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| title | chunk | source | category | tags | date_saved | instance |
|---|---|---|---|---|---|---|
| Cryogenic electron microscopy | 2/3 | https://en.wikipedia.org/wiki/Cryogenic_electron_microscopy | reference | science, encyclopedia | 2026-05-05T10:04:06.678811+00:00 | kb-cron |
Scanning electron cryomicroscopy (cryoSEM) is a scanning electron microscopy technique with a scanning electron microscope's cold stage in a cryogenic chamber.
=== Technique of use and data analysis ===
==== Electron cryotomography ====
In electron cryotomography (cyro-ET), many pictures of a sample are taken from different angles using a tilting mechanism. The images are combined to create a 3D model (map) of ~1–4 nm resolution.
==== Single particle analysis ====
SPA or single-particle cyro-EM is the method used to obtain near-atomic resolution (<1 nm) models of biomolecules. It is what the 2017 Nobel Prize refers to. In SPA, a large collection of cyro-TEM images are automatically sorted into classes. Within each class, the images are combined to reduce noise and to create a 3D model of the class of particles, a 3D "map". The main innovation compared to cyro-ET is the combination of images from similar objects. When combined with a knowledge of time progression, the result is time-resolved cyro-TEM.
===== Comparisons to X-ray crystallography =====
Traditionally, X-ray crystallography has been the most popular technique for determining the 3D structures of biological molecules. However, the aforementioned improvements in cryo-EM have increased its popularity as a tool for examining the details of biological molecules. Since 2010, yearly cryo-EM structure deposits have outpaced X-ray crystallography. Though X-ray crystallography has drastically more total deposits due to a decades-longer history, total deposits of the two methods are projected to eclipse around 2035. The resolution of X-ray crystallography is limited by crystal homogeneity, and coaxing biological molecules with unknown ideal crystallization conditions into a crystalline state can be very time-consuming, in extreme cases taking months or even years. To contrast, sample preparation in cryo-EM may require several rounds of screening and optimization to overcome issues such as protein aggregation and preferred orientations, but it does not require the sample to form a crystal, rather samples for cryo-EM are flash-frozen and examined in their near-native states.
According to Proteopedia, the median resolution achieved by X-ray crystallography (as of May 19, 2019) on the Protein Data Bank is 2.05 Å, and the highest resolution achieved on record (as of September 30, 2022) is 0.48 Å. As of 2020, the majority of the protein structures determined by cryo-EM (single particle analysis) are at a lower resolution of 3–4 Å. However, as of 2020, the best cryo-EM resolution has been recorded at 1.22 Å, making it a competitor in resolution in some cases.
==== Electron crystallography ====
Similar to X-ray crystallography used to determine the crystal structure of molecules of different sizes (from small molecules to large biomolecular complexes) using the X-ray diffraction pattern, electrons can also produce a electron diffraction pattern from a crystal. Work in this area has a long history dating back to early work such as the determination of the positions of hydrogen atoms in NH4Cl crystals by W. E. Laschkarew and I. D. Usykin in 1933, Cyro-EC is typically done with 3D crystals, but it has also been used in analysis of two-dimensional crystals and analysis of helical filaments or tubes. Microcrystal electron diffraction (MicroED) is a version of electron crystallography that works with crystals a billion times smaller than what X-ray diffraction requires. It has been used to determine the structure of large biomolecules (proteins, nucleic acids, their complexes). It is also very useful in studying small molecules, from peptides to simpler compounds.
== Specimen handling for imaging == (This section does not apply to electron crystallography.)
=== 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 radiosensitive, so they must be imaged with low-dose techniques (usefully, the low temperature of transmission electron cryomicroscopy 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 using the technique known as single particle analysis. This approach in general requires that the things being averaged are identical, although some limited conformational heterogeneity can now be studied (e.g. ribosome). Three-dimensional reconstructions from CryoTEM images of protein complexes and viruses have been solved to sub-nanometer or near-atomic resolution, allowing new insights into the structure and biology of these large assemblies. 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 (cryofixation) 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 transmission electron cryomicroscopy of vitreous sections (CEMOVIS) or transmission electron cryomicroscopy of frozen-hydrated sections.
=== Material specimens === In addition to allowing vitrified biological samples to be imaged, CryoTEM can also be used to image material specimens that are too volatile in vacuum to image using standard, room temperature electron microscopy. For example, vitrified sections of liquid-solid interfaces can be extracted for analysis by CryoTEM, and sulfur, which is prone to sublimation in the vacuum of electron microscopes, can be stabilized and imaged in CryoTEM.