Cryogenic electron microscopy

In the 1960s, the use of transmission electron microscopy for structure determination methods was limited because of the radiation damage due to high energy electron beams. Scientists hypothesized that examining specimens at low temperatures would reduce beam-induced radiation damage.{{cite journal | vauthors = Dubochet J, Knapek E | title = Ups and downs in early electron cryo-microscopy | journal = PLOS Biology | volume = 16 | issue = 4 | pages = e2005550 | date = April 2018 | pmid = 29672565 | pmc = 5929567 | doi = 10.1371/journal.pbio.2005550 | doi-access = free }} Both liquid helium (−269 °C or 4 K or −452.2 °F) and liquid nitrogen (−195.79 °C or 77 K or −320 °F) were considered as cryogens. In 1980, Erwin Knapek and Jacques Dubochet published comments on beam damage at cryogenic temperatures sharing observations that:

Thin crystals mounted on carbon film were found to be from 30 to 300 times more beam-resistant at 4 K than at room temperature... Most of our results can be explained by assuming that cryoprotection in the region of 4 K is strongly dependent on the temperature.{{cite journal | vauthors = Knapek E, Dubochet J | title = Beam damage to organic material is considerably reduced in cryo-electron microscopy | journal = Journal of Molecular Biology | volume = 141 | issue = 2 | pages = 147–161 | date = August 1980 | pmid = 7441748 | doi = 10.1016/0022-2836(80)90382-4 }}

However, these results were not reproducible and amendments were published in Nature just two years later informing that the beam resistance was less significant than initially anticipated. The protection gained at 4 K was closer to "tenfold for standard samples of L-valine",{{Cite journal|title=Cryo-transmission microscopy Fading hopes| vauthors = Newmark P |date=30 September 1982|journal=Nature|volume=299|issue=5882|pages=386–387|bibcode=1982Natur.299..386N|doi=10.1038/299386c0|doi-access=free}} than what was previously stated.

In 1981, Alasdair McDowall and Jacques Dubochet, scientists at the European Molecular Biology Laboratory, reported the first successful implementation of cryo-EM.{{Cite journal| vauthors = Dubochet J, McDowall AW |title=Vitrification of Pure Water for Electron Microscopy|date=December 1981|journal=Journal of Microscopy|language=en|volume=124|issue=3|pages=3–4|doi=10.1111/j.1365-2818.1981.tb02483.x|doi-access=free}} McDowall and Dubochet vitrified pure water in a thin film by spraying it onto a hydrophilic carbon film that was rapidly plunged into cryogen (liquid propane or liquid ethane cooled to 77 K). The thin layer of amorphous ice was less than 1 μm thick and an electron diffraction pattern confirmed the presence of amorphous/vitreous ice. In 1984, Dubochet's group demonstrated the power of cryo-EM in structural biology with analysis of vitrified adenovirus type 2, T4 bacteriophage, Semliki Forest virus, Bacteriophage CbK, and Vesicular-Stomatitis-Virus.{{cite journal | vauthors = Adrian M, Dubochet J, Lepault J, McDowall AW | title = Cryo-electron microscopy of viruses | journal = Nature | volume = 308 | issue = 5954 | pages = 32–36 | date = March 1984 | pmid = 6322001 | doi = 10.1038/308032a0 | s2cid = 4319199 | bibcode = 1984Natur.308...32A | url = https://serval.unil.ch/notice/serval:BIB_BEC796503260 }} 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, by far, high enough that covalent bonds of organic and biological samples can be broken in an inelastic scattering interaction. 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.{{cite journal |vauthors=Callaway E |date=September 2015 |title=The revolution will not be crystallized: a new method sweeps through structural biology |journal=Nature |volume=525 |issue=7568 |pages=172–4 |bibcode=2015Natur.525..172C |doi=10.1038/525172a |pmid=26354465 |doi-access=free}}

