Electron Microscopy

Cryo Electron Tomography of Intracellular Structures

Cryo ET of vitrified subcellular structures in 3D at nanometer resolution.

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

HeLa cell imaged with fluorescence imaging and cryo electron tomography.
Fluorescence image (left) and tomography structure (right) of a HeLa cell. The fluorescence image shows nuclei (blue), actin filaments (red) and mitochondria (green), but without cellular context. Cryo electorn tomography provides a close up containing vital contextual information.

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.

Excitatory synapse of a neuron imaged with cryo electron tomography.
Excitatory synapse of hippocampal neuron. Courtesy of Guoqiang Bi of USTC and Hong Zhou of UCLA. Data segmentation and visualization by Thermo Scientific Amira Software.

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.)

 

Cryo Tomography Workflow

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.

WebinarCellBiologyCryoTomo_282x286

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.

Watch on-demand webinar ›

Golgi apparatus imaged with cryo electron tomography.
In situ cryo-electron tomography reveals the molecular architecture of the Chlamydomonas Golgi apparatus with native morphology. Data courtesy of Dr. Benjamin Engel, Helmholtz Zentrum München. Data segmentation and visualization by Thermo Scientific Amira Software.
ALS/FTD poly-Gly-Ala peptides aggregate into a dense network of twisted ribbons using cryo-tomography
In neurons, ALS/FTD poly-Gly-Ala peptides aggregate into a dense network of twisted ribbons.The ribbons sequester a large fraction of the cells’ proteasomes. Courtesy of Q. Guo, Max Planck Institute - Martensreid.
Conical protective envelope of the virus genome
Insights into HIV in unprecedented resolution: The green and red CA protein structures form the conical protective envelope of the virus genome. Courtesy of Simone Mattei, EMBL.

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.

WebinarCellBiologyCryoTomo_282x286

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.

Watch on-demand webinar ›

Golgi apparatus imaged with cryo electron tomography.
In situ cryo-electron tomography reveals the molecular architecture of the Chlamydomonas Golgi apparatus with native morphology. Data courtesy of Dr. Benjamin Engel, Helmholtz Zentrum München. Data segmentation and visualization by Thermo Scientific Amira Software.
ALS/FTD poly-Gly-Ala peptides aggregate into a dense network of twisted ribbons using cryo-tomography
In neurons, ALS/FTD poly-Gly-Ala peptides aggregate into a dense network of twisted ribbons.The ribbons sequester a large fraction of the cells’ proteasomes. Courtesy of Q. Guo, Max Planck Institute - Martensreid.
Conical protective envelope of the virus genome
Insights into HIV in unprecedented resolution: The green and red CA protein structures form the conical protective envelope of the virus genome. Courtesy of Simone Mattei, EMBL.

Applications

Infectious_Disease_Research_App_w274x180

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.

Life_Sciences_Application_274x180_144DPI

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_Application_274x180_144_DPI

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).


Samples


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.

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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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Products

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Krios G4 Cryo-TEM for
Life Sciences

  • Improved ergonomics
  • Fits more easily into new and existing labs
  • Maximized productivity and automation
  • Best image quality for high-resolution 3D reconstruction

Aquilos 2 Cryo-FIB for Life Sciences

  • Automation enables production of multiple lamellas
  • Target and extract your structure of interest with lift-out nano-manipulator
  • 3D visualization for high-resolution tomography

Vitrobot System

  • Fully Automated Sample vitrification
  • Blotting Device
  • Semi-Automated Grid Transfer
  • High Sample Throughput

Amira Software for Life Sciences

  • Import and process image data
  • Auto-segmentation, object separation, labeling
  • Analyze and quantify with automated statistics
  • Create images, animations and videos

Auto Slice and View 4.0 Software

  • Serial sectioning for 3D STEM images
  • Imaging plus analytical data (EDS, EBSD)
  • On-the-fly editing capabilities
  • New algorithms – easier to use

Maps Software

  • Acquire high resolution images over large areas
  • Easily find regions of interest
  • Automate image acquisition process
  • Correlate data from different sources

Tomography 4.0
Software

  • On-the-fly Reconstruction Algorithm
  • Fully Automatic TEM and STEM Acquisition
  • One-Time Calibration
  • Easy workflow from data to structure

Falcon 4 Detector

  • Leading detective quantum efficiency
  • 10x shorter exposure time than its predecessor
  • Fully embedded in Thermo Scientific software
  • Built in data management
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