Cryogenic electron tomography

File:Slice from electron cryotomogram of Bdellovibrio bacteriovorus cell.jpg cell. Scale bar 200 nm.|left]]

In electron microscopy (EM), samples are imaged in a high vacuum. Such a vacuum is incompatible with biological samples such as cells; the water would boil off, and the difference in pressure would explode the cell. In room-temperature EM techniques, samples are therefore prepared by fixation and dehydration. Another approach to stabilize biological samples, however, is to freeze them (cryo-electron microscopy or cryoEM). As in other electron cryomicroscopy techniques, samples for cryoET (typically small cells such as Bacteria, Archaea, or viruses) are prepared in standard aqueous media and applied to an EM grid. The grid is then plunged into a cryogen, for example liquid ethane, with sufficiently large specific heat that the rate of cooling is rapid enough that water molecules do not have time to rearrange into a crystalline lattice. The resulting water state is called low-density amorphous ice, or commonly "vitreous ice" for its glass like nature. This form of ice preserves native cellular structures, such as lipid membranes, that would normally be disrupted by hexagonal or other ordered ice upon slower freezing. Plunge-frozen samples are subsequently kept at liquid-nitrogen temperatures through storage and imaging so that the water never warms enough to crystallize.

Samples are imaged in a transmission electron microscope (TEM). As in other electron tomography techniques, the sample is tilted to different angles relative to the electron beam (typically every 2-3 degrees from about −60° to +60°), and an image is acquired at each angle.{{cite journal|title=Electron tomography for functional nanomaterials|year=2020|author1=R. Hovden |author2=D. A. Muller|journal=MRS Bulletin|volume=45|issue=4|pages=298–304|doi=10.1557/mrs.2020.87|arxiv=2006.01652|bibcode=2020MRSBu..45..298H |s2cid=216522865 }} This tilt-series of images can then be computationally reconstructed into a three-dimensional view of the object of interest.{{Cite journal|title = Cryo-electron tomography: the challenge of doing structural biology in situ|journal = The Journal of Cell Biology|date = 2013-08-05|issn = 1540-8140|pmc = 3734081|pmid = 23918936|pages = 407–419|volume = 202|issue = 3|doi = 10.1083/jcb.201304193|first1 = Vladan|last1 = Lučič|first2 = Alexander|last2 = Rigort|first3 = Wolfgang|last3 = Baumeister}} This is called a tomogram, or tomographic reconstruction.

One of the most commonly cited benefits of cryoET is the ability to reconstruct 3D volumes of individual objects (proteins, cells, etc.) rather than necessitating multiple copies of the sample in crystallographic methods or in other cryoEM imaging methods like single particle analysis. CryoET is considered to be an in situ method when used on an unperturbed cell or other system since plunge-freezing of sufficiently thin samples fixes the specimen in place fast enough to cause minimal changes to atomic positioning.{{Cite journal |last=Adrian |first=Marc |last2=Dubochet |first2=Jacques |last3=Lepault |first3=Jean |last4=McDowall |first4=Alasdair W. |date=March 1984 |title=Cryo-electron microscopy of viruses |url=https://www.nature.com/articles/308032a0 |journal=Nature |language=en |volume=308 |issue=5954 |pages=32–36 |doi=10.1038/308032a0 |issn=1476-4687}} Thick samples, greater than ~500 nm, require additional conditions like high-pressure to promote vitrification throughout the sample.

In transmission electron microscopy (TEM), because electrons interact strongly with matter, samples must be kept very thin to not cause samples to darken due to multiple elastic scattering events. Therefore, in cryoET, samples are generally less than ~500 nm thick. For this reason, most cryoET studies have focused on purified macromolecular complexes, viruses, or small cells such as those of many species of Bacteria and Archaea. For example, cryoET has been used to understand encapsulation of 12 nm size protein cage nanoparticles inside 60 nm sized virus-like nanoparticles.{{cite journal | vauthors = Waghwani HK, Uchida M, Douglas, T | title = Virus-Like Particles (VLPs) as a Platform for HierarchicalCompartmentalization | journal = Biomacromolecules | volume = 21 | issue = 6| pages = 2060–2072 | date = April 2020 | doi = 10.1021/acs.biomac.0c00030 | pmid = 32319761 | doi-access = free }}