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.{{cite journal |vauthors=Murata K, Wolf M |date=Feb 2018 |title=Cryo-electron microscopy for structural analysis of dynamic biological macromolecules |journal=Biochimica et Biophysica Acta (BBA) - General Subjects |volume=1862 |issue=2 |pages=324–334 |doi=10.1016/j.bbagen.2017.07.020 |pmid=28756276 |doi-access=free}} 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){{cite journal |vauthors=Khoshouei M, Radjainia M, Baumeister W, Danev R |date=June 2017 |title=Cryo-EM structure of haemoglobin at 3.2 Å determined with the Volta phase plate |journal=Nature Communications |volume=8 |pages=16099 |bibcode=2017NatCo...816099K |doi=10.1038/ncomms16099 |pmc=5497076 |pmid=28665412}} and with resolutions up to 1.8 Å.{{cite journal |vauthors=Merk A, Bartesaghi A, Banerjee S, Falconieri V, Rao P, Davis MI, Pragani R, Boxer MB, Earl LA, Milne JL, Subramaniam S |date=June 2016 |title=Breaking Cryo-EM Resolution Barriers to Facilitate Drug Discovery |journal=Cell |volume=165 |issue=7 |pages=1698–1707 |doi=10.1016/j.cell.2016.05.040 |pmc=4931924 |pmid=27238019}} In 2019, cryo-EM structures represented 2.5% of structures deposited in the Protein Data Bank,{{Cite web |title=PDB Data Distribution by Experimental Method and Molecular Type |url=https://www.rcsb.org/stats/summary |access-date=2019-12-03 |website=www.rcsb.org}} and this number continues to grow.{{Cite web |title=PDB Statistics: Growth of Structures from 3DEM Experiments Released per Year |url=https://www.rcsb.org/stats/growth/em |access-date=2018-12-22 |website=www.rcsb.org}} 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 2010s were marked with drastic advancements of electron cameras. Notably, the improvements made to direct electron detectors have led to a "resolution revolution"{{Cite journal |last=Kühlbrandt |first=Werner |date=2014-03-28 |title=The Resolution Revolution |url=https://www.science.org/doi/10.1126/science.1251652 |journal=Science |language=en |volume=343 |issue=6178 |pages=1443–1444 |doi=10.1126/science.1251652 |pmid=24675944 |bibcode=2014Sci...343.1443K |s2cid=35524447 |issn=0036-8075|url-access=subscription }} pushing the resolution barrier beneath the crucial ~2-3 Å limit to resolve amino acid position and orientation.{{Cite journal |last1=Kuster |first1=Daniel J. |last2=Liu |first2=Chengyu |last3=Fang |first3=Zheng |last4=Ponder |first4=Jay W. |last5=Marshall |first5=Garland R. |date=2015-04-20 |title=High-Resolution Crystal Structures of Protein Helices Reconciled with Three-Centered Hydrogen Bonds and Multipole Electrostatics |journal=PLOS ONE |language=en |volume=10 |issue=4 |pages=e0123146 |doi=10.1371/journal.pone.0123146 |doi-access=free |issn=1932-6203 |pmc=4403875 |pmid=25894612|bibcode=2015PLoSO..1023146K }}

Henderson (MRC Laboratory of Molecular Biology, Cambridge, UK) formed a consortium with engineers at the Rutherford Appleton Laboratory and scientists at the Max Planck Society to fund and develop a first prototype. The consortium then joined forces with the electron microscope manufacturer FEI to roll out and market the new design. At about the same time, Gatan Inc. of Pleasanton, California came out with a similar detector designed by Peter Denes (Lawrence Berkeley National Laboratory) and David Agard (University of California, San Francisco). A third type of camera was developed by Nguyen-Huu Xuong at the Direct Electron company (San Diego, California).

