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Transmission electron cryomicroscopy (CryoTEM), commonly known as cryo-EM, is a form of
cryogenic electron microscopy Cryogenic electron microscopy (cryo-EM) is a cryomicroscopy technique applied on samples cooled to cryogenic temperatures. For biological specimens, the structure is preserved by embedding in an environment of vitreous ice. An aqueous sample so ...
, more specifically a type of
transmission electron microscopy Transmission electron microscopy (TEM) is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a g ...
(TEM) where the sample is studied at
cryogenic In physics, cryogenics is the production and behaviour of materials at very low temperatures. The 13th IIR International Congress of Refrigeration (held in Washington DC in 1971) endorsed a universal definition of “cryogenics” and “cr ...
temperatures (generally liquid-nitrogen temperatures). Cryo-EM is gaining popularity in
structural biology Structural biology is a field that is many centuries old which, and as defined by the Journal of Structural Biology, deals with structural analysis of living material (formed, composed of, and/or maintained and refined by living cells) at every le ...
. 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 X-ray crystallography is the experimental science determining the atomic and molecular structure of a crystal, in which the crystalline structure causes a beam of incident X-rays to diffract into many specific directions. By measuring the angles ...
, 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
virus A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria and archaea. Since Dmitri Ivanovsky's 1 ...
es,
ribosome Ribosomes ( ) are macromolecular machines, found within all cells, that perform biological protein synthesis (mRNA translation). Ribosomes link amino acids together in the order specified by the codons of messenger RNA (mRNA) molecules to ...
s,
mitochondria A mitochondrion (; ) is an organelle found in the Cell (biology), cells of most Eukaryotes, such as animals, plants and Fungus, fungi. Mitochondria have a double lipid bilayer, membrane structure and use aerobic respiration to generate adenosi ...
,
ion channel Ion channels are pore-forming membrane proteins that allow ions to pass through the channel pore. Their functions include establishing a resting membrane potential, shaping action potentials and other electrical signals by gating the flow of io ...
s, and
enzyme Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...
complexes. Starting in 2018, cryo-EM could applied to structures as small as
hemoglobin Hemoglobin (haemoglobin BrE) (from the Greek word αἷμα, ''haîma'' 'blood' + Latin ''globus'' 'ball, sphere' + ''-in'') (), abbreviated Hb or Hgb, is the iron-containing oxygen-transport metalloprotein present in red blood cells (erythrocyte ...
(64
kDa The dalton or unified atomic mass unit (symbols: Da or u) is a non-SI unit of mass widely used in physics and chemistry. It is defined as of the mass of an unbound neutral atom of carbon-12 in its nuclear and electronic ground state and at ...
) and with resolutions up to 1.8 Å. In 2019, cryo-EM structures represented 2.5% of structures deposited in the
Protein Data Bank The Protein Data Bank (PDB) is a database for the three-dimensional structural data of large biological molecules, such as proteins and nucleic acids. The data, typically obtained by X-ray crystallography, NMR spectroscopy, or, increasingly, cry ...
, and this number continues to grow. An application of cryo-EM is
cryo-electron tomography Electron cryotomography (CryoET) 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. CryoET is a specialized application of t ...
(cryo-ET), where a 3D reconstruction of the sample is created from tilted 2D images.


