User:CryingEM/Cryo-Electron tomography

Cryo-electron tomography (cryo-ET) is an imaging technique used to produce high-resolution (~1–4 nm) three-dimensional views of samples, often (but not limited to) biological macromolecules and cells. cryo-ET is a specialized application of transmission electron cryomicroscopy (CryoTEM) in which samples are imaged as they are tilted, resulting in a series of 2D images that can be combined to produce a 3D reconstruction, similar to a CT scan of the human body. In contrast to other electron tomography techniques, samples are imaged under cryogenic conditions (< −150 °C). For cellular material, the structure is immobilized in non-crystalline, vitreous ice, allowing them to be imaged without dehydration or chemical fixation, which would otherwise disrupt or distort biological structures.

Description of technique
In electron microscopy (EM), samples are imaged in a high vacuum. Such a vacuum is incompatible with biological samples such as cells; the water would boil off, and the difference in pressure would explode the cell. In room-temperature EM techniques, samples are therefore prepared by fixation and dehydration. Another approach to stabilize biological samples, however, is to freeze them (electron cryomicroscopy). As in other electron cryomicroscopy techniques, samples for cryo-ET (typically small cells such as Bacteria, Archaea, or viruses) are prepared in standard aqueous media and applied to an EM grid. The grid is then plunged into a cryogen (typically liquid ethane) so efficiently such that water molecules do not have time to rearrange into a crystalline lattice. The resulting water state is called "vitreous ice" and preserves native cellular structures, such as lipid membranes, that would normally be destroyed by freezing. Plunge-frozen samples are subsequently stored and imaged at liquid-nitrogen temperatures so that the water never warms enough to crystallize.

Samples are imaged in a transmission electron microscope (TEM). As in other electron tomography techniques, the sample is tilted to different angles relative to the electron beam (typically every 1 or 2 degrees from about −60° to +60°), and an image is acquired at each angle. This tilt-series of images can then be computationally reconstructed into a three-dimensional view of the object of interest. This is called a tomogram, or tomographic reconstruction.

Process to construct tomogram and Subtomogram averaging
Cryo-ET is heavily depends on computational analysis. It yields significantly different data light microscopy data. The micrograph that has been collected from the electron microscope is not readable right away. The micrograph is in 2D format and various types of software available to stacked all 2D tilt series into a 3D tomogram. There is several different software exist to construct a 3D tomogram from 2D tilt series, such as IMOD, eTomo, AreTomo etc. Briefly stating, after collecting tilt series data from different angle, the user stacked them altogether one after another. The number of collecting tilt series can be varied and which tilt series comes after which is determined by fiducial markers.

Fiducial markers are tiny gold beads that were added while preparing sample. Since gold nanoparticle is very inert, it will not interact to the biomolecular substances, but guide the user to keep track of tilt series. The stacks of tilt series then can easily converted to tomogram using IMOD or eTomo software. Converting tilt series into 3D tomogram is a highly manual process until the existence of Tomography 5 software developed by ThermoFisher Scientific. This software can collect tilt series and simultaneously construct 3D tomogram and researchers can decide based on matrix value which data set to keep for further analysis.

Tomogram can be connected to SPA (single particle CryoEM) in a sense that one can do subtomogram averaging from the 3D tomogram and leaning towards to build an atomic model if the resolution is high enough. In subtomogram averaging process, user basically zoom out into the tomogram to look for low-resolution features that is distinguishable and present in multiple copies throughout the tomogram. That specific entity can be targeted and resolve into atomic model similar to single particle model building process.

Combining Cryo-ET to light microscopy
Cryo-ET can be bridged to fluorescence light microscopy by the new emerging technique called CLEM (Correlative-light electron microscopy) where the limitation for both light and electron microscopy can be compensated. Light microscope cannot resolve less than 200 nm and even though electron microscopy can resolve at angstrom level, it is sometimes difficult to localize cellular entity. This is where fluorescence tags come in and helps to locate. When both fluorescence light microscopy data and electron microscopy data is superimposed, some cellular entity an be tracked down, yet this technique is still in new phase and more work needs to be done to exploit this combined techniques but it comes with great potential for in-situ cellular tomography.

Applications
In transmission electron microscopy (TEM), because electrons interact strongly with matter, resolution is limited by the thickness of the sample. Also, the thickness of the sample increases as the sample is tilted, and thicker samples can then completely block the electron beam, making the image dark or completely black. Therefore, for cryo-ET, samples should be less than ~500 nm thick to achieve "macromolecular" resolution (~4 nm). For this reason, most ECT studies have focused on purified macromolecular complexes, viruses, or small cells such as those of many species of Bacteria and Archaea. Cryotomography was used to understand encapsulation of 12 nm size protein cage nanoparticles inside 60 nm sized virus-like nanoparticles.

Larger cells, and even tissues, can be prepared for cryo-ET by thinning, either by cryo-sectioning or by focused ion beam (FIB) milling. In cryo-sectioning, frozen blocks of cells or tissue are sectioned into thin samples with a cryo-microtome. In FIB-milling, plunge-frozen samples are exposed to a focused beam of ions, typically gallium, that precisely whittle away material from the top and bottom of a sample, leaving a thin lamella suitable for ECT imaging.

The strong interaction of electrons with matter also results in an anisotropic resolution effect. As the sample is tilted during imaging, the electron beam interacts with a relatively greater cross-sectional area at higher tilt angles. In practice, tilt angles greater than approximately 60–70° do not yield much information and are therefore not used. This results in a "missing wedge" of information in the final tomogram that decreases resolution parallel to the electron beam.

For structures that are present in multiple copies in one or multiple tomograms, higher resolution (even ≤1 nm) can be obtained by subtomogram averaging. Similar to single particle analysis, subtomogram averaging computationally combines images of identical objects to increase the signal-to-noise ratio.

A major obstacle in cryo-ET is identifying structures of interest within complicated cellular environments. One solution is to apply correlated cryo-fluorescence light microscopy, and even super-resolution light microscopy (e.g. cryo-PALM ), and cryo-ET. In these techniques, a sample containing a fluorescently-tagged protein of interest is plunge-frozen and first imaged in a light microscope equipped with a special stage to allow the sample to be kept at sub-crystallization temperatures (< −150 °C). The location of the fluorescent signal is identified and the sample is transferred to the CryoTEM, where the same location is then imaged at high resolution by cryo-ET.

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