In recognition of the impact cryo-EM has had on biochemistry, three scientists, Jacques Dubochet, Joachim Frank and Richard Henderson, were awarded the Nobel Prize in Chemistry "for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution."{{Cite web |title=The Nobel Prize in Chemistry 2017 |url=https://www.nobelprize.org/prizes/chemistry/2017/press-release/ |access-date=2022-09-30 |website=NobelPrize.org |language=en-US}}

{{main|X-ray crystallography}}

Traditionally, X-ray crystallography has been the most popular technique for determining the 3D structures of biological molecules.{{cite journal | vauthors = Smyth MS, Martin JH | title = x ray crystallography | journal = Molecular Pathology | volume = 53 | issue = 1 | pages = 8–14 | date = February 2000 | pmid = 10884915 | pmc = 1186895 | doi = 10.1136/mp.53.1.8 }} 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.{{Cite journal |last1=Chiu |first1=Wah |last2=Schmid |first2=Michael F. |last3=Pintilie |first3=Grigore D. |last4=Lawson |first4=Catherine L. |date=January 2021 |title=Evolution of standardization and dissemination of cryo-EM structures and data jointly by the community, PDB, and EMDB |journal=Journal of Biological Chemistry |volume=296 |pages=100560 |doi=10.1016/j.jbc.2021.100560 |doi-access=free |issn=0021-9258 |pmc=8050867 |pmid=33744287}} 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,{{Cite web|title=Resolution - Proteopedia, life in 3D|url=https://proteopedia.org/wiki/index.php/Resolution#Confusion_of_high_vs._low_resolution|access-date=2020-10-27|website=proteopedia.org}} 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.{{cite journal |vauthors=Callaway E |date=February 2020 |title=Revolutionary cryo-EM is taking over structural biology |journal=Nature |volume=578 |issue=7794 |pages=201 |bibcode=2020Natur.578..201C |doi=10.1038/d41586-020-00341-9 |pmid=32047310 |doi-access=free}} 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,{{Cite journal |last=Lyumkis |first=Dmitry |date=2019-03-29 |title=Challenges and opportunities in cryo-EM single-particle analysis |journal=Journal of Biological Chemistry |volume=294 |issue=13 |pages=5181–5197 |doi=10.1074/jbc.rev118.005602 |doi-access=free |issn=0021-9258 |pmc=6442032 |pmid=30804214 }}{{cite journal | vauthors = Nakane T, Kotecha A, Sente A, McMullan G, Masiulis S, Brown PM, Grigoras IT, Malinauskaite L, Malinauskas T, Miehling J, Uchański T, Yu L, Karia D, Pechnikova EV, de Jong E, Keizer J, Bischoff M, McCormack J, Tiemeijer P, Hardwick SW, Chirgadze DY, Murshudov G, Aricescu AR, Scheres SH | display-authors = 6 | title = Single-particle cryo-EM at atomic resolution | journal = Nature | volume = 587 | issue = 7832 | pages = 152–156 | date = November 2020 | pmid = 33087931 | pmc = 7611073 | doi = 10.1038/s41586-020-2829-0 | bibcode = 2020Natur.587..152N }} 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.{{cite journal | vauthors = Wang HW, Wang JW | title = How cryo-electron microscopy and X-ray crystallography complement each other | journal = Protein Science | volume = 26 | issue = 1 | pages = 32–39 | date = January 2017 | pmid = 27543495 | pmc = 5192981 | doi = 10.1002/pro.3022 }}

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.{{cite journal | vauthors = Schmidt A, Teeter M, Weckert E, Lamzin VS | title = Crystal structure of small protein crambin at 0.48 Å resolution | journal = Acta Crystallographica. Section F, Structural Biology and Crystallization Communications | volume = 67 | issue = Pt 4 | pages = 424–428 | date = April 2011 | pmid = 21505232 | pmc = 3080141 | doi = 10.1107/S1744309110052607 }} As of 2020, the majority of the protein structures determined by cryo-EM are at a lower resolution of 3–4 Å.{{cite journal | vauthors = Yip KM, Fischer N, Paknia E, Chari A, Stark H | title = Atomic-resolution protein structure determination by cryo-EM | journal = Nature | volume = 587 | issue = 7832 | pages = 157–161 | date = November 2020 | pmid = 33087927 | doi = 10.1038/s41586-020-2833-4 | s2cid = 224823207 | bibcode = 2020Natur.587..157Y }} 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.

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.

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.