Development

The original rationale for CryoTEM was as a means to fight
radiation damage Radiation damage is the effect of ionizing radiation on physical objects including non-living structural materials. It can be either detrimental or beneficial for materials. Radiobiology is the study of the action of ionizing radiation on living ...
for biological specimens. The amount of radiation required to collect an image of a specimen in the
electron microscope An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons, electron microscopes have a hi ...
is high enough to be a potential source of specimen damage for delicate structures. In addition, the
high vacuum ''High Vacuum'' is a science fiction novel by Charles Eric Maine. It was first published in 1957 by Hodder & Stoughton. Synopsis The first crewed Moon ship, ''Alpha'', runs out of fuel just before landing in the Mare Imbrium and crashes, killin ...
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 stain In microscopy, negative staining is an established method, often used in diagnostic microscopy, for contrasting a thin specimen with an optically opaque fluid. In this technique, the background is stained, leaving the actual specimen untouched, an ...
s but even with negative stains biological samples are prone to structural collapse upon
dehydration In physiology, dehydration is a lack of total body water, with an accompanying disruption of metabolic processes. It occurs when free water loss exceeds free water intake, usually due to exercise, disease, or high environmental temperature. Mil ...
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 can destroy the structure of anything that is embedded in it. In the early 1980s, several groups studying solid state physics were attempting to produce
vitreous ice Amorphous ice (non-crystalline or "vitreous" ice) is an amorphous solid form of water. Common ice is a crystalline material wherein the molecules are regularly arranged in a hexagonal lattice, whereas amorphous ice has a lack of long-range order ...
by different means, such as high pressure freezing or flash freezing. In a seminal paper in 1984, the group led by
Jacques Dubochet Jacques Dubochet (born 8 June 1942) is a retired Swiss biophysicist. He is a former researcher at the European Molecular Biology Laboratory in Heidelberg, Germany, and an honorary professor of biophysics at the University of Lausanne in Switzerl ...
at the
European Molecular Biology Laboratory The European Molecular Biology Laboratory (EMBL) is an intergovernmental organization dedicated to molecular biology research and is supported by 27 member states, two prospect states, and one associate member state. EMBL was created in 1974 and ...
showed images of
adenovirus Adenoviruses (members of the family ''Adenoviridae'') are medium-sized (90–100 nm), nonenveloped (without an outer lipid bilayer) viruses with an icosahedral nucleocapsid containing a double-stranded DNA genome. Their name derives from the ...
embedded in a vitrified layer of water. This paper is generally considered to mark the origin of Cryo-EM, and the technique has been developed to the point of becoming routine at numerous laboratories throughout the world. The energy of the electrons used for imaging (80–300 kV) is high enough that
covalent bond A covalent bond is a chemical bond that involves the sharing of electrons to form electron pairs between atoms. These electron pairs are known as shared pairs or bonding pairs. The stable balance of attractive and repulsive forces between atoms ...
s can be broken. When imaging specimens are vulnerable to radiation damage, it is necessary to limit the electron exposure used to acquire the image. These low exposures require that the images of thousands or even millions of identical frozen molecules be selected, aligned, and averaged to obtain high-resolution maps, using specialized software. A significant improvement in structural features was achieved in 2012 by the introduction of direct electron detectors and better computational algorithms. In 2015,
Bridget Carragher Bridget Olivia Carragher (born 17 June 1957) is a South African physicist specializing in electron microscopy. She is an adjunct professor at the Columbia University (New York City, NY), and alongside Clint Potter, is also the director of the Nati ...
and colleagues at the Scripps National Resource for Automated Molecular Microscopy used techniques she and
Clint Potter Clint is both a given name and a surname. Notable people with the name include: Given name: *Clint Alberta (1970–2002), Canadian filmmaker *Clint Albright (1926–1999), Canadian ice hockey player *Clint Alfino (born 1968), South African basebal ...
developed to determine the first cryo-EM structure with a resolution finer than 3 Å, thereby elevating CryoTEM as a tool comparable to and potentially superior to traditional x-ray crystallography techniques. Since then, higher resolutions have been achieved, including a 2.2 Å structure of bacterial enzyme
β-galactosidase β-Galactosidase (EC 3.2.1.23, lactase, beta-gal or β-gal; systematic name β-D-galactoside galactohydrolase), is a glycoside hydrolase enzyme that catalyst, catalyzes hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosi ...
in 2015 and a 1.8 Å structure of
glutamate dehydrogenase Glutamate dehydrogenase (GLDH, GDH) is an enzyme observed in both prokaryotes and eukaryotic mitochondria. The aforementioned reaction also yields ammonia, which in eukaryotes is canonically processed as a substrate in the urea cycle. Typical ...
in 2016. Cryo-EM has also been used to determine the structure of various viruses, including the
Zika virus ''Zika virus'' (ZIKV; pronounced or ) is a member of the virus family ''Flaviviridae''. It is spread by daytime-active ''Aedes'' mosquitoes, such as '' A. aegypti'' and '' A. albopictus''. Its name comes from the Ziika Forest of Uganda, whe ...
, and has been applied to large complexes such as the
spliceosome A spliceosome is a large ribonucleoprotein (RNP) complex found primarily within the nucleus of eukaryotic cells. The spliceosome is assembled from small nuclear RNAs (snRNA) and numerous proteins. Small nuclear RNA (snRNA) molecules bind to specifi ...
. In 2017, the
Nobel Prize in Chemistry ) , image = Nobel Prize.png , alt = A golden medallion with an embossed image of a bearded man facing left in profile. To the left of the man is the text "ALFR•" then "NOBEL", and on the right, the text (smaller) "NAT•" then "M ...
was awarded jointly to
Jacques Dubochet Jacques Dubochet (born 8 June 1942) is a retired Swiss biophysicist. He is a former researcher at the European Molecular Biology Laboratory in Heidelberg, Germany, and an honorary professor of biophysics at the University of Lausanne in Switzerl ...
,
Joachim Frank Joachim Frank () (born September 12, 1940) is a German-American biophysicist at Columbia University and a Nobel laureate. He is regarded as the founder of single-particle cryo-electron microscopy (cryo-EM), for which he shared the Nobel Prize i ...
and Richard Henderson, "for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution".


