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Cryo-ET: A Window to the Miniature Universe Within Cells

Updated: Apr 26

Why is everyone talking about cryo-EM?

Now-a-days in cell biology research, the terminology of "Cryo-EM" (Cryo Electron Microscopy) and "Cryo-ET" (Cryo Electron Tomography) are commonly encountered (Eisenstein, 2023). The three-dimensional data obtained through this technique has a remarkable (sub-nanometer-) resolution and reveals cell structures at unprecedented detail, unattainable by alternative imaging methodologies. It is, therefore, unsurprising that many cell biologists are interested in integrating this methodology into their research topics. However, many are unaware about the amount of resources required for generating such data. It is important to note that "Cryo-EM" constitutes an umbrella term encompassing all work conducted within a cryo-electron microscope. However, the scope of this article is centred around Cryo-ET—a method in which the sample is imaged from different angles, after which a three-dimensional dataset thereof can be reconstructed.

To understand why cryo-ET is such a revolutionizing technique, it is important to look at the history of Electron Microscopy, which has been around for over 80 years. Traditionally, the preparation of cell samples necessitated a sequence of chemical fixation, resin embedding, sectioning, and subsequent imaging steps. For room temperature EM, cells are flash-frozen (vitrified), followed by freeze substitution, which is a process to replace water with an organic solvent. Staining agents are then introduced to enhance the contrast, and a resin is added which hardens to form a block that can be cut into thin sections (<100 nm), allowing penetration by the electron beam. This traditional approach carries both merits and limitations but still remains in practice today to answer various research questions. However, it has attracted criticism for introducing artefacts and alterations to cellular ultrastructure through chemical treatment.

On the contrary, Cryo-EM brings a revolutionary upgrade to the criticism encountered in room-temperature EM. Here, cells are directly grown on fragile EM-grids (3.05 mm diameter), which are vitrified and theoretically need no further processing. If the sample is maintained at cryogenic temperatures (below -150°C, typically in a liquid nitrogen environment), it remains in a near-native state, virtually frozen in time and space within the ice. However, the main challenges in Cryo-EM are the low intrinsic contrast of the cell-sample, the thickness limitation for electron beam, and the low throughput. Some of these challenges were only recently surmounted (Turk & Baumeister, 2020), a feat recognized through a Nobel Prize in 2017 (, 2017). One high impact example is the development of direct electron detectors, which drastically improved the quality of acquired images for low dose acquisitions. But cell biologists wanted to go further, looking deep inside the cell in areas which cannot be penetrated by the electron beam. Here sectioning, either by knife (CEMOVIS) or by ion beam (FIB-SEM), helped to overcome this limitation. Especially the development of Focused Ion Beam (FIB) milling within Cryo conditions has enabled cell biologists to efficiently and reproducibly thin their cells to 100-200 nm slices (Figure 1), as show very recently where this pipeline was used to characterize SARS-CoV-2 replication cycle in situ (Klein et al., 2020).

Foremost among the advantages, is the ability to observe samples in near-native states with extraordinary precision. The technique affords resolutions below 10 nanometres, enabling the resolution of myriad structural components within the cell, ranging from larger organelle-type structures to intermediary structures such as microtubules and actin filaments. In addition to that, one can combine a single particle analysis (SPA) pipeline, in which particles are picked and averaged to resolve protein structures, with tomography, where particles within the tomogram can be picked, averaged and their 3D structure reconstructed. This ground-breaking approach is known as sub-tomogram averaging (STA) and allows the attainment of protein structures at nanometer resolution, in situ.

To overcome the throughput issue, researchers are incorporating interdisciplinary ideas to develop protocols to facilitate the process. It is noteworthy to mention micropatterning of the grids which is a strategy to coat the grid with a material which cells do not like, to then remove part of it, to position the cells in desired location. Another approach is to use fluorescence to guide the user to the structure of interest. This approach is called cryo-correlated light and electron microscopy (cryo-CLEM). This does add an extra step to the pipeline but provides the benefit of combining the specificity of fluorescence microscopy with the structural information obtained by EM. A high-resolution cryo-CLEM approach (i.e. cryo-confocal microscopy or cryo-SIM), is possible too, but at present, the lack of cryo-immersion objectives, among others, limits the achievable resolution drastically.

As mentioned before, one challenge of cryo-EM is the need for thin samples. This means, investigating tissues is even more complex, as it is multi-layered and a section which can provide valuable information of a more biologically representative sample, can be hundreds of microns thick. In this case FIB-sectioning is not possible. To tackle this, a very different approach, termed cryo-lift-out, has been deployed in recent years. This technique requires a different freezing strategy called high-pressure freezing (HPF), followed by cutting and lifting out a block using a cryo-needle, which is then placed on a different sample holder for further thinning (Schaffer et al., 2019). Recently this has even evolved to the organism scale, where an entire C. Elegans has been cut into 40 sections, to be imaged by tomography (Schiøtz et al., 2023), however at present this remains a challenging technique and has not yet been replicated anywhere.

