{{Short description|Electron microscopy technique}} {{Copy edit|for=grammar and sentence structure|date=November 2025}}
[[File:Titan Krios University of Leeds.jpg|thumb|Titan Krios at the University of Leeds]]
'''Cryogenic electron microscopy''' ('''cryo-EM''') is a transmission electron microscopy technique applied to samples cooled to cryogenic temperatures. Developed in the 1970s, advances in detector technology and software allow biomolecular structures to be imaged at near-atomic resolution.<ref>{{cite journal | vauthors = Cheng Y, Grigorieff N, Penczek PA, Walz T | title = A primer to single-particle cryo-electron microscopy | journal = Cell | volume = 161 | issue = 3 | pages = 438–449 | date = April 2015 | pmid = 25910204 | pmc = 4409659 | doi = 10.1016/j.cell.2015.03.050 }}</ref> The approach has become a popular alternative to X-ray crystallography or NMR spectroscopy in structural biology.<ref name="Stoddart">{{cite journal | vauthors = Stoddart C |title=Structural biology: How proteins got their close-up |journal=Knowable Magazine |date=1 March 2022 |doi=10.1146/knowable-022822-1|s2cid=247206999 |doi-access=free |url=https://knowablemagazine.org/article/living-world/2022/structural-biology-how-proteins-got-their-closeup |access-date=25 March 2022|url-access=subscription }}</ref>
When scanning biological specimens, sample structure is preserved by embedding the specimens in vitreous ice. An aqueous sample solution is applied to a grid-mesh and plunge-frozen in liquid ethane or a mixture of liquid ethane and propane.<ref>{{cite journal | vauthors = Tivol WF, Briegel A, Jensen GJ | title = An improved cryogen for plunge freezing | journal = Microscopy and Microanalysis | volume = 14 | issue = 5 | pages = 375–379 | date = October 2008 | pmid = 18793481 | pmc = 3058946 | doi = 10.1017/S1431927608080781 | bibcode = 2008MiMic..14..375T }}</ref>
The 2017 Nobel Prize in Chemistry was awarded to Jacques Dubochet, Joachim Frank, and Richard Henderson "for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution."<ref name="Cressey 2017" /> ''Nature Methods'' also named cryo-EM as the "Method of the Year" in 2015.<ref>{{Cite journal| vauthors = Doerr A |date=January 2017|title=Cryo-electron tomography|journal=Nature Methods |volume=14 |issue=1 |page=34 |doi=10.1038/nmeth.4115 |s2cid=27162203 |issn=1548-7091}}</ref>
== History == === Early development === In the 1960s, transmission electron microscopy of biological samples was limited because of radiation damage from the high energy electron beams. Scientists hypothesized that examining specimens at low temperatures would reduce beam-induced radiation damage.<ref>{{cite journal | vauthors = Dubochet J, Knapek E | title = Ups and downs in early electron cryo-microscopy | journal = PLOS Biology | volume = 16 | issue = 4 | article-number = e2005550 | date = April 2018 | pmid = 29672565 | pmc = 5929567 | doi = 10.1371/journal.pbio.2005550 | doi-access = free }}</ref> Both liquid helium (−269 °C or 4 K or −452 °F) and liquid nitrogen (−195.79 °C or 77 K or −320 °F) were considered as cryogens,<ref>{{cite journal |vauthors=Venables JA |year=1963 |title=Liquid helium cooled tilting stage for an electron microscope |journal=Review of Scientific Instruments |volume=34 |issue=5 |pages=582–583}}</ref> however high stability was never achieved. In 1980, Erwin Knapek and Jacques Dubochet published comments on beam damage at cryogenic temperatures sharing observations that:
<blockquote>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.<ref>{{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 }}</ref></blockquote>
However, these results were not reproduced. Amendments were published two years later,<ref>{{cite journal |last1=Lepault |first1=J. |last2=Dubochet |first2=J. |last3=Dietrich |first3=I. |last4=Knapek |first4=E. |last5=Zeitler |first5=E. |title=Amendment to: Electron beam damage to organic specimens at liquid helium temperature |journal=Journal of Molecular Biology |date=January 1983 |volume=163 |issue=3 |page=511 |doi=10.1016/0022-2836(83)90073-6}}</ref> along with a commentary in ''Nature'',<ref name='Nat_LH'>{{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}}</ref> indicating that the beam resistance was less significant than anticipated. The protection gained at 4 K was closer to "tenfold for standard samples of L-valine" than what was previously stated.