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 Ethane ( , ) is an organic chemical compound with chemical formula . At standard temperature and pressure, ethane is a colorless, odorless gas. Like many hydrocarbons, ethane is isolated on an industrial scale from natural gas and as a petr ...
near
liquid nitrogen Liquid nitrogen—LN2—is nitrogen in a liquid state at low temperature. Liquid nitrogen has a boiling point of about . It is produced industrially by fractional distillation of liquid air. It is a colorless, low viscosity liquid that is wide ...
temperature. By maintaining specimens at liquid nitrogen temperature or colder, they can be introduced into the high-
vacuum A vacuum is a space devoid of matter. The word is derived from the Latin adjective ''vacuus'' for "vacant" or "void". An approximation to such vacuum is a region with a gaseous pressure much less than atmospheric pressure. Physicists often dis ...
of the
electron microscope An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons, electron microscopes have a hi ...
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 Single particle analysis is a group of related computerized image processing techniques used to analyze images from transmission electron microscopy (TEM). These methods were developed to improve and extend the information obtainable from TEM imag ...
. This approach in general requires that the things being averaged are identical, although some limited conformational heterogeneity can now be studied (e.g.
ribosome Ribosomes ( ) are macromolecular machines, found within all cells, that perform biological protein synthesis (mRNA translation). Ribosomes link amino acids together in the order specified by the codons of messenger RNA (mRNA) molecules to ...
). Three-dimensional reconstructions from CryoTEM images of protein complexes and
virus A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria and archaea. Since Dmitri Ivanovsky's 1 ...
es 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 A crystal or crystalline solid is a solid material whose constituents (such as atoms, molecules, or ions) are arranged in a highly ordered microscopic structure, forming a crystal lattice that extends in all directions. In addition, macrosc ...
of
transmembrane proteins A transmembrane protein (TP) is a type of integral membrane protein that spans the entirety of the cell membrane. Many transmembrane proteins function as gateways to permit the transport of specific substances across the membrane. They frequentl ...
or
helical Helical may refer to: * Helix, the mathematical concept for the shape * Helical engine, a proposed spacecraft propulsion drive * Helical spring, a coilspring * Helical plc, a British property company, once a maker of steel bar stock * Helicoil A t ...
arrays of proteins, also allows a kind of averaging which can provide high-resolution information about the specimen. This technique is called
electron crystallography Electron crystallography is a method to determine the arrangement of atoms in solids using a transmission electron microscope (TEM). Comparison with X-ray crystallography It can complement X-ray crystallography for studies of very small crystals ...
.


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 Cryofixation is a technique for fixation or stabilisation of biological materials as the first step in specimen preparation for electron microscopy and cryo-electron microscopy. Typical specimens for cryofixation include small samples of plant or an ...
) 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.