[[File:Cardiac sarcomere tomogram.jpg|thumb|(a) Tomographic slice of a cardiac sarcomere. Scale bar, 50 nm. (b) Reconstructed filaments mapped into a tomogram. Scale bar, 50 nm (c) Structure of the thick filament from the M-band to the C-zone.{{Citation |last=Tamborrini |first=Davide |title=Structure of the native myosin filament in the relaxed cardiac sarcomere. |journal = Nature |date=2023-12-23 |url=https://www.nature.com/articles/s41586-023-06690-5 |language=en |doi=10.1038/s41586-023-06690-5

|last2=Wang |first2=Zhexin |last3=Wagner |first3=Thorsten |last4=Tacke |first4=Sebastian |last5=Stabrin |first5=Markus |last6=Grange |first6=Michael |last7=Kho |first7=Ay Lin |last8=Rees |first8=Martin |last9=Bennett |first9=Pauline|last10=Gautel |first10=Mathias|last11=Raunser |first11=Stefan|pmc=10665186 }}]]

Larger cells, and even tissues, can be prepared for cryoET by thinning, either by cryo-sectioning or by focused ion beam (FIB) milling. In cryo-sectioning, frozen blocks of cells or tissue are sectioned into thin samples with a cryo-microtome.{{Cite journal|title = Cryo-electron microscopy of vitreous sections|journal = The EMBO Journal|date = 2004-09-15|issn = 0261-4189|pmc = 517607|pmid = 15318169|pages = 3583–3588|volume = 23|issue = 18|doi = 10.1038/sj.emboj.7600366|first1 = Ashraf|last1 = Al-Amoudi|first2 = Jiin-Ju|last2 = Chang|first3 = Amélie|last3 = Leforestier|first4 = Alasdair|last4 = McDowall|first5 = Laurée Michel|last5 = Salamin|first6 = Lars P. O.|last6 = Norlén|first7 = Karsten|last7 = Richter|first8 = Nathalie Sartori|last8 = Blanc|first9 = Daniel|last9 = Studer}} In FIB-milling, plunge-frozen samples are exposed to a focused beam of ions, typically gallium, that precisely whittle away material from the top and bottom of a sample, leaving a thin lamella suitable for cryoET imaging.{{Cite journal|last1=Villa|first1=Elizabeth|author-link=Elizabeth Villa|last2=Schaffer|first2=Miroslava|last3=Plitzko|first3=Jürgen M.|last4=Baumeister|first4=Wolfgang|date=2013-10-01|title=Opening windows into the cell: focused-ion-beam milling for cryo-electron tomography|journal=Current Opinion in Structural Biology|volume=23|issue=5|pages=771–777|doi=10.1016/j.sbi.2013.08.006|issn=1879-033X|pmid=24090931}}

For structures that are present in multiple copies in one or multiple tomograms, higher resolution (even ≤1 nm) can be obtained by subtomogram averaging.{{Cite journal|title = Structural biology in situ—the potential of subtomogram averaging|journal = Current Opinion in Structural Biology|date = 2013-04-01|issn = 1879-033X|pmid = 23466038|pages = 261–267|volume = 23|issue = 2|doi = 10.1016/j.sbi.2013.02.003|first = John A. G.|last = Briggs}}{{Cite journal|title = The Structure of Immature Virus-Like Rous Sarcoma Virus Gag Particles Reveals a Structural Role for the p10 Domain in Assembly|journal = Journal of Virology|date = 2015-10-15|issn = 1098-5514|pmc = 4580193|pmid = 26223638|pages = 10294–10302|volume = 89|issue = 20|doi = 10.1128/JVI.01502-15|first1 = Florian K. M.|last1 = Schur|first2 = Robert A.|last2 = Dick|first3 = Wim J. H.|last3 = Hagen|first4 = Volker M.|last4 = Vogt|first5 = John A. G.|last5 = Briggs}} Similar to single particle analysis, subtomogram averaging computationally combines images of identical objects to increase the signal-to-noise ratio.