<ref name='Nat_LH'/> While cryo-EM samples are routinely collected at liquid nitrogen temperatures,<ref name='pnas_CR'>{{cite journal |last1=Dickerson |first1=JL |last2=Naydenova |first2=K |last3=Peet |first3=MJ |last4=Wilson |first4=H |last5=Nandy |first5=B |last6=McMullan |first6=G |last7=Morrison |first7=R |last8=Russo |first8=CJ |title=Reducing the effects of radiation damage in cryo-EM using liquid helium temperatures. |journal=Proceedings of the National Academy of Sciences of the United States of America |date=29 April 2025 |volume=122 |issue=17 |article-number=e2421538122 |doi=10.1073/pnas.2421538122 |pmid=40261934|pmc=12054821 }}</ref> work has continued to understand sample behavior at liquid helium temperatures.<ref>{{cite journal |last1=Pfeil-Gardiner |first1=Olivia |last2=Mills |first2=Deryck J. |last3=Vonck |first3=Janet |last4=Kuehlbrandt |first4=Werner |title=A comparative study of single-particle cryo-EM with liquid-nitrogen and liquid-helium cooling |journal=IUCrJ |date=1 November 2019 |volume=6 |issue=6 |pages=1099–1105 |doi=10.1107/S2052252519011503 |doi-access=free|pmc=6830223 }}</ref><ref>{{cite journal |last1=Dickerson |first1=Joshua L |last2=Russo |first2=Christopher J |title=A Physical Theory For Cryo-EM At Liquid-Helium Temperatures |journal=Microscopy and Microanalysis |date=25 July 2025 |volume=31 |issue=Supplement_1 |doi=10.1093/mam/ozaf048.514}}</ref><ref name='pnas_CR' />
In 1981 scientists at the European Molecular Biology Laboratory reported the first successful cryo-EM.<ref>{{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}}</ref> Researchers sprayed pure water 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 formed on the film was less than 1 μm thick and an electron diffraction pattern confirmed the presence of amorphous/vitreous ice. In 1984 the group demonstrated the power of cryo-EM in structural biology by analysing vitrified adenovirus type 2, T4 bacteriophage, Semliki Forest virus, Bacteriophage CbK, and Vesicular-Stomatitis-Virus.<ref>{{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 }}</ref> The paper marked the origin of Cryo-EM, and the technique has become routine in laboratories throughout the world.
The energy of the electrons used for imaging (80–300 kV) can break covalent bonds in organic and biological samples.<ref>{{Cite journal |last=Garman |first=Elspeth F. |last2=Owen |first2=Robin Leslie |date=2006-01-01 |title=Cryocooling and radiation damage in macromolecular crystallography |url=https://journals.iucr.org/paper?S0907444905034207 |journal=Acta Crystallographica Section D Biological Crystallography |volume=62 |issue=1 |pages=32–47 |doi=10.1107/S0907444905034207 |issn=0907-4449|doi-access=free }}</ref> Imaging biological specimens requires minimising electron exposure. Low exposures require images of thousands or millions of frozen molecules be selected, aligned, and averaged to obtain high-resolution maps, using specialized software. The 2012 introduction of direct electron detectors and better computational algorithms significantly improved structural features.<ref name="nat1">{{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}}</ref>
=== Recent advancements === Direct Electron Detectors, and more powerful imaging algorithms allow macromolecular structures to be determined at near-atomic resolution.<ref>{{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}}</ref> Imaged macromolecules include viruses, ribosomes, mitochondria, ion channels, and enzyme complexes. Starting in 2018, cryo-EM could be applied to structures as small as hemoglobin (64 kDa)<ref>{{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 |article-number=16099 |bibcode=2017NatCo...816099K |doi=10.1038/ncomms16099 |pmc=5497076 |pmid=28665412}}</ref> with resolutions up to 1.8 Å.<ref>{{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}}</ref> In 2019, cryo-EM structures grew to 2.5% of structures deposited in the Protein Data Bank.<ref>{{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}}</ref><ref>{{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}}</ref> Cryo-EM can be used for cryo-electron tomography (cryo-ET), creating 3D reconstructions of samples from tilted 2D images.
