Cryogenic electron microscopy

Electron microscopy technique

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. For biological specimens, the structure is preserved by embedding in an environment of 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.[1] While development of the technique began in the 1970s, recent advances in detector technology and software algorithms have allowed for the determination of biomolecular structures at near-atomic resolution.[2] This has attracted wide attention to the approach as an alternative to X-ray crystallography or NMR spectroscopy in the structural biology field.[3]

In 2017, the 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]

## History

[edit]

### Early development

[edit]

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.[6] 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.[7]

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",[8] 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.[9] 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.[10] 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.[11]

### Recent advancements

[edit]

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.[12] 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)[13] and with resolutions up to 1.8 Å.[14] In 2019, cryo-EM structures represented 2.5% of structures deposited in the Protein Data Bank,[15] and this number continues to grow.[16] 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"[17] pushing the resolution barrier beneath the crucial ~2-3 Å limit to resolve amino acid position and orientation.[18]

> 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).[17]

More recently, advancements in the use of protein-based imaging scaffolds are helping to solve the problems of sample orientation bias and size limit. Proteins smaller than ~50 kDa generally have too low a signal-to-noise ratio (SNR) to be able to resolve protein particles in the image, making 3D reconstruction difficult or impossible.[19] The SNR of smaller proteins can be improved by binding them to an imaging scaffold. The Yeates group at UCLA was able to create a clearer image of three variants of KRAS (roughly 19 kDa in size) by utilising a rigid imaging scaffold, and using DARPins as modular binding domains between the scaffold and the protein of interest.[20]

### 2017 Nobel Prize in Chemistry

[edit]

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]

## Comparisons to X-ray crystallography

[edit]

Main article: X-ray crystallography

Traditionally, X-ray crystallography has been the most popular technique for determining the 3D structures of biological molecules.[21] 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.[22] 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.[22]

The resolution of X-ray crystallography is limited by crystal homogeneity,[23] 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.[24] 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,[25][26] 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.[27]

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,[23] and the highest resolution achieved on record (as of September 30, 2022) is 0.48 Å.[28] As of 2020, the majority of the protein structures determined by cryo-EM are at a lower resolution of 3–4 Å.[29] However, as of 2020, the best cryo-EM resolution has been recorded at 1.22 Å,[26] making it a competitor in resolution in some cases.

## Biological specimens

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### Thin film

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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

[edit]

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

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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,[30] and sulfur, which is prone to sublimation in the vacuum of electron microscopes, can be stabilized and imaged in CryoTEM.[31]

## Image processing in cryo-TEM

[edit]

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.[32] There are two popular approaches that are widely used nowadays in cryo-EM image processing, the maximum likelihood approach that was discovered in 1998[33] and relatively recently adapted Bayesian approach.[34]

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.[35] 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.[36]

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.[35] 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.[34]

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,[37][38] 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.[39][40]

## Techniques

[edit]

A variety of techniques can be used in CryoTEM.[41] Popular techniques include:

1. Single particle analysis (SPA)
1. Time-resolved CryoTEM[42][43][44]
2. Electron cryotomography (cryoET)
3. Electron crystallography
1. Analysis of two-dimensional crystals
2. Analysis of helical filaments or tubes
3. Microcrystal Electron Diffraction (MicroED)[45][46][47][48]

## Correlative light cryo-TEM and cryo-ET

[edit]

Main article: Correlative light-electron microscopy

In 2019, correlative light cryo-TEM and cryo-ET were used to observe tunnelling nanotubes (TNTs) in neuronal cells.[49]

## Scanning electron cryomicroscopy

[edit]

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.

## Cryogenic transmission electron microscopy

[edit]

Main article: 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.

## Centers

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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.[50] 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.[51]

The Danish National cryo-EM Facility also known as 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).

Single particle analysis workflow

## Advanced methods

[edit]

* 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.[52]
* Electron crystallography, method to determine the arrangement of atoms in solids using a TEM
* MicroED,[53] method to determine the structure of proteins, peptides, organic molecules, and inorganic compounds using electron diffraction from 3D crystals[54][55][56]
* Single particle analysis cryo-EM, an averaging method to determine protein structure from monodisperse samples.[57]

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

* 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)[58]

## See also

[edit]

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

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[edit]

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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
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Microscopes|

| EM|

* Cryo-EM
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SEM|

* Environmental SEM
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* SEM-XRF

TEM|

* Cryo-TEM
* Cryo-ET
* EFTEM
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* STEM
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Techniques|

* 4D STEM
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* EDS
* EBSD
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* 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
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* Nion Company
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* Zeiss

Software|

* CASINO
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* Direct methods
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