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Cryo-Tomography

In Situ Structural Biology Workflow will expose the Molecular Machinery of Cells

Studying the molecular machinery of cells from atomic details to the cellular context and beyond is a great challenge for cell biology. It requires the integration of dynamic and structural information obtained using different ranges of spatial resolution.

In order for this to be accomplished, a comprehensive workflow is needed. This workflow must cover vitreous freezing, cryo-fluorescence microscopy, targeted sample thinning by focused ion beam, and high-resolution cryo-electron tomography (Cryo-ET).

Challenges in Cryo-Tomography

Structural biologists have made great progress in unraveling the structures of individual proteins and protein complexes. Much of these recent results have been made using Single Particle Analysis (SPA), which has turned 3D reconstructions of protein (complexes) into an almost routine technique.

As SPA requires large amounts of isolated and purified proteins, the contextual information is lost in the process.

The ultimate goal for these structural and cell biologists is to derive the structures of molecular machineries of all living systems within their natural, functional contexts, where their interactions with other components can be observed. 

 

Cryo-TEM advantages:

  • Allow for nanometer-scale 3D models of biological structures in their native, cellular context

Visualization of the nuclear periphery of a HeLa cell revealed by cryo‐electron tomography. The interphase HeLa cellwas thinned by cryo‐focused ion beam (cryo‐FIB) milling. The 3D segmentation of the tomographic data shows microtubules (orange), actin filaments (yellow), endoplasmic reticulum (green), nuclear pore complexes (light blue),large (dark blue) and small (brown) ribosomal subunits.


Data courtesy of Dr. J. Mahamid, Department of Molecular Structural Biology, Max Planck Institute for Biochemistry,Martinsried, Germany.

Integrative Structural Biology

Mass spectrometry offers the possibilities of a complete lab and an abundance of information for structural biology. The only thing missing is an actual structure to match the information against. Several techniques are capable of providing this complementing structure; however, especially for the focus on large protein complexes, one technique is simply superior to the others: cryo-electron microscopy (cryo-EM). Are you aware of how the integration of mass spectrometry and cryo-EM can benefit your structural biology research? 

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Cryo-Tomography Workflow

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Step one: Sample Preparation

The first step in the Cryo-Tomography workflow is key in order to produce the highest quality 3D protein complex structures.

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Step two: Cryo-LM

Maintain sample integrity while transferring to an imaging platform for identifying structures of interest.

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Step three: Cryo-FIB Milling

After cryo-LM imaging, regions of interest are identified and the locations are stored in a uniform software platform that will allow retrieval of this data in the cryo-FIB. From the cryo-LM, the sample is transferred to the cryo-FIB without contamination.

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Step four: Cryo-TEM Tomography

A series of 2D projection images is captured using very low electron dose.

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Step five: Reconstruction & Visualization

Small subsets of data, containing the structures of interest can be computationally extracted from the 3D tomogram and subjected to image processing methods.

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Step one: Sample Preparation

Sample Vitrification

Cells prepared by routine culture methods are surrounded by physiological solution and are grown on carbon-coated gold electron microscopy (EM) grids. Excess solution must be blotted away before the sample grid is plunge-frozen in a cryogenically cooled fluid, typically liquid ethane. The water in the sample freezes so rapidly that it does not crystallize, thus avoiding the molecular-scale disruption (by formed ice crystals) that would occur with a normal slow freezing process.

Products & Solutions

Vitrification with Vitrobot

Create high quality vitrified samples for single particle analysis or cryo-tomography research applications with Vitrobot. Vitrobot offers fully automated vitrification, fast and easy. It performs the cryo-fixation process at constant physical and mechanical conditions like temperature, relative humidity, blotting conditions and freezing velocity. This ensures high quality cryo-fixation results and a high sample preparation throughput prior to cryo-TEM observation.

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Step two: Cryo-LM

The sample is transferred to a cryo-fluorescence light microscope, where structures of interest are identified by fluorescent tags. A dedicated cryo-LM stage keeps the sample in its vitrified state during cryo-fluorescence imaging.

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Step three: Cryo-FIB Milling

Within the cryo-FIB, the LM data will be correlated using MAPS software, allowing quick identification of the region of interest with the cryo-FIB. On these regions of interest, a thin, uniform lamella must be prepared for cryo-ET. The dedicated cryo-FIB will allow for the sample to be milled at the proper temperature (approximately -170C), and it also prevents contamination of the sample.

Before milling, the sample is protected with a micrometer-thick metal layer (Pt) using the GIS. This layer prevents beam erosion on the sample. After milling, the lamella is sputtered with a nanometer-thin metal coating to render the sample conductive for TEM imaging. The cryo lamella is then transferred to a high-end TEM autoloader.

Products & Solutions

Aquilos Cryo-FIB

The Thermo Scientific™ Aquilos™ Cryo-FIB is the first cryo-DualBeam™ (focused ion beam/scanning electron microscope) system dedicated to preparation of frozen, thin lamella samples from biological specimens for high-resolution tomographic imaging in a cryo-transmission electron microscope (cryo-TEM).

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Step four: Cryo-TEM Tomography

The samples are placed within the autoloader in the TEM. Per cryo-lamella, a series of 2D images is acquired from different perspectives as the sample is tilted up to +/- 70 degrees.

