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Concept
Transmission electron cryomicroscopy
Résumé
Transmission electron cryomicroscopy (CryoTEM), commonly known as cryo-EM, is a form of cryogenic electron microscopy, more specifically a type of transmission electron microscopy (TEM) where the sample is studied at cryogenic temperatures (generally liquid-nitrogen temperatures). Cryo-EM is gaining popularity in structural biology.
The utility of transmission electron cryomicroscopy stems from the fact that it allows the observation of specimens that have not been stained or fixed in any way, showing them in their native environment. This is in contrast to X-ray crystallography, which requires crystallizing the specimen, which can be difficult, and placing them in non-physiological environments, which can occasionally lead to functionally irrelevant conformational changes.
Advances in electron detector technology, particularly DED (Direct Electron Detectors) as well as more powerful software imaging algorithms have allowed for the determination of macromolecular structures at near-atomic resolution. Imaged macromolecules include viruses, ribosomes, mitochondria, ion channels, and enzyme complexes. Starting in 2018, cryo-EM could applied to structures as small as hemoglobin (64 kDa) and with resolutions up to 1.8 Å. In 2019, cryo-EM structures represented 2.5% of structures deposited in the Protein Data Bank, and this number continues to grow. An application of cryo-EM is cryo-electron tomography (cryo-ET), where a 3D reconstruction of the sample is created from tilted 2D images.
The original rationale for CryoTEM was as a means to fight radiation damage for biological specimens. The amount of radiation required to collect an image of a specimen in the electron microscope is high enough to be a potential source of specimen damage for delicate structures. In addition, the high vacuum required on the column of an electron microscope makes the environment for the sample quite harsh.
The problem of the vacuum was partially solved by the introduction of negative stains but even with negative stains biological samples are prone to structural collapse upon dehydration of the specimen.
Source officielle
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