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Cellular cryo-electron tomography (cryo-ET) allows you to visualize macromolecular structures in situ, inside the cell. Vitreous frozen cells are first thinned with a focused ion beam (FIB) microscope and then imaged in three dimensions using a transmission electron microscope (TEM). This transformative method has the power to revolutionize our understanding of cell biology, revealing the native cellular architecture with molecular clarity. The Thermo Scientific cryo-electron tomography (cryo-ET) workflow covers the entire process, from flash freezing of cells to final 3D visualization. Focused ion beam milling instruments can also provide 3D insights into sample structures (3D electron microscopy) by selectively removing or milling material and sequentially imaging with SEM at cryogenic conditions or at room temperature. Cryo-focused ion beam microscopes (cryo-FIBs) can also be used to thin protein microcrystals for microcrystal electron diffraction (MicroED), a technique that produces high-resolution 3D molecular structures of small chemical compounds or biological macromolecules.
Cryo-FIB-SEM technology
Thermo Fisher Scientific is the industry leader in focused ion beam scanning electron microscopy (FIB-SEM) with 30 years of experience as part of our Thermo Scientific DualBeam product line. We offer a broad product portfolio and advanced automation capabilities for a range of applications, including transmission electron microscopy sample preparation as well as subsurface and 3D characterization. The addition of cryo-FIB technology has allowed the integrity of flash-frozen (vitrified) samples to be maintained for a variety of biological and life-science applications.
Cryo-FIB sample preparation instruments
Aquilos 2 Cryo-FIB | Hydra Bio Plasma-FIB | Arctis Cryo-Plasma-FIB |
|---|---|---|
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| Dedicated cryo-FIB for cellular cryo-electron tomography sample preparation | Versatile plasma-FIB for multi-application labs | Automated cryo-plasma-FIB for throughput and connectivity in the cryo-electron tomography workflow |
| The Thermo Scientific Aquilos 2 Cryo-FIB produces high-quality lamellae for cryo-ET. Key steps can be automated through user-friendly milling recipes. | The Thermo Scientific Hydra Bio Plasma-FIB provides high-quality results for large volume microscopy and TEM lamella preparation, both at cryogenic and room temperatures. | The Thermo Scientific Arctis Cryo-Plasma-FIB automates high-throughput TEM lamellae production and features Autoloader connectivity for the cryo-ET workflow. |
Key Features
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| Key Features
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Cryo-FIB applications | ||
| Applications: Cryo-electron tomography, MicroED | Applications: Cryo-electron tomography, MicroED, 3D electron microscopy | Applications: Cryo-electron tomography, MicroED |
3D visualization of a Golgi apparatus from Chlamydomonas. The unicellular alga was flash-frozen without any artificial stains or fixatives. Data courtesy of Benjamin Engel. |
Contextual volume imaging of high-pressure frozen mouse brain with no added stains or fixatives viewed with Thermo Scientific Auto Slice and View Software. Sample courtesy of MRC-LMB. |
Reconstructed tomogram of a synapse in a murine neuronal cell prepared by xenon plasma-ion milling. Sample courtesy of MRC-LMB. |
| Learn more | Learn more | Learn more |
Cryo-focused ion beam milling
Following localization by correlative microscopy, the focused ion beam is used to prepare a thin, electron-transparent lamella by removing material above and below the target region. This lamella contains the region of interest and can be milled as thin as 100–200 nanometers. There is no mechanical sectioning; instead, the vitrified sample is thinned with the help of a focused beam of gallium ions that is scanned across the frozen sample surface, removing surface atoms in a layer-by-layer fashion (called sputtering or ion beam milling). Sample thinning is essential for the tomography workflow because the electron beam in the TEM can only pass through samples that are thin enough to transmit 200–300 keV electrons. Cryo-FIB thinning is a straightforward and more manageable method compared to cryo-ultramicrotomy, and it avoids intrinsic cutting artifacts of mechanical sectioning at cryo-temperatures. TheAquilos 2 Cryo-FIBwith the iFLM Correlative System is a dedicated cryo-FIB for preselection and targeting with fluorescence microscopy.
Preparation of cells for cryo-electron tomography with the Thermo Scientific Aquilos 2.
Cryo-plasma focused ion beam milling
High-quality lamellae with consistent thickness can also be prepared with a cryo-PFIB. Lamellae milling for cryo-ET is similar to cryo-FIB milling but PFIB microscopes offer multiple milling ion species. It is possible to use the different milling properties of the multiple plasma ion beams to produce a high-quality lamella without gallium implantation effects. Also, plasma ion beam systems mill at higher throughput rates than gallium-ion systems. Thermo Scientific cryo-PFIB systems include the Arctis Cryo-PFIB and the Hydra Bio PFIB, both of which feature the iFLM Correlative System for targeting of regions of interest.
Introducing Arctis Cryo-PFIB for high-throughput, automated cryo-lamellae preparation
Cryo-electron tomography workflow
Cryo-lamellae preparation with the cryo-electron tomography workflow
The Thermo Scientific cryo-electron tomography (cryo-ET) workflow is an end-to-end workflow spanning from flash freezing of cells to final 3D visualization. A cryo-TEM is used to image interior cellular regions at nanoscale resolution using cryo-lamellae that are precisely prepared with cryo-FIB/PFIB microscopes
Fluorescence localization with the iFLM Correlative System
During the cryo-ET workflow, correlative microscopy is used to identify structures of interest. A dedicated cryo-FLM stage keeps the sample in its vitrified state during fluorescence imaging. All Thermo Scientific cryo-FIBs and cryo-PFIBs can be equipped with an iFLM Correlative System, allowing samples to be imaged directly within the high vacuum without additional transfer steps to/from an external cryo-light microscope. The iFLM Correlative System features its own software interface that controls the fluorescence microscope and allows the user to switch between electron and light microscope imaging positions.
MicroED - Microcrystal electron diffraction workflow
The Thermo Scientific microcrystal electron diffraction workflow (MicroED), is an end-to-end workflow for the structural determination of small molecules and proteins. With this method, atomic details can be extracted from individual nanocrystals (<200 nm in size), even in a heterogeneous mixture. If crystals are too thick for MicroED, crystals can be thinned using one of our cryo-FIB/PFIB microscopes.
Cryo-focused ion beam milling for MicroED
Thermo Scientific Cryo-FIBs/PFIBs perform ion-beam thinning of the crystals directly on the grid and the sample can remain there for subsequent diffraction analysis in the cryo-TEM. This “on-the-grid” preparation method avoids further manipulation steps such as lift-out of the thin and fragile cryo-lamella.
3D electron microscopy
Traditional microscopy is a valuable tool for high-resolution 2D imaging, but the inherent disadvantage is that these images require expert interpretation to extrapolate 3D structures. For example, a single cross-section of a microtubule may appear to be a simple circle or an ellipse. The user must evaluate the 2D image and then decide which section of the tubule it represents. With modern 3D electron microscopy, the entire volume is captured through the stacking of sequential images and the true 3D structure of the specimen can be observed. The Helios Hydra PFIB can provide 3D insights into sample structures by selectively removing/milling material and sequentially imaging with SEM at cryogenic conditions or room temperature.
Cryo-tomography webinars
Watch our latest webinars to discover how cryo-tomography has been used to provide critical data for life science research.
Service and support for Cryo-Tomography Tools
Our service and support options ensure that you maximize your instrument uptime and data quality.
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