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,{{cite journal |vauthors=Zachman MJ, Asenath-Smith E, Estroff LA, Kourkoutis LF |date=December 2016 |title=Site-Specific Preparation of Intact Solid-Liquid Interfaces by Label-Free In Situ Localization and Cryo-Focused Ion Beam Lift-Out |journal=Microscopy and Microanalysis |volume=22 |issue=6 |pages=1338–1349 |bibcode=2016MiMic..22.1338Z |doi=10.1017/S1431927616011892 |pmid=27869059 |doi-access=free}} and sulfur, which is prone to sublimation in the vacuum of electron microscopes, can be stabilized and imaged in CryoTEM.{{cite journal |vauthors=Levin BD, Zachman MJ, Werner JG, Sahore R, Nguyen KX, Han Y, Xie B, Ma L, Archer LA, Giannelis EP, Wiesner U, Kourkoutis LF, Muller DA |date=February 2017 |title=Characterization of Sulfur and Nanostructured Sulfur Battery Cathodes in Electron Microscopy Without Sublimation Artifacts |url=https://zenodo.org/record/889883 |journal=Microscopy and Microanalysis |volume=23 |issue=1 |pages=155–162 |bibcode=2017MiMic..23..155L |doi=10.1017/S1431927617000058 |pmid=28228169 |s2cid=6801783}}

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.{{Cite journal |last=Cheng |first=Yifan |date=2018-08-31 |title=Single-particle cryo-EM—How did it get here and where will it go |journal=Science |language=en |volume=361 |issue=6405 |pages=876–880 |bibcode=2018Sci...361..876C |doi=10.1126/science.aat4346 |issn=0036-8075 |pmc=6460916 |pmid=30166484}} There are two popular approaches that are widely used nowadays in cryo-EM image processing, the maximum likelihood approach that was discovered in 1998{{Cite journal |last=Sigworth |first=F.J. |date=1998 |title=A Maximum-Likelihood Approach to Single-Particle Image Refinement |journal=Journal of Structural Biology |language=en |volume=122 |issue=3 |pages=328–339 |doi=10.1006/jsbi.1998.4014 |pmid=9774537 |doi-access=free}} and relatively recently adapted Bayesian approach.{{Cite journal |last=Scheres |first=Sjors H.W. |date=January 2012 |title=A Bayesian View on Cryo-EM Structure Determination |journal=Journal of Molecular Biology |language=en |volume=415 |issue=2 |pages=406–418 |doi=10.1016/j.jmb.2011.11.010 |pmc=3314964 |pmid=22100448}}

The maximum likelihood estimation 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.{{Cite journal |last1=Nogales |first1=Eva |last2=Scheres |first2=Sjors H.W. |date=May 2015 |title=Cryo-EM: A Unique Tool for the Visualization of Macromolecular Complexity |url=http://dx.doi.org/10.1016/j.molcel.2015.02.019 |journal=Molecular Cell |volume=58 |issue=4 |pages=677–689 |doi=10.1016/j.molcel.2015.02.019 |issn=1097-2765 |pmc=4441764 |pmid=26000851}} 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.{{Cite journal |last=Sigworth |first=Fred J. |date=2016-02-01 |title=Principles of cryo-EM single-particle image processing |url=https://doi.org/10.1093/jmicro/dfv370 |journal=Microscopy |volume=65 |issue=1 |pages=57–67 |doi=10.1093/jmicro/dfv370 |issn=2050-5698 |pmc=4749045 |pmid=26705325}}

