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Electron cryomicroscopy (cryo-EM or sometimes cryo-electron microscopy) 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.
The popularity of cryoelectron microscopy 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, in contrast to X-ray crystallography, which generally requires placing the samples in non-physiological environments, which arguably can lead to undesirable conformational changes.
The original rationale for cryoelectron microscopy 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 comparable to placing the sample about 20m away from a thermonuclear device. In addition, the high vacuum required on the column of an electron microscope makes the environment for the sample to be quite harsh.
The problem of the vacuum was partially solved by the introduction of negative stains such as uranium salts (uranyl acetate is perhaps the most common negative stain), but even with negative stains, biological samples are prone to structural collapse upon dehydration of the specimen. Embedding the samples in ice below the sublimation temperature was a possibility that was contemplated early on, but water tends to arrange into a crystalline lattice of lower density upon freezing and this tends to destroy the structure of anything that is embedded in it.
In the early 80's, several groups studying solid state physics were attempting to produce vitreous ice by different means, such as high pressure freezing or flash freezing. Theoretical calculations deemed this task impossible, but in a seminal paper in 1982, the group led by Jacques Dubochet at the European Molecular Biology Laboratory showed images of adenovirus embedded in a vitrified layer of water. This paper is generally considered to mark the birth of cryoelectron microscopy, and the technique has been developed to the point of becoming routine at several laboratories throughout the world.
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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Biological specimens
Thin film
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.
Vitreous sections
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.
See also
References
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This article includes a list of references, related reading or external links, but its sources remain unclear because it lacks inline citations. Please improve this article by introducing more precise citations where appropriate. (March 2008) |
- Frank, Joachim (2006). Three-Dimensional Electron Microscopy of Macromolecular Assemblies. New York: Oxford University Press. ISBN 0-19-518218-9.
External links
- The Fine Structure of a Frozen Virus - Sophisticated single-particle electron cryomicroscopy reveals unprecedented details in a virus's protein coat, Technology Review, March 19, 2008
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