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Electron cryomicroscopy (More often called cryo-EM or sometimes cryo-electron microscopy, although it is the microscope and specimen stage and not the electrons that are cold) is a form of electron microscopy (EM) where the sample is studied at cryogenic temperatures (generally liquid nitrogen temperatures). CryoEM is developing popularity in structural biology.
A version of electron cryomicroscopy is cryo-electron tomography (CET) where a 3D reconstruction of a sample is created from tilted 2D images, again at cryogenic temperatures (either liquid nitrogen or helium).
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The biological material is spread on an electron microscopy grid and is preserved in a frozen-hydrated state by rapid freezing, usually in liquid ethane near liquid nitrogen temperature. By maintaining specimens at liquid nitrogen temperature or colder, they can be introduced into the high-vacuum of the electron microscope column. Most biological specimens are extremely radiation sensitive, so they must be imaged with low-dose techniques (usefully, the low temperature of cryo-electron microscopy provides an additional protective factor against radiation damage).
Consequently, the images are extremely noisy. For some biological systems it is possible to average images to increase the signal to noise ratio and retrieve high-resolution information about the specimen. This approach requires that the things being averaged are identical (e.g. ribosome particles). Analysis of ordered arrays of protein, such as 2-D crystals of transmembrane proteins or helical arrays of proteins, also allows a kind of averaging which can provide high-resolution information about the specimen. This technique is called electron crystallography.
The thin film method is limited to thin specimens (typically < 500 nm) because the electrons cannot cross thicker samples without multiple scattering events. Thicker specimens can be vitrified by plunge freezing in ethane (up to tens of μm in thickness) or more commonly by high pressure freezing (up to hundreds of μm). They can then be cut in thin sections (40 to 200 nm thick) with a diamond knife in a cryoultramicrotome at temperatures lower than -135 °C (devitrification temperature). The sections are collected on an electron microscope grid and are imaged in the same manner as specimen vitrified in thin film. This technique is called cryo-electron microscopy of vitreous sections (CEMOVIS) or cryo-electron microscopy of frozen-hydrated sections.
|This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "Cryo-electron_microscopy". A list of authors is available in Wikipedia.|