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 there is a fixed prior probability 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,{{Cite journal |last=Scheres |first=Sjors H. W. |date=2012-12-01 |title=RELION: Implementation of a Bayesian approach to cryo-EM structure determination |journal=Journal of Structural Biology |language=en |volume=180 |issue=3 |pages=519–530 |doi=10.1016/j.jsb.2012.09.006 |issn=1047-8477 |pmc=3690530 |pmid=23000701}}{{cite web |date=27 October 2023 |title=RELION: Image-processing software for cryo-electron microscopy |url=https://github.com/3dem/relion |access-date=27 October 2023 |website=GitHub |publisher=3dem}} 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 language and executes the usual tasks of 2D/3D model classifications or creating de novo models.{{Cite journal |last1=Bai |first1=Xiao-chen |last2=McMullan |first2=Greg |last3=Scheres |first3=Sjors H.W |date=January 2015 |title=How cryo-EM is revolutionizing structural biology |url=http://dx.doi.org/10.1016/j.tibs.2014.10.005 |journal=Trends in Biochemical Sciences |volume=40 |issue=1 |pages=49–57 |doi=10.1016/j.tibs.2014.10.005 |issn=0968-0004 |pmid=25544475 |s2cid=19727349|url-access=subscription }}{{Cite journal |last1=Zivanov |first1=Jasenko |last2=Nakane |first2=Takanori |last3=Forsberg |first3=Björn O |last4=Kimanius |first4=Dari |last5=Hagen |first5=Wim JH |last6=Lindahl |first6=Erik |last7=Scheres |first7=Sjors HW |date=2018-11-09 |editor-last=Egelman |editor-first=Edward H |editor2-last=Kuriyan |editor2-first=John |title=New tools for automated high-resolution cryo-EM structure determination in RELION-3 |journal=eLife |volume=7 |pages=e42166 |doi=10.7554/eLife.42166 |issn=2050-084X |pmc=6250425 |pmid=30412051 |doi-access=free}}

A variety of techniques can be used in CryoTEM.[http://pharmaxchange.info/press/2011/01/cryoelectron-microscopy/ Presentation on Cryoelectron Microscopy | PharmaXChange.info] Popular techniques include:

1. Single particle analysis (SPA)
2. Time-resolved CryoTEM{{cite journal |vauthors=Fu Z, Kaledhonkar S, Borg A, Sun M, Chen B, Grassucci RA, Ehrenberg M, Frank J |date=December 2016 |title=Key Intermediates in Ribosome Recycling Visualized by Time-Resolved Cryoelectron Microscopy |journal=Structure |volume=24 |issue=12 |pages=2092–2101 |doi=10.1016/j.str.2016.09.014 |pmc=5143168 |pmid=27818103}}{{cite journal |vauthors=Feng X, Fu Z, Kaledhonkar S, Jia Y, Shah B, Jin A, Liu Z, Sun M, Chen B, Grassucci RA, Ren Y, Jiang H, Frank J, Lin Q |date=April 2017 |title=A Fast and Effective Microfluidic Spraying-Plunging Method for High-Resolution Single-Particle Cryo-EM |journal=Structure |volume=25 |issue=4 |pages=663–670.e3 |doi=10.1016/j.str.2017.02.005 |pmc=5382802 |pmid=28286002}}{{cite journal |vauthors=Chen B, Kaledhonkar S, Sun M, Shen B, Lu Z, Barnard D, Lu TM, Gonzalez RL, Frank J |date=June 2015 |title=Structural dynamics of ribosome subunit association studied by mixing-spraying time-resolved cryogenic electron microscopy |journal=Structure |volume=23 |issue=6 |pages=1097–105 |doi=10.1016/j.str.2015.04.007 |pmc=4456197 |pmid=26004440}}
3. Electron cryotomography (cryoET)
4. Electron crystallography
5. Analysis of two-dimensional crystals
6. Analysis of helical filaments or tubes
7. Microcrystal Electron Diffraction (MicroED){{cite journal |vauthors=Shi D, Nannenga BL, Iadanza MG, Gonen T |date=November 2013 |title=Three-dimensional electron crystallography of protein microcrystals |journal=eLife |volume=2 |pages=e01345 |doi=10.7554/eLife.01345 |pmc=3831942 |pmid=24252878 |doi-access=free}}{{cite journal |vauthors=Nannenga BL, Shi D, Leslie AG, Gonen T |date=September 2014 |title=High-resolution structure determination by continuous-rotation data collection in MicroED |journal=Nature Methods |volume=11 |issue=9 |pages=927–930 |doi=10.1038/nmeth.3043 |pmc=4149488 |pmid=25086503}}{{cite journal |vauthors=Shi D, Nannenga BL, de la Cruz MJ, Liu J, Sawtelle S, Calero G, Reyes FE, Hattne J, Gonen T |date=May 2016 |title=The collection of MicroED data for macromolecular crystallography |journal=Nature Protocols |volume=11 |issue=5 |pages=895–904 |doi=10.1038/nprot.2016.046 |pmc=5357465 |pmid=27077331}}{{cite journal |vauthors=de la Cruz MJ, Hattne J, Shi D, Seidler P, Rodriguez J, Reyes FE, Sawaya MR, Cascio D, Weiss SC, Kim SK, Hinck CS, Hinck AP, Calero G, Eisenberg D, Gonen T |date=February 2017 |title=Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED |journal=Nature Methods |volume=14 |issue=4 |pages=399–402 |doi=10.1038/nmeth.4178 |pmc=5376236 |pmid=28192420}}

