Cryogenic electron microscopy

Short description: Electron microscopy technique

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File:Titan Krios University of Leeds.jpgTitan 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.[1] The approach has become a popular alternative to X-ray crystallography or NMR spectroscopy in structural biology.[2]

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.[3]

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."[4] Nature Methods also named cryo-EM as the "Method of the Year" in 2015.[5]

## Contents

* 1 History
* 1.1 Early development
* 1.2 Recent advancements
* 1.3 2017 Nobel Prize in Chemistry
* 2 Techniques
* 2.1 Mode of electron microscopy
* 2.1.1 Cryogenic transmission electron microscopy
* 2.1.2 Correlative light cryo-TEM and cryo-ET
* 2.1.3 Scanning electron cryomicroscopy
* 2.2 Technique of use and data analysis
* 2.2.1 Electron cryotomography
* 2.2.2 Single particle analysis
* 2.2.2.1 Comparisons to X-ray crystallography
* 2.2.3 Electron crystallography
* 3 Specimen handling for imaging
* 3.1 Biological specimens
* 3.1.1 Thin film
* 3.1.2 Vitreous sections
* 3.2 Material specimens
* 4 Image processing in cryo-TEM
* 5 Gallery
* 6 See also
* 7 References

## 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.[6] 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,[7] however high stability was never achieved. 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.[8]

However, these results were not reproduced and just two years later amendments were published,[9] along with a commentary in Nature,[10] 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.[10] While cryo-EM samples are routinely collected at liquid nitrogen temperatures,[11] work has continued to understand sample behavior at liquid helium temperatures.[12][13][11]

In 1981 scientists at the European Molecular Biology Laboratory, reported the first successful cryo-EM.[14] Researchers used a thin film of vitrified pure water 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 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.[15] 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.[16] 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.[17]

### Recent advancements

Direct Electron Detectors, and more powerful imaging algorithms allow macromolecular structures to be determined at near-atomic resolution.[18] 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)[19] with resolutions up to 1.8 Å.[20] In 2019, cryo-EM structures grew to 2.5% of structures deposited in the Protein Data Bank.[21][22] 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"[23] pushing the resolution barrier beneath the crucial ~2-3 Å limit to resolve amino acid position and orientation.[24]

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).[23]

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.[25][26] Multiple techniques have been reported to improve SNR when determining the structures of small proteins.[27][28] Based on high-affinity DARPins, nanobodies, antibody fragments,[29] 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."[4]

## Techniques

### Mode of electron microscopy

#### Cryogenic transmission electron microscopy

Main article: Biology: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 article: Engineering:Correlative light-electron microscopy

In 2019, correlative light cryo-TEM and cryo-ET were used to observe tunnelling nanotubes (TNTs) in neuronal cells.[30]

#### Scanning electron cryomicroscopy

Main article: 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 article: Biology: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.[31]

#### Single particle analysis

Main article: Biology:Single particle analysis

File:Cryogenic electron microscopy workflow.svgSingle 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.[32]

When combined with a knowledge of time progression, the result is time-resolved cyro-TEM.[33][34][35]

##### Comparisons to X-ray crystallography

Main article: Physics:X-ray crystallography

Traditionally, X-ray crystallography has been the most popular technique for determining the 3D structures of biological molecules.[36] 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.[37] 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.[37]

The resolution of X-ray crystallography is limited by crystal homogeneity,[38] 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.[39] 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,[40][41] 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.[42]

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,[38] and the highest resolution achieved on record (as of September 30, 2022) is 0.48 Å.[43] 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.[44] However, as of 2020, the best cryo-EM resolution has been recorded at 1.22 Å,[41] making it a competitor in resolution in some cases.

#### Electron crystallography

Main article: Physics:Electron crystallography

Similar to X-ray crystallography used to determine the crystal structure of molecules of different sizes (from small molecules to large biomolecular complexes) using the X-ray diffraction pattern, electrons can also produce a electron diffraction pattern from a crystal. Work in this area has a long history dating back to early work such as the determination of the positions of hydrogen atoms in NH4Cl crystals by W. E. Laschkarew and I. D. Usykin in 1933,[45]

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).[46] It is also very useful in studying small molecules, from peptides to simpler compounds.[47]

## Specimen handling for imaging

(This section does not apply to electron crystallography.)