To elaborate on the challenges associated with Cryo-ET, it is essential to address three factors: cost, time, and labour. A single week of microscope access can easily amass costs exceeding €10,000, dependent on the specific equipment employed. Furthermore, the collection and processing of data for merely one or two grids, may extend over weeks or even months before yielding results. Important to note is also that depending on the project, a single good dataset might be sufficient to support previously, through other techniques, obtained data, while in other cases multiple grids need to be imaged to obtain any statistically relevant results. This timeline assumes a dedicated expert —a point that leads to the third facet, labour. Expertise in cryo-ET is attained with a steep learning curve hence a dedicated person is recommended, yet after all, it still might take several iterations to obtain the desired results. Nevertheless, the data that can be obtained is often worth the time and effort

To conclude, cryo-ET is an immensely powerful technique that can definitely be worth the investment, however it does require appropriate resources and should not be considered as an “easy extra picture” for the next publication. Therefore, it is worth discussing the needs for the project with an expert of the technique, as cryo-ET is not the answer to all questions. More often than not, cryo-ET leads to new questions. Additionally, and depending on the research question, there might be other, more suitable techniques. Single Molecule localization techniques, like PALM and STORM, can reach similar resolutions and have much higher specificity due to the use of fluorescent markers (Lelek et al., 2022). Soft X-ray tomography (cryo-SXT) has a lower resolution (30 nm) but allows imaging of cryo-samples up to 10 microns thick, with a much larger field of view and without the need for staining (Groen et al., 2019). Furthermore, other EM techniques, be it at room temperature or in cryo, can provide better answers to certain questions, like for example volume-EM (Collinson et al., 2023). Nonetheless, cryo-EM, specifically cryo-ET, is revolutionary in and of itself, revealing new and never seen before structures on a regular basis (i.e. Laughlin et al., 2022). Furthermore, cryo-ET is a technique in its infancy and more developments can be expected in the future, opening even more doors into the miniature universe within cells.


  1. Collinson, L. M., Bosch, C., Bullen, A., Burden, J. J., Carzaniga, R., Cheng, C., Darrow, M. C., Fletcher, G., Johnson, E., Narayan, K., Peddie, C. J., Winn, M., Wood, C., Patwardhan, A., Kleywegt, G. J., & Verkade, P. (2023). Volume EM: a quiet revolution takes shape. In Nature Methods (Vol. 20, Issue 6).

  2. Eisenstein, M. (2023). Catching proteins at play: the method revealing the cell’s inner mysteries. Nature, 621(7979), 646–648.

  3. Groen, J., Conesa, J. J., Valcárcel, R., & Pereiro, E. (2019). The cellular landscape by cryo soft X-ray tomography. Biophysical Reviews, 11(4).

  4. Klein, Steffen, Mirko Cortese, Sophie L. Winter, Moritz Wachsmuth-Melm, Christopher J. Neufeldt, Berati Cerikan, Megan L. Stanifer, Steeve Boulant, Ralf Bartenschlager, and Petr Chlanda, 2020, SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography. Nat Commun 11, 5885.

  5. Laughlin, T.G., Deep, A., Prichard, A.M., Seitz, C., Gu, Y., Enustun, E., Suslov, S., Khanna, K., Birkholz, E.A., Armbruster, E. and McCammon, J.A., 2022. Architecture and self-assembly of the jumbo bacteriophage nuclear shell. Nature, 608(7922), pp.429-435.

  6. Lelek, M., Gyparaki, M. T., Beliu, G., Schueder, F., Griffié, J., Manley, S., Jungmann, R., Sauer, M., Lakadamyali, M., & Zimmer, C. (2022). Author Correction: Single-molecule localization microscopy. Nature Reviews Methods Primers, 2(1), 70.

  7. (2017). The Nobel Prize in Chemistry 2017.

  8. Schaffer, M., Pfeffer, S., Mahamid, J., Kleindiek, S., Laugks, T., Albert, S., Engel, B. D., Rummel, A., Smith, A. J., Baumeister, W., & Plitzko, J. M. (2019). A cryo-FIB lift-out technique enables molecular-resolution cryo-ET within native Caenorhabditis elegans tissue. Nature Methods, 16(8).

  9. Schiøtz, O.H., Kaiser, C.J., Klumpe, S., Morado, D.R., Poege, M., Schneider, J., Beck, F., Klebl, D.P., Thompson, C. and Plitzko, J.M., 2023. Serial Lift-Out: sampling the molecular anatomy of whole organisms. Nature Methods, pp.1-9.

  10. Turk, M., & Baumeister, W. (2020). The promise and the challenges of cryo‐electron tomography. FEBS Letters, 594(20), 3243–3261.

This article was specialist edited by Marie Prevost.

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