The 2010s saw drastic advancements of electron cameras, including to direct electron detectors, causing a "resolution revolution"<ref name=":3">{{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 }}</ref> pushing the resolution barrier beneath the crucial ~2-3 Å limit to resolve amino acid position and orientation.<ref>{{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 |article-number=e0123146 |doi=10.1371/journal.pone.0123146 |doi-access=free |issn=1932-6203 |pmc=4403875 |pmid=25894612|bibcode=2015PLoSO..1023146K }}</ref>
Richard 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).<ref name=":3" />
Recent advancements in protein-based imaging scaffolds assist with sample orientation bias and size limit. Though the minimum size for Cryo-EM remains undetermined, proteins smaller than ~50 kDa generally have too low a signal-to-noise ratio (SNR) to resolve protein particles, making 3D reconstruction difficult or impossible.<ref>{{cite journal |last1=Henderson |first1=Richard |title=The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules |journal=Quarterly Reviews of Biophysics |date=May 1995 |volume=28 |issue=2 |pages=171–193 |doi=10.1017/s003358350000305x}}</ref><ref>{{Cite journal |last1=Herzik |first1=Mark A. |last2=Wu |first2=Mengyu |last3=Lander |first3=Gabriel C. |date=2019-03-04 |title=High-resolution structure determination of sub-100 kDa complexes using conventional cryo-EM |journal=Nature Communications |language=en |volume=10 |issue=1 |page=1032 |doi=10.1038/s41467-019-08991-8 |issn=2041-1723 |pmc=6399227 |pmid=30833564|bibcode=2019NatCo..10.1032H }}</ref> Multiple techniques have been reported to improve SNR when determining the structures of small proteins.<ref>{{cite journal |last1=Wentinck |first1=Koen |last2=Gogou |first2=Christos |last3=Meijer |first3=Dimphna H. |title=Putting on molecular weight: Enabling cryo-EM structure determination of sub-100-kDa proteins |journal=Current Research in Structural Biology |date=2022 |volume=4 |pages=332–337 |doi=10.1016/j.crstbi.2022.09.005|pmc=9562432 }}</ref><ref>{{cite journal |last1=Gilman |first1=Morgan S. A. |last2=Skiba |first2=Meredith A. |title=Hidden gems bring proteins into view |journal=Nature Chemical Biology |date=15 August 2025 |doi=10.1038/s41589-025-01959-4}}</ref> Based on high-affinity DARPins, nanobodies, antibody fragments,<ref>{{Unbulleted list citebundle | {{cite journal |last1=Wu |first1=Shenping |last2=Avila-Sakar |first2=Agustin |last3=Kim |first3=JungMin |last4=Booth |first4=David S. |last5=Greenberg |first5=Charles H. |last6=Rossi |first6=Andrea |last7=Liao |first7=Maofu |last8=Li |first8=Xueming |last9=Alian |first9=Akram |last10=Griner |first10=Sarah L. |last11=Juge |first11=Narinobu |last12=Yu |first12=Yadong |last13=Mergel |first13=Claudia M. |last14=Chaparro-Riggers |first14=Javier |last15=Strop |first15=Pavel |last16=Tampé |first16=Robert |last17=Edwards |first17=Robert H. |last18=Stroud |first18=Robert M. |last19=Craik |first19=Charles S. |last20=Cheng |first20=Yifan |title=Fabs Enable Single Particle cryoEM Studies of Small Proteins |journal=Structure |date=April 2012 |volume=20 |issue=4 |pages=582–592 |doi=10.1016/j.str.2012.02.017|pmc=3322386 }}{{cite journal |last1=Mukherjee |first1=Somnath |last2=Erramilli |first2=Satchal K. |last3=Ammirati |first3=Mark |last4=Alvarez |first4=Frances J. D. |last5=Fennell |first5=Kimberly F. |last6=Purdy |first6=Michael D. |last7=Skrobek |first7=Blazej M. |last8=Radziwon |first8=Katarzyna |last9=Coukos |first9=John |last10=Kang |first10=Yanyong |last11=Dutka |first11=Przemysław |last12=Gao |first12=Xiang |last13=Qiu |first13=Xiayang |last14=Yeager |first14=Mark |last15=Eric Xu |first15=H. |last16=Han |first16=Seungil |last17=Kossiakoff |first17=Anthony A.