Products & Solutions

Imaging Platforms

Glacios Cryo-TEM

The new Thermo Scientific™ Glacios™ Cryo Transmission Electron Microscope (Cryo-TEM) delivers a complete and affordable Cryo-EM solution to a broad range of scientists. It features 200 kV XFEG optics, the industry-leading Autoloader (cryogenic sample manipulation robot) and the same innovative automation for ease of use as on the Krios G3i Cryo-TEM. The Glacios Cryo-TEM bundles all this into a small footprint that simplifies installation.

Krios G3i Cryo-TEM

The new Thermo Scientific™ Krios™ G3i Cryo Transmission Electron Microscope (Cryo-TEM) enables life science researchers to unravel life at the molecular level—easier, faster, and more reliably than ever before. Its highly stable 300 kV TEM platform and industry-leading Autoloader (cryogenic sample manipulation robot) are designed for automated applications, such as single particle analysis (SPA) and cryo-tomography. Designed-in connectivity ensures a robust and risk-free pathway throughout the entire workflow, from sample preparation and optimization to image acquisition and data processing.

Talos™ Arctica

The Talos Arctica is a 200kV FEG Transmission and Scanning Electron Microscope (STEM). It is a powerful, stable, and versatile system for delivering high-resolution 3D characterization of biological and biomaterials samples in cell biology, structural biology, and nanotechnology research. Talos enables scientists to quickly obtain better insight and understanding of macromolecular structures, cellular components, cells, and tissues in three dimensions.

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Step five: Reconstruction & Visualization

In cases where multiple identical structures are present in the sample (e.g., pores in the cell’s nuclear membrane), the reconstruction and analysis software can combine data from individual structures, averaging out noise and increasing contrast to create a composite model with higher resolution. The operator can segment, color, and cross-section the model in a variety of ways to enhance its display and presentation.

Featured Video: Cryo-TEM Lamella Preparation

Watch how, after vitrification of the sample, a DualBeam FIB-SEM is used for in situ lamella milling. This on-the-grid thinning prepares the sample for automated loading into a cryo-TEM for imaging.

Featured Document

Publication: Assembly of a Nucleus-like Structure During Viral Replication in Bacteria

We observed the assembly of a nucleus-like structure in bacteria during viral infection. Using fluorescence microscopy and cryo-electron tomography, we showed that Pseudomonas chlororaphis phage 201ɸ2-1 assembled a compartment that separated viral DNA from the cytoplasm. The phage compartment was centered by a bipolar tubulin-based spindle, and it segregated phage and bacterial proteins according to function. Proteins involved in DNA replication and transcription localized inside the compartment, whereas proteins involved in translation and nucleotide synthesis localized outside. Later during infection, viral capsids assembled on the cytoplasmic membrane and moved to the surface of the compartment for DNA packaging. Ultimately, viral particles were released from the compartment and the cell lysed. These results demonstrate that phages have evolved a specialized structure to compartmentalize viral replication.

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Products for Cryo-Tomography

Krios G3i Cryo-TEM for Life Sciences
The new Thermo Scientific™ Krios™ G3i Cryo Transmission Electron Microscope (Cryo-TEM) enables life science researchers to unravel life at the molecular level—easier, faster, and more reliably than ever before. Its highly stable 300 kV TEM platform and industry-leading Autoloader (cryogenic sample manipulation robot) are designed for automated applications, such as single particle analysis (SPA) and cryo-tomography. Designed-in connectivity ensures a robust and risk-free pathway throughout the entire workflow, from sample preparation and optimization to image acquisition and data processing.
Aquilos Cryo-FIB for Life Sciences

The Thermo Scientific™ Aquilos™ Cryo-FIB is the first cryo-DualBeam™ (focused ion beam/scanning electron microscope) system dedicated to preparation of frozen, thin lamella samples from biological specimens for high-resolution tomographic imaging in a cryo-transmission electron microscope (cryo-TEM). 

Glacios Cryo-TEM for Life Sciences

The new Thermo Scientific™ Glacios™ Cryo Transmission Electron Microscope (Cryo-TEM) delivers a complete and affordable Cryo-EM solution to a broad range of scientists. It features 200 kV XFEG optics, the industry-leading Autoloader (cryogenic sample manipulation robot) and the same innovative automation for ease of use as on the Krios G3i Cryo-TEM. The Glacios Cryo-TEM bundles all this into a small footprint that simplifies installation.

Scios 2 DualBeam for Materials Science

The Thermo Scientific™ Scios™ 2 DualBeam™ is an ultra-high-resolution analytical FIB-SEM system that provides outstanding sample preparation and 3D characterization performance for the widest range of samples, including magnetic and non-conductive materials. 

Talos Arctica TEM for Life Sciences
The Thermo Scientific™ Talos™ Arctica is a 200kV FEG Transmission and Scanning Electron Microscope (S/TEM). It is a powerful, stable, and versatile system for delivering high-resolution 3D characterization of biological and biomaterials samples in cell biology, structural biology, and nanotechnology research. The Talos S/TEM enables scientists to quickly obtain better insight and understanding of macromolecular structures, cellular components, cells, and tissues in three dimensions.
Vitrobot for Life Sciences
Vitrobot completely automates the vitrification process to provide fast, easy, reproducible sample preparation - the first step in obtaining high quality images and repeatable experimental results.
MAPS Software
Navigate, tile, stitch, correlate and analyse data from large areas of high resolution images 
Tomography 4.0 software
The fast, easy and complete solution for electron tomography

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