{{Main|Correlative light-electron microscopy}}

In 2019, correlative light cryo-TEM and cryo-ET were used to observe tunnelling nanotubes (TNTs) in neuronal cells.{{cite journal | vauthors = Sartori-Rupp A, Cordero Cervantes D, Pepe A, Gousset K, Delage E, Corroyer-Dulmont S, Schmitt C, Krijnse-Locker J, Zurzolo C | display-authors = 6 | title = Correlative cryo-electron microscopy reveals the structure of TNTs in neuronal cells | journal = Nature Communications | volume = 10 | issue = 1 | pages = 342 | date = January 2019 | pmid = 30664666 | pmc = 6341166 | doi = 10.1038/s41467-018-08178-7 | bibcode = 2019NatCo..10..342S }}

{{main|Scanning electron cryomicroscopy}}

Scanning electron cryomicroscopy (cryoSEM) is a scanning electron microscopy technique with a scanning electron microscope's cold stage in a cryogenic chamber.

{{main|Transmission electron cryomicroscopy}}

Cryogenic transmission electron microscopy (cryo-TEM) is a transmission electron microscopy technique that is used in structural biology and materials science. Colloquially, the term "cryogenic electron microscopy" or its shortening "cryo-EM" refers to cryogenic transmission electron microscopy by default, as the vast majority of cryo-EM is done in transmission electron microscopes, rather than scanning electron microscopes.

The Federal Institute of Technology, the University of Lausanne and the University of Geneva opened the Dubochet Center For Imaging (DCI) at the end of November 2021, for the purposes of applying and further developing cryo-EM.{{Cite web|title=Inauguration of the Dubochet Center for Imaging (DCI) on the campuses of UNIGE, UNIL and EPFL|url=https://biologie.unige.ch/en/2021/11/inauguration-of-the-dubochet-center-for-imaging-dci-on-the-campuses-of-unige-unil-and-epfl/|date=2021-11-30|access-date=2022-04-30|website=unige.ch}} Less than a month after the first identification of the SARS-CoV-2 Omicron variant, researchers at the DCI were able to define its structure, identify the crucial mutations to circumvent individual vaccines and provide insights for new therapeutic approaches.{{Cite web|title=Scientists uncover Omicron variant mysteries using microscopes|url=https://www.swissinfo.ch/eng/scientists-uncover-omicron-variant-mysteries-using-microscopes/47228078|date=2021-12-30|access-date=2022-04-30|website=swissinfo.ch}}

The Danish National cryo-EM Facility also known as [https://embion.au.dk EMBION] was inaugurated on December 1, 2016. EMBION is a cryo-EM consortium between Danish Universities (Aarhus University host and University of Copenhagen co-host).

File:Cryogenic electron microscopy workflow.svg workflow]]

