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Concept
Cryotomographie électronique
Résumé
Electron cryotomography (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.
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.
Source officielle
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