Since its integration into cell biology in the 1940s, transmission electron microscopy (TEM) has revolutionized the field by offering unprecedented resolution, far surpassing that of light microscopy. However, traditional TEM processes, including fixation, dehydration, resin embedding and heavy metal staining, can introduce artifacts that distort the biological sample (Mollenhauer, 1993, Dubochet and Sartori Blanc, 2001). To address these limitations, cryotechniques have emerged. These methods involve rapidly freezing cells or tissues to prevent the formation of ice crystals that can damage subcellular structures, thereby preserving the samples in a lifelike, frozen-hydrated state (Dubochet, Adrian et al. 1988).
Although TEM generates 2D projections, electron tomography has been developed to provide three-dimensional views of cellular structures (Koster, Grimm et al. 1997). This approach requires incrementally tilting the sample in the electron microscope within a range of +/- 70º. Each projection is captured as the sample is tilted, resulting in a series of images that are subsequently reconstructed into 3D tomograms (Fig. 1A). Cryo-electron tomography (cryo-ET) combines electron tomography with cryotechniques (Medalia et al., 2002, McIntosh et al., 2005, Ng and Gan, 2020). Unlike single-particle analysis (SPA) in cryo-electron microscopy (cryo-EM), which achieves high-resolution structures of purified biomolecules, cryo-ET enables the study of complex and heterogeneous assemblies within their native environment, achieving at least molecular resolution (2-4 nm).
Besides cryo-ET, emerging 3D cryogenic imaging techniques include cryo-scanning transmission electron microscopy tomography (CSTET), soft X-ray cryo-tomography, and cryo-focused ion beam scanning electron microscopy (cryo-FIB-SEM) volume imaging. Compared to cryo-ET, CSTET studies samples up to three times thicker, albeit at a lower resolution (Wolf and Elbaum, 2019, Kirchweger et al., 2023). Soft X-ray cryo-tomography images whole cells up to several tens of micrometers thick at a mesoscopic resolution ranging from 36-70 nm (Groen et al., 2019, Loconte et al., 2023, Fahy et al., 2024). Cryo-FIB-SEM volume imaging models subcellular structures at a resolution of 20-50 nm in specimens that are at least tens of micrometers thick (Schertel et al., 2013, Vidavsky et al., 2016, Dumoux et al., 2023). However, these techniques cannot resolve molecular substructures, making cryo-ET the preferred method for high-resolution 3D imaging of macromolecules within biological specimens (Gan et al., 2011, Beck and Baumeister, 2016).
Cellular cryo-ET not only reveals the in situ structures of macromolecules but also enables precise determination of their organization and interactions in their life-like states. By visualizing molecular processes, cell biologists can use cryo-ET, along with other imaging methods, to directly test existing models or develop new ones. Therefore, cellular cryo-ET is a bridge between structural biology and cell biology. The workflow for cellular cryo-ET begins by rapidly freezing samples to preserve them in a frozen-hydrated state, thus preserving their lifelike structures. The target of interest is then localized and, if necessary, thinned using the methods discussed in the following section. The frozen-hydrated sample is then introduced into the TEM for tilt-series data collection. These tilt series images are then computationally processed to reconstruct cryotomograms. Within these tomograms, subcellular membrane structures can be segmented for visualization purposes. Ultimately, sub-volumes containing specific macromolecules of interest are extracted and averaged to generate higher-resolution density maps through a process known as subtomogram averaging (STA) (Fig. 1B). These density maps are then integrated back into the tomograms or 3D segmented view, offering detailed spatial information on the distribution and organization of macromolecules within the cellular context.
In this review, we provide an overview of recent advancements in cellular cryo-ET at each critical stage of the process, including sample preparation, data collection, and image analysis. We emphasize cryo-preparation for cellular and tissue samples, as it remains the primary bottleneck in the workflow. Finally, we provide a brief outlook on the potential and forthcoming developments of cryo-ET.