• Cryogenic electron tomography (cryo-ET), a specialized application where many images are taken of individual samples at various tilt angles, resulting in a 3D reconstruction of a single sample.{{Cite journal |last1=Bäuerlein |first1=Felix J. B. |last2=Baumeister |first2=Wolfgang |date=2021-10-01 |title=Towards Visual Proteomics at High Resolution |journal=Journal of Molecular Biology |series=From Protein Sequence to Structure at Warp Speed: How Alphafold Impacts Biology |volume=433 |issue=20 |pages=167187 |doi=10.1016/j.jmb.2021.167187 |pmid=34384780 |issn=0022-2836|doi-access=free }}
• Electron crystallography, method to determine the arrangement of atoms in solids using a TEM
• MicroED,{{cite journal | vauthors = Nannenga BL, Shi D, Leslie AG, Gonen T | title = High-resolution structure determination by continuous-rotation data collection in MicroED | journal = Nature Methods | volume = 11 | issue = 9 | pages = 927–930 | date = September 2014 | pmid = 25086503 | pmc = 4149488 | doi = 10.1038/nmeth.3043 }} method to determine the structure of proteins, peptides, organic molecules, and inorganic compounds using electron diffraction from 3D crystals{{cite journal | vauthors = Jones CG, Martynowycz MW, Hattne J, Fulton TJ, Stoltz BM, Rodriguez JA, Nelson HM, Gonen T | display-authors = 6 | title = The CryoEM Method MicroED as a Powerful Tool for Small Molecule Structure Determination | journal = ACS Central Science | volume = 4 | issue = 11 | pages = 1587–1592 | date = November 2018 | pmid = 30555912 | pmc = 6276044 | doi = 10.1021/acscentsci.8b00760 }}{{cite journal | vauthors = de la Cruz MJ, Hattne J, Shi D, Seidler P, Rodriguez J, Reyes FE, Sawaya MR, Cascio D, Weiss SC, Kim SK, Hinck CS, Hinck AP, Calero G, Eisenberg D, Gonen T | display-authors = 6 | title = Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED | journal = Nature Methods | volume = 14 | issue = 4 | pages = 399–402 | date = February 2017 | pmid = 28192420 | pmc = 5376236 | doi = 10.1038/nmeth.4178 }}{{cite journal | vauthors = Gruene T, Wennmacher JT, Zaubitzer C, Holstein JJ, Heidler J, Fecteau-Lefebvre A, De Carlo S, Müller E, Goldie KN, Regeni I, Li T, Santiso-Quinones G, Steinfeld G, Handschin S, van Genderen E, van Bokhoven JA, Clever GH, Pantelic R | display-authors = 6 | title = Rapid Structure Determination of Microcrystalline Molecular Compounds Using Electron Diffraction | journal = Angewandte Chemie | volume = 57 | issue = 50 | pages = 16313–16317 | date = December 2018 | pmid = 30325568 | pmc = 6468266 | doi = 10.1002/anie.201811318 }}
• Single particle analysis cryo-EM, an averaging method to determine protein structure from monodisperse samples.{{cite journal | vauthors = Cheng Y | title = Single-particle cryo-EM-How did it get here and where will it go | journal = Science | volume = 361 | issue = 6405 | pages = 876–880 | date = August 2018 | pmid = 30166484 | pmc = 6460916 | doi = 10.1126/science.aat4346 | bibcode = 2018Sci...361..876C }}

File:Cryoem groel.jpg|Cryo-EM image of GroEL suspended in amorphous ice at {{val|50000}}× magnification

File:Structure-of-Alcohol-Oxidase-from-Pichia-pastoris-by-Cryo-Electron-Microscopy-pone.0159476.s006.ogv|Structure of alcohol oxidase from Pichia pastoris by Cryo-EM

File:25K15pA9Def4sec Arman 4 Box1.png|Cryo-EM image of an intact ARMAN cell from an Iron Mountain biofilm. Image width is 576 nm.

File:CroV TEM (cropped).jpg|Cryo-EM image of the CroV giant marine virus
(scale bar represents 200 nm)Xiao, C., Fischer, M.G., Bolotaulo, D.M., Ulloa-Rondeau, N., Avila, G.A., and Suttle, C.A. (2017) "Cryo-EM reconstruction of the Cafeteria roenbergensis virus capsid suggests novel assembly pathway for giant viruses". Scientific Reports, 7: 5484. {{doi|10.1038/s41598-017-05824-w}}.

{{Wikibook|Software Tools For Molecular Microscopy}}

• Cryogenic scanning electron microscopy
• EM Data Bank
• Resolution (electron density)
• Single particle analysis
• Cryofixation
• Cryo bio-crystallography
• Electron tomography (ET)
• Virus crystallisation

{{reflist}}

{{Electron microscopy}}

Category:Electron microscopy techniques

Category:Cell biology

Category:Protein structure

Category:Scientific techniques