Image Processing in Cryo-TEM

Even though in the majority of approaches in electron microscopy one tries to get the best resolution image of the material, it is not always the case in cryo-TEM. Besides all the benefits of high resolution images, the signal to noise ratio remains the main hurdle that prevents assigning orientation to each particle. For example, in macromolecule complexes, there are several different structures that are being projected from 3D to 2D during imaging and if they are not distinguished the result of image processing will be a blur. That is why the probabilistic approaches become more powerful in this type of investigation. There are two popular approaches that are widely used nowadays in cryo-EM image processing, the maximum likelihood approach that was discovered in 1998 and relatively recently adapted Bayesian approach. The
maximum likelihood estimation In statistics, maximum likelihood estimation (MLE) is a method of estimating the parameters of an assumed probability distribution, given some observed data. This is achieved by maximizing a likelihood function so that, under the assumed statis ...
approach comes to this field from the statistics. Here, all the possible orientations of particles are summed up to get the resulting probability distribution. We can compare this to a typical least square estimation where particles get exact orientations per image. This way, the particles in the sample get "fuzzy" orientations after calculations, weighted by corresponding probabilities. The whole process is iterative and with each next iteration the model gets better. The good conditions for making the model that closely represent the real structure is when the data does not have too much noise and the particles do not have any preferential direction. The main downside of maximum likelihood approach is that the result depends on the initial guess and model optimization can sometimes get stuck at local minimum. The Bayesian approach that is now being used in cryo-TEM is empirical by nature. This means that the distribution of particles is based on the original dataset. Similarly, in the usual
Bayesian method Bayesian inference is a method of statistical inference in which Bayes' theorem is used to update the probability for a hypothesis as more evidence or information becomes available. Bayesian inference is an important technique in statistics, and ...
there is a fixed
prior probability In Bayesian statistical inference, a prior probability distribution, often simply called the prior, of an uncertain quantity is the probability distribution that would express one's beliefs about this quantity before some evidence is taken into ...
that is changed after the data is observed. The main difference from the maximum likelihood estimation lies in special reconstruction term that helps smoothing the resulting maps while also decreasing the noise during reconstruction. The smoothing of the maps occurs through assuming prior probability to be a Gaussian distribution and analyzing the data in the Fourier space. Since the connection between the prior knowledge and the dataset is established, there is less chance for human factor errors which potentially increases the objectivity of image reconstruction. With emerging new methods of cryo-TEM imaging and image reconstruction the new software solutions appear that help to automate the process. After the empirical Bayesian approach have been implemented in the open source computer program RELION (REgularized LIkelihood OptimizatioN) for 3D reconstruction, the program became widespread in the cryo-TEM field. It offers a range of corrections that improve the resolution of reconstructed images, allows implementing versatile scripts using
python Python may refer to: Snakes * Pythonidae, a family of nonvenomous snakes found in Africa, Asia, and Australia ** ''Python'' (genus), a genus of Pythonidae found in Africa and Asia * Python (mythology), a mythical serpent Computing * Python (pro ...
language and executes the usual tasks of 2D/3D model classifications or creating '' de novo'' models.


Techniques

A variety of techniques can be used in CryoTEM. Popular techniques include: #
Electron crystallography Electron crystallography is a method to determine the arrangement of atoms in solids using a transmission electron microscope (TEM). Comparison with X-ray crystallography It can complement X-ray crystallography for studies of very small crystals ...
## Analysis of two-dimensional crystals ## Analysis of helical filaments or tubes ##
Microcrystal Electron Diffraction Microcrystal electron diffraction, or MicroED, is a CryoEM method that was developed by the Gonen laboratory in late 2013 at the Janelia Research Campus of the Howard Hughes Medical Institute. MicroED is a form of electron crystallography where t ...
(MicroED) #
Single particle analysis Single particle analysis is a group of related computerized image processing techniques used to analyze images from transmission electron microscopy (TEM). These methods were developed to improve and extend the information obtainable from TEM imag ...
(SPA) ##Time-resolved CryoTEM #
Electron cryotomography Electron cryotomography (CryoET) 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. CryoET is a specialized application of tra ...
(cryoET)


See also

*
Cryogenic scanning electron microscopy Scanning electron cryomicroscopy (CryoSEM) is a form of electron microscopy where a hydrated but cryogenically fixed sample is imaged on a scanning electron microscope's cold stage in a cryogenic chamber. The cooling is usually achieved with liquid ...
*
EM Data Bank The EM Data Bank or Electron Microscopy Data Bank (EMDB) collects 3D EM maps and associated experimental data determined using electron microscopy of biological specimens. It was established in 2002 at the MSD/PDBe group of the European Bioinformat ...
*
Resolution (electron density) Resolution in terms of electron density is a measure of the resolvability in the electron density map of a molecule. In X-ray crystallography, resolution is the highest resolvable peak in the diffraction pattern, while resolution in cryo-electron m ...
*
Single particle analysis Single particle analysis is a group of related computerized image processing techniques used to analyze images from transmission electron microscopy (TEM). These methods were developed to improve and extend the information obtainable from TEM imag ...


References


Further reading

* *


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
Getting Started in Cryo-EM
– Online course from Caltech, Professor Grant Jensen


EMstats
Trends and distributions of maps in EMDB, e.g. resolution trends {{Portal bar, Science Cell biology Protein structure Electron microscopy