### Biological specimens

#### Thin film

The biological material is spread on an electron microscopy grid and is preserved in a frozen-hydrated state by rapid freezing, usually in liquid ethane near liquid nitrogen temperature. By maintaining specimens at liquid nitrogen temperature or colder, they can be introduced into the high-vacuum of the electron microscope column. Most biological specimens are extremely radiosensitive, so they must be imaged with low-dose techniques (usefully, the low temperature of transmission electron cryomicroscopy provides an additional protective factor against radiation damage).

Consequently, the images are extremely noisy. For some biological systems it is possible to average images to increase the signal-to-noise ratio and retrieve high-resolution information about the specimen using the technique known as single particle analysis. This approach in general requires that the things being averaged are identical, although some limited conformational heterogeneity can now be studied (e.g. ribosome). Three-dimensional reconstructions from CryoTEM images of protein complexes and viruses have been solved to sub-nanometer or near-atomic resolution, allowing new insights into the structure and biology of these large assemblies.

Analysis of ordered arrays of protein, such as 2-D crystals of transmembrane proteins or helical arrays of proteins, also allows a kind of averaging which can provide high-resolution information about the specimen. This technique is called electron crystallography.

#### Vitreous sections

The thin film method is limited to thin specimens (typically < 500 nm) because the electrons cannot cross thicker samples without multiple scattering events. Thicker specimens can be vitrified by plunge freezing (cryofixation) in ethane (up to tens of μm in thickness) or more commonly by high pressure freezing (up to hundreds of μm). They can then be cut in thin sections (40 to 200 nm thick) with a diamond knife in a cryoultramicrotome at temperatures lower than −135 °C (devitrification temperature). The sections are collected on an electron microscope grid and are imaged in the same manner as specimen vitrified in thin film. This technique is called transmission electron cryomicroscopy of vitreous sections (CEMOVIS) or transmission electron cryomicroscopy of frozen-hydrated sections.

### Material specimens

In addition to allowing vitrified biological samples to be imaged, CryoTEM can also be used to image material specimens that are too volatile in vacuum to image using standard, room temperature electron microscopy. For example, vitrified sections of liquid-solid interfaces can be extracted for analysis by CryoTEM,[48] and sulfur, which is prone to sublimation in the vacuum of electron microscopes, can be stabilized and imaged in CryoTEM.[49]

## 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.[50] There are two popular approaches that are widely used nowadays in cryo-EM image processing, the maximum likelihood approach that was discovered in 1998[51] and relatively recently adapted Bayesian approach.[52]

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.[53] 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.[54]

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.[53] 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.[52]

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,[55][56] 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.[57][58]

## Gallery

* Cryo-EM image of GroEL suspended in amorphous ice at 50000× magnification

* Structure of alcohol oxidase from Pichia pastoris by Cryo-EM

Structure of alcohol oxidase from Pichia pastoris by Cryo-EM

* Cryo-EM image of an intact ARMAN cell from an Iron Mountain biofilm. Image width is 576 nm.

* Cryo-EM image of the CroV giant marine virus
(scale bar represents 200 nm)[59]

## See also

| Wikibooks has a book on the topic of: 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

