unique blend of data-driven and hand-on services will help you quickly achieve your desired outcomes.
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).
Neurodegenerative Disease Research
Cryo-EM can determine the structural features of protein aggregates implicated in neurodegenerative diseases, allowing scientists to address how they form, interact with the cellular environment, and alter brain function.
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.