Electron microscopy is known to swiftly decay biological samples compared to samples in materials science and physics due to radiation damage.{{Cite journal |last=Saha |first=Ambarneil |last2=Nia |first2=Shervin S. |last3=Rodríguez |first3=José A. |date=2022-09-14 |title=Electron Diffraction of 3D Molecular Crystals |url=https://pubs.acs.org/doi/10.1021/acs.chemrev.1c00879 |journal=Chemical Reviews |language=en |volume=122 |issue=17 |pages=13883–13914 |doi=10.1021/acs.chemrev.1c00879 |issn=0009-2665 |pmc=9479085 |pmid=35970513}} In most other electron microscopy-based methods for imaging biological samples, combining the signal from many different sample copies has been the general way of surpassing this problem (e.g. crystallography, single particle analysis). In cryoET, instead of taking many images of different sample copies, many images are taken of one area. Consequentially, the fluence (number of electrons imparted per unit area) on the sample is around 2-5x more than in single particle analysis.{{Citation |last=Schmid |first=Michael F. |title=Chapter 2 - Single-particle electron cryotomography (cryoET) |date=2011-01-01 |url=https://www.sciencedirect.com/science/article/pii/B9780123865076000026 |work=Advances in Protein Chemistry and Structural Biology |volume=82 |pages=37–65 |editor-last=Ludtke |editor-first=Steven J. |access-date=2024-01-07 |series=Recent Advances in Electron Cryomicroscopy, Part B |publisher=Academic Press |doi=10.1016/b978-0-12-386507-6.00002-6 |editor2-last=Venkataram Prasad |editor2-first=B. V.|url-access=subscription }} Tomography on materials much more resilient allows drastically higher resolution than typical biological imaging, suggesting that radiation damage is the greatest limitation to cryoET of biological samples.{{Cite journal |last=Bäuerlein |first=Felix J. B. |last2=Baumeister |first2=Wolfgang |date=2021-10-01 |title=Towards Visual Proteomics at High Resolution |url= |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 |issn=0022-2836|doi-access=free }}{{Cite journal |last=Scott |first=M. C. |last2=Chen |first2=Chien-Chun |last3=Mecklenburg |first3=Matthew |last4=Zhu |first4=Chun |last5=Xu |first5=Rui |last6=Ercius |first6=Peter |last7=Dahmen |first7=Ulrich |last8=Regan |first8=B. C. |last9=Miao |first9=Jianwei |date=March 2012 |title=Electron tomography at 2.4-ångström resolution |url=https://www.nature.com/articles/nature10934 |journal=Nature |language=en |volume=483 |issue=7390 |pages=444–447 |doi=10.1038/nature10934 |issn=1476-4687}}

The strong interaction of electrons with matter also results in an anisotropic resolution effect. As the sample is tilted during imaging, the electron beam interacts with a thicker apparent sample along the optical axis of the microscope at higher tilt angles. In practice, tilt angles greater than approximately 60–70° do not yield much information and are therefore not used. This results in a "missing wedge" of information in the final tomogram that decreases resolution parallel to the electron beam.

File:CryoET dose symmetric collection scheme.jpg

The term "missing wedge" originates from the view of the Fourier transform of the tomogram, where an empty wedge is apparent due to not tilting the sample to 90°. The missing wedge results in a lack of resolution in sample depth, as the missing information is mostly along the z-axis. The missing wedge is also a problem in 3D electron crystallography, where it is usually solved by merging multiple datasets that overlap each other or through symmetry expansion where possible. Both of these solutions are due to the nature of crystallography, and so neither can be applied to tomography.

A major obstacle in cryoET is identifying structures of interest within complicated cellular environments. Solutions such as correlated cryo-fluorescence light microscopy,{{Cite journal|title = Correlative cryo-electron tomography and optical microscopy of cells|journal = Current Opinion in Structural Biology|date = 2013-10-01|issn = 1879-033X|pmc = 3812453|pmid = 23962486|pages = 763–770|volume = 23|issue = 5|doi = 10.1016/j.sbi.2013.07.017|first = Peijun|last = Zhang}} and super-resolution light microscopy (e.g. cryo-PALM{{Cite journal|title = Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography|journal = Nature Methods|date = 2014-07-01|issn = 1548-7105|pmc = 4081473|pmid = 24813625|pages = 737–739|volume = 11|issue = 7|doi = 10.1038/nmeth.2961|first1 = Yi-Wei|last1 = Chang|first2 = Songye|last2 = Chen|first3 = Elitza I.|last3 = Tocheva|first4 = Anke|last4 = Treuner-Lange|first5 = Stephanie|last5 = Löbach|first6 = Lotte|last6 = Søgaard-Andersen|first7 = Grant J.|last7 = Jensen}}) can be integrated with cryoET. In these techniques, a sample containing a fluorescently-tagged protein of interest is plunge-frozen and first imaged in a light microscope equipped with a special stage to allow the sample to be kept at sub-crystallization temperatures (< −150 °C). The location of the fluorescent signal is identified and the sample is transferred to the CryoTEM, where the same location is then imaged at high resolution by cryoET.

• Electron microscopy
• Electron tomography
• Transmission electron cryomicroscopy
• Transmission electron microscopy

{{Reflist}}

• [http://cryo-em-course.caltech.edu/ Getting started in cryo-EM course (Caltech)]

{{Electron microscopy}}

Category:Cell biology

Category:Electron microscopy techniques