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46. ↑ "The collection of MicroED data for macromolecular crystallography". Nature Protocols 11 (5): 895–904. May 2016. doi:10.1038/nprot.2016.046. PMID 27077331.
47. ↑ "The CryoEM Method MicroED as a Powerful Tool for Small Molecule Structure Determination". ACS Central Science 4 (11): 1587–1592. November 2018. doi:10.1021/acscentsci.8b00760. PMID 30555912.
48. ↑ "Site-Specific Preparation of Intact Solid-Liquid Interfaces by Label-Free In Situ Localization and Cryo-Focused Ion Beam Lift-Out". Microscopy and Microanalysis 22 (6): 1338–1349. December 2016. doi:10.1017/S1431927616011892. PMID 27869059. Bibcode: 2016MiMic..22.1338Z.
49. ↑ "Characterization of Sulfur and Nanostructured Sulfur Battery Cathodes in Electron Microscopy Without Sublimation Artifacts". Microscopy and Microanalysis 23 (1): 155–162. February 2017. doi:10.1017/S1431927617000058. PMID 28228169. Bibcode: 2017MiMic..23..155L. https://zenodo.org/record/889883.
50. ↑ Cheng, Yifan (2018-08-31). "Single-particle cryo-EM—How did it get here and where will it go" (in en). Science 361 (6405): 876–880. doi:10.1126/science.aat4346. ISSN 0036-8075. PMID 30166484. Bibcode: 2018Sci...361..876C.
51. ↑ Sigworth, F.J. (1998). "A Maximum-Likelihood Approach to Single-Particle Image Refinement" (in en). Journal of Structural Biology 122 (3): 328–339. doi:10.1006/jsbi.1998.4014. PMID 9774537.
52. ↑ 52.0 52.1 Scheres, Sjors H.W. (January 2012). "A Bayesian View on Cryo-EM Structure Determination" (in en). Journal of Molecular Biology 415 (2): 406–418. doi:10.1016/j.jmb.2011.11.010. PMID 22100448.
53. ↑ 53.0 53.1 Nogales, Eva; Scheres, Sjors H.W. (May 2015). "Cryo-EM: A Unique Tool for the Visualization of Macromolecular Complexity". Molecular Cell 58 (4): 677–689. doi:10.1016/j.molcel.2015.02.019. ISSN 1097-2765. PMID 26000851.
54. ↑ Sigworth, Fred J. (2016-02-01). "Principles of cryo-EM single-particle image processing". Microscopy 65 (1): 57–67. doi:10.1093/jmicro/dfv370. ISSN 2050-5698. PMID 26705325.
55. ↑ Scheres, Sjors H. W. (2012-12-01). "RELION: Implementation of a Bayesian approach to cryo-EM structure determination" (in en). Journal of Structural Biology 180 (3): 519–530. doi:10.1016/j.jsb.2012.09.006. ISSN 1047-8477. PMID 23000701.
56. ↑ "RELION: Image-processing software for cryo-electron microscopy". 3dem. 27 October 2023. https://github.com/3dem/relion.
57. ↑ Bai, Xiao-chen; McMullan, Greg; Scheres, Sjors H.W (January 2015). "How cryo-EM is revolutionizing structural biology". Trends in Biochemical Sciences 40 (1): 49–57. doi:10.1016/j.tibs.2014.10.005. ISSN 0968-0004. PMID 25544475.
58. ↑ Zivanov, Jasenko; Nakane, Takanori; Forsberg, Björn O; Kimanius, Dari; Hagen, Wim JH; Lindahl, Erik; Scheres, Sjors HW (2018-11-09). Egelman, Edward H; Kuriyan, John. eds. "New tools for automated high-resolution cryo-EM structure determination in RELION-3". eLife 7. doi:10.7554/eLife.42166. ISSN 2050-084X. PMID 30412051.
59. ↑ 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.

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Electron microscopy

Basics|

* Electron microscope
* History
* Micrograph
* Microscope
* Timeline of microscope technology

Electron interaction
with matter|

* Auger effect
* Bremsstrahlung
* Electron diffraction
* Electron scattering
* Kikuchi lines
* Secondary electrons
* X-ray fluorescence

Instrumentation|

* Detectors for transmission electron microscopy
* Electron gun
* Everhart–Thornley detector
* Field electron emission
* Field emission gun
* Magnetic lens
* Stigmator

Microscopes|

| EM|

* Cryo-EM
* EPMA
* Liquid-Phase EM
* Low-energy EM
* Photoemission EM

|

SEM|

* Environmental SEM
* CryoSEM
* Confocal SEM
* SEM-XRF

TEM|

* Cryo-TEM
* Cryo-ET
* EFTEM
* HRTEM
* STEM
* 4D STEM
* Aberration-Corrected TEM

Techniques|

* 4D STEM
* Annular dark-field imaging
* Cathodoluminescence
* Charge contrast imaging
* CBED
* cryoEM
* Dark-field microscopy
* EDS
* EBSD
* TKD
* ECCI
* EELS
* EBIC
* Electron holography
* Electron tomography
* FIB
* FEM
* Immune electron microscopy
* Geometric phase analysis
* PINEM
* Precession electron diffraction
* Serial block-face scanning electron microscopy
* WDXS
* WBDF

Others|

| Developers|

* Albert Crewe
* Bodo von Borries
* Dennis Gabor
* Ernst G. Bauer
* Ernst Ruska
* Gerasimos Danilatos
* Harald Rose
* James Hillier
* Manfred von Ardenne
* Max Knoll
* Maximilian Haider
* Nestor J. Zaluzec
* Ondrej Krivanek
* Thomas Eugene Everhart
* Vernon Ellis Cosslett
* Vladimir K. Zworykin

|

Manufacturers|

* FEI Company
* Hitachi High-Technologies
* JEOL
* Leica
* Nion Company
* TESCAN
* Thermo Fisher Scientific
* Zeiss

Software|

* CASINO
* CrysTBox
* EM Data Bank
* EMsoft
* Digital Micrograph
* Direct methods
* IUCr
* MTEX
* Multislice

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