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Cryo electron tomography (cryoET) is a groundbreaking technology for 3D visualization and analysis of biomolecules in the context of cellular structures. The use of this technology enables a better understanding of the structural basis of cellular processes, which is essential for understanding cell functions. Unlike other biological imaging techniques, cryoET is a label-free cryogenic imaging technique that does not require chemical fixation or staining. Instead, cells are preserved in their native state by flash freezing (vitrification). In this way, structural information about organelles and individual proteins is linked with their exact spatial arrangement within the cell. This unique capability holds enormous potential for cell biology, making cryo electron tomography a highly promising technology.
Cryo-Tomography workflow
Step 1: Sample preparation by vitrification
Cells prepared by routine culture methods are grown on carbon-coated gold electron microscopy (EM) grids. In order to preserve the native cellular environment, cells are not stained but flash-frozen directly on the EM grid. The cryogenic freezing process is so fast that liquid water forms non-crystalline vitreous ice, thus avoiding the damage caused by the formation of crystals at slower freezing rates. This process preserves the ultrastructure of the flash-frozen cell. Sample freezing is performed in a semi-automated manner using a Thermo Scientific Vitrobot System.
Step 2: Localization by fluorescence
Locating the structure of interest can be difficult in the vast complexity of the natural cellular environment. However, using cryo-correlative microscopy, the structures of interest are identified in the cryo-fluorescence light microscope, the Leica Cryo CLEM system. A dedicated cryo stage keeps the sample in its vitrified state during cryo-fluorescence imaging. The vitrified sample, along with the coordinates of the target regions for milling, is transferred to the Thermo Scientific Aquilos Cryo-FIB (cryo-focused ion beam electron microscope) in a dedicated cartridge system that safeguards the sample from contamination.
Step 3: Thinning by milling
After preselection and targeting in the Leica Cryo CLEM system, the sample is transferred to the Thermo Scientific Aquilos Cryo-FIB, a dedicated Cryo-DualBeam electron microscope for thinning. The Aquilos Cryo-FIB combines a scanning electron beam (SEM) and a focused ion beam (FIB). The electron beam is used for imaging the sample, and the ion beam ensures precise removal of material from vitrified cells. 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. The cryo-lamella contains the region of interest and can be milled as thin as 100–200 nanometers. There is no mechanical sectioning with the Aquilos Cryo-FIB. 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 in a process called sputtering (also referred to as 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 under cryo-temperatures (e.g. compression in the cutting direction).
Step 4: Electron cryo-tomography
After the milling step, thin cryo-lamellas are transferred to the Thermo Scientific Krios Cryo-TEM (cryo-transmission electron microscope), where the actual tomographic image acquisition takes place. The images in the tomographic series are acquired by tilting the sample in known increments. Individual projection images are then computationally combined in a procedure known as back-projection, which creates the 3D tomographic volume.
Step 5: Visualization and structural analysis
The 3D tomogram that features cellular structures can be segmented and colored in a variety of ways to enhance its display and presentation by using Thermo Scientific Amira Software, a universal 2D-5D platform for advanced visualization and analysis. From the tomogram, small subsets of data that contain the structures of interest can be computationally extracted and subjected to image-processing methods. Image processing allows researchers to achieve a resolution in the nanometer range while maintaining the best possible structure preservation.
Cryo Electron Tomography - How it works
Cryo Electron Tomography - How it works
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To ensure optimal system performance, we provide you access to a world-class network of field service experts, technical support, and certified spare parts.