|author17-link=Anthony Kossiakoff |title=Synthetic antibodies against BRIL as universal fiducial marks for single−particle cryoEM structure determination of membrane proteins |journal=Nature Communications |date=27 March 2020 |volume=11 |issue=1 |doi=10.1038/s41467-020-15363-0|pmc=7101349 }}{{cite journal |last1=Xie |first1=Pujun |last2=Li |first2=Yan |last3=Lamon |first3=Gaëlle |last4=Kuang |first4=Huihui |last5=Wang |first5=Da-Neng |last6=Traaseth |first6=Nathaniel J. |title=A fiducial-assisted strategy compatible with resolving small MFS transporter structures in multiple conformations using cryo-EM |journal=Nature Communications |date=2 January 2025 |volume=16 |issue=1 |doi=10.1038/s41467-024-54986-5|pmc=11695964 }}{{cite journal |display-authors=6 |vauthors=Castells-Graells R, Meador K, Arbing MA, Sawaya MR, Gee M, Cascio D, Gleave E, Debreczeni JÉ, Breed J, Leopold K, Patel A, Jahagirdar D, Lyons B, Subramaniam S, Phillips C, Yeates TO |date=September 2023 |title=Cryo-EM structure determination of small therapeutic protein targets at 3 Å-resolution using a rigid imaging scaffold |journal=Proceedings of the National Academy of Sciences of the United States of America |volume=120 |issue=37 |article-number=e2305494120 |doi=10.1073/pnas.2305494120 |pmc=10500258 |pmid=37669364|bibcode=2023PNAS..12005494C }}{{cite journal |last1=Ackle |first1=Fabian |last2=Thavarasah |first2=Sujani |last3=Earp |first3=Jennifer C. |last4=Seeger |first4=Markus A. |title=Rigid enlargement of sybodies with antibody fragments for cryo-EM analyses of small membrane proteins |journal=Scientific Reports |date=19 March 2025 |volume=15 |issue=1 |doi=10.1038/s41598-025-92950-5|pmc=11923154 }}{{cite journal |last1=Wu |first1=Xudong |last2=Rapoport |first2=Tom A. |title=Cryo-EM structure determination of small proteins by nanobody-binding scaffolds (Legobodies) |journal=Proceedings of the National Academy of Sciences |date=12 October 2021 |volume=118 |issue=41 |doi=10.1073/pnas.2115001118|pmc=8521671 }}{{cite journal |last1=Yi |first1=Gangshun |last2=Mamalis |first2=Dimitrios |last3=Ye |first3=Mingda |last4=Carrique |first4=Loic |last5=Fairhead |first5=Michael |last6=Li |first6=Huanyu |last7=Duerr |first7=Katharina L. |last8=Zhang |first8=Peijun |last9=Sauer |first9=David B. |last10=von Delft |first10=Frank |last11=Davis |first11=Benjamin G. |last12=Gilbert |first12=Robert J. C. |title=Covalently constrained 'Di-Gembodies' enable parallel structure solutions by cryo-EM |journal=Nature Chemical Biology |date=15 August 2025 |doi=10.1038/s41589-025-01972-7|doi-access=free }}{{cite journal |last1=Kung |first1=Jennifer E. |last2=Johnson |first2=Matthew C. |last3=Tegunov |first3=Dimitry |last4=Jao |first4=Christine C. |last5=Wu |first5=Ping |last6=Oh |first6=Angela |last7=Lin |first7=May |last8=Daria |first8=Jose M. |last9=Koth |first9=Christopher M. |last10=Arthur |first10=Christopher P. |last11=Rohou |first11=Alexis |last12=Sudhamsu |first12=Jawahar |title=Disulfide-constrained Fabs overcome target size limitation for high-resolution single particle cryoEM |journal=Nature Communications |date=30 September 2025 |volume=16 |issue=1 |doi=10.1038/s41467-025-63766-8|pmc=12484979 }} }}</ref> these methods rigidly bind the target protein and thereby increase the effective particle size and introduce symmetry to improve SNR for Cryo-EM map reconstruction. An advantage of Cryo-EM over crystallization is that it requires much less sample material. This makes it easier to determine structures of proteins that cannot be isolated with high yield.
=== 2017 Nobel Prize in Chemistry === 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."<ref name="Cressey 2017">{{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}}</ref>
== Techniques ==
=== Mode of electron microscopy === ==== Cryogenic transmission electron microscopy ==== {{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.
==== Correlative light cryo-TEM and cryo-ET ==== {{Main|Correlative light-electron microscopy}}
In 2019, correlative light cryo-TEM and cryo-ET were used to observe tunnelling nanotubes (TNTs) in neuronal cells.<ref>{{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 | page = 342 | date = January 2019 | pmid = 30664666 | pmc = 6341166 | doi = 10.1038/s41467-018-08178-7 | bibcode = 2019NatCo..10..342S }}</ref>
==== Scanning electron cryomicroscopy ==== {{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.
=== Technique of use and data analysis ===
==== Electron cryotomography ==== {{main|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.<ref>{{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 |article-number=167187 |doi=10.1016/j.jmb.2021.167187 |pmid=34384780 |issn=0022-2836|doi-access=free }}</ref>
==== Single particle analysis ==== {{main|Single particle analysis}}
[[File:Cryogenic electron microscopy workflow.svg|thumb|Single particle analysis workflow]] 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.<ref>{{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 }}</ref>
When combined with a knowledge of time progression, the result is time-resolved cyro-TEM.<ref>{{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}}</ref><ref>{{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}}</ref><ref>{{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}}</ref>
===== Comparisons to X-ray crystallography ===== {{main|X-ray crystallography}}
Traditionally, X-ray crystallography has been the most popular technique for determining the 3D structures of biological molecules.<ref>{{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 }}</ref> 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.<ref name=":4">{{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 |article-number=100560 |doi=10.1016/j.jbc.2021.100560 |doi-access=free |issn=0021-9258 |pmc=8050867 |pmid=33744287}}</ref> 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.<ref name=":4" />
The resolution of X-ray crystallography is limited by crystal homogeneity,<ref name=":1">{{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}}</ref> 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.<ref name=":0">{{cite journal |vauthors=Callaway E |date=February 2020 |title=Revolutionary cryo-EM is taking over structural biology |journal=Nature |volume=578 |issue=7794 |page=201 |bibcode=2020Natur.578..201C |doi=10.1038/d41586-020-00341-9 |pmid=32047310 |doi-access=free}}</ref> 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,<ref>{{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 }}</ref><ref name="Bhella 515–519">{{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 }}</ref> 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.<ref name=":2">{{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 }}</ref>
{{hatnote|1=1 nm = 10 Å}} 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,<ref name=":1" /> and the highest resolution achieved on record (as of September 30, 2022) is 0.48 Å.<ref>{{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 }}</ref> 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.<ref>{{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 }}</ref> However, as of 2020, the best cryo-EM resolution has been recorded at 1.22 Å,<ref name="Bhella 515–519" /> making it a competitor in resolution in some cases.
==== Electron crystallography ==== {{Main|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 NH<sub>4</sub>Cl crystals by W. E. Laschkarew and I. D. Usykin in 1933,<ref>{{Cite journal |last1=Laschkarew |first1=W. E. |last2=Usyskin |first2=I. D. |date=1933 |title=Die Bestimmung der Lage der Wasserstoffionen im NH4Cl-Kristallgitter durch Elektronenbeugung |url=http://link.springer.com/10.1007/BF01331003 |journal=Zeitschrift für Physik |language=de |volume=85 |issue=9–10 |pages=618–630 |bibcode=1933ZPhy...85..618L |doi=10.1007/BF01331003 |issn=1434-6001 |s2cid=123199621|url-access=subscription }}</ref>
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).<ref>{{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}}</ref> It is also very useful in studying small molecules, from peptides to simpler compounds.<ref name="Jones 2018">{{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 | doi-access = free}}</ref> == Specimen handling for imaging ==
(This section does not apply to electron crystallography.)
=== Biological specimens === {{More citations needed section|date=October 2025}}
==== 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,<ref>{{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}}</ref> and sulfur, which is prone to sublimation in the vacuum of electron microscopes, can be stabilized and imaged in CryoTEM.<ref>{{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}}</ref>
== 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.<ref>{{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}}</ref> There are two popular approaches that are widely used nowadays in cryo-EM image processing, the maximum likelihood approach that was discovered in 1998<ref>{{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}}</ref> and relatively recently adapted Bayesian approach.<ref name="Scheres 406–418">{{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}}</ref>
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.<ref name=":02">{{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 |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}}</ref> 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.<ref>{{Cite journal |last=Sigworth |first=Fred J. |date=2016-02-01 |title=Principles of cryo-EM single-particle image processing |journal=Microscopy |volume=65 |issue=1 |pages=57–67 |doi=10.1093/jmicro/dfv370 |issn=2050-5698 |pmc=4749045 |pmid=26705325}}</ref>
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.<ref name=":02" /> 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.<ref name="Scheres 406–418" />
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,<ref>{{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}}</ref><ref>{{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}}</ref> 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.<ref>{{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 |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}}</ref><ref>{{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 |article-number=e42166 |doi=10.7554/eLife.42166 |issn=2050-084X |pmc=6250425 |pmid=30412051 |doi-access=free}}</ref>
== Gallery == <gallery widths="200" heights="200"> 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<br />(scale bar represents 200 nm)<ref>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}}.</ref> </gallery>
== See also == {{Wikibooks|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
== References == {{reflist}} {{Electron microscopy}}
Category:Electron microscopy techniques Category:Cell biology Category:Protein structure Category:Scientific techniques