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Published in final edited form as: Curr Opin Struct Biol. 2025 Jun 4;93:103076. doi: 10.1016/j.sbi.2025.103076

In situ cryo-electron microscopy and tomography of cellular and organismal samples

Parijat Majumder 1, Peijun Zhang 1,2
PMCID: PMC12374797  NIHMSID: NIHMS2105404  PMID: 40472449

Abstract

As cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) continue to advance, the ability to visualize cellular and organismal structures with unprecedented clarity is redefining the landscape of structural biology. Breakthroughs in imaging technology, sample preparation and image processing now enable the detailed elucidation of cellular architecture, macromolecular organization, and dynamic biological processes at sub-nanometer resolution. Recent methodological advances have propelled the field to new frontiers, facilitating the investigation of complex biological questions across scales—from macromolecular complexes to organism-wide structural insights. This review explores rapidly emerging trends, highlights key innovations that are pushing the boundaries of in situ structural biology, and addresses persistent challenges in expanding the applicability of cryo-EM and cryo-ET across diverse biological systems.

Introduction

Cryo-EM and cryo-ET have emerged as indispensable tools in structural biology, that enable the determination of three-dimensional (3D) structures of biological systems in near-native states. The “resolution revolution” in cryo-EM has enabled visualization of isolated biomolecules in unprecedented detail. Simultaneously, cryo-ET has earned powerful capabilities for elucidating the internal architecture of cells and tissues [1,2].

Over the past decade, these techniques have been significantly enhanced by the integration of complementary approaches [2]. Focused ion beam (FIB) milling has facilitated the preparation of thin lamellae from thick biological samples, enabling high-resolution imaging [3]. Correlative light and electron microscopy (CLEM) has bridged functional and structural imaging, combining fluorescence-based localization with cryo-EM and cryo-ET [4,5]. Furthermore, advancements in deep learning have powered data processing and structural reconstruction [6]. These innovations have significantly expanded the scope of cryo-EM and cryo-ET in cellular and organismal research, supporting applications across diverse fields, spanning organelle dynamics, structural virology, host–pathogen interactions, neurodegeneration, and phase separation.

This review provides an overview of the methods (Figure 1) and latest applications (Figure 2) of cryo-EM and cryo-ET in cellular and organismal research. By examining recent developments and addressing current challenges, it highlights the transformative potential of these techniques for in situ structural studies.

Figure 1.

Figure 1

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Overview of the cryo-EM and cryo-ET methods for cellular and organismal samples

(Vitrification) Cellular and organismal samples vary in size, ranging from less than a hundred nanometres to several hundred millimetres. Representative specimens, from left to right, include viruses, bacteria, yeast, animal and plant cells, worms, fruit flies, and the human brain. The selection of vitrification methods is dictated by specimen size. Plunge freezing is suitable for samples with a thickness of less than ~10 μm, whereas high-pressure freezing is more effective for thicker specimens, currently up to a few hundred micrometres

(Thinning) This step is necessary for samples that are thicker than ~ 1 μm. Focused ion beam (FIB) milling can be used for on-grid lamella preparation, waffle lamellae and for lift-out, with the choice of method dictated by sample geometry. Cryo-sectioning (CEMOVIS) offers an alternative thinning route for high pressure frozen samples

(Localization) Cryo-fluorescence imaging of vitrified specimens enables the identification and localization of regions of interest, guiding targeted thinning or transmission electron microscopy (TEM) imaging

(TEM imaging) Cryo-EM involves acquisition of 2D projection images at a single angle, while cryo-ET involves acquisition of 2D images over a range of sample tilt angles.

(Analysis) In cryo-EM, 2D projections are conventionally processed using the single-particle analysis pipeline to achieve high-resolution 3D reconstructions. In cryo-ET, tilt projection series are reconstructed into 3D volumes or tomograms, enabling ultrastructural elucidation through segmentation or the generation of 3D macromolecular reconstructions via subtomogram averaging.

(This figure is partially prepared in BioRender).

Figure 2.

Figure 2

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Latest examples of in situ cryo-ET applications

I. Ultrastructural Elucidation. A representative tomographic slice (left panel) and the corresponding segmented volume (middle and right panels) illustrate the nuclear import of wild-type HIV-1 cores [41]. Three cores are identified and marked with purple arrowheads and numbers: No.1, an imported tube-shaped core with discernible surrounding densities (enlarged in the inset); No.2, a docked cone-shaped core with its wide end positioned on the nuclear pore complex (NPC); and No.3, a cone-shaped core traversing the NPC, with its narrow end inside the nucleus. The NPC, ribosomes, nucleosomes, and prominent nuclear factors are labelled. The nucleus, nuclear envelope (NE), and membranes are annotated accordingly. Scale bar: 100 nm.

II. Spatial and Functional Context. A representative tomographic slice of a heat shock-exposed E. coli cell (left panel) and the corresponding three-dimensional rendering of macromolecular complexes (right panel) illustrate the spatial organization of GroEL–GroES complexes and ribosomes in situ [64]. GroEL–GroES complexes are indicated by black open circles. Scale bar: 100 nm. A schematic representation of asymmetrical (EL-ES1) and symmetrical (EL-ES2) GroEL–GroES complexes is shown in the top-left inset. To the right of the tomographic slice, a gallery presents central subtomogram slices of EL-ES1 and EL-ES2 complexes in a side view. Scale bar: 10 nm. The right panel shows subtomogram averages of GroEL–GroES complexes in different conformations and stoichiometries.

III. Macromolecular Structures In Situ. A representative tomographic slice (left panel) of a nuclear copia cluster in Drosophila ovarian cells shows virus-like particles (VLPs) [78]. Scale bar, 200 nm. Rectangles highlight a putative immature VLP and a putative mature VLP, for which the corresponding zoomed-in views are shown in the right inset. Scale bar, 20 nm. The right panel (top) displays a 7.7 Å resolution subtomogram average of the entire copia CA, alongside an AlphaFold-Multimer and MDFF-based model of the complete CA structure. The right panel (bottom) presents a rendering of a repeat unit from a nuclear copia cluster, along with schematic representations of different environments within an icosahedral lattice.

Methods for Cryo-EM and Cryo-ET of cellular and organismal samples

Sample preparation techniques

For high-resolution imaging, samples must be vitrified to preserve their biological structures in a near-native state [7]. Vitrification involves rapid freezing, typically achieved through immersion in cryogens such as liquid ethane or ethane-propane mixtures. This process prevents ice crystal formation, resulting in amorphous, glass-like ice that retains the structural properties of liquid water [7].

Cellular samples can be broadly categorized as adherent (e.g., mammalian cells) or suspended (e.g., bacteria, yeast, algae, or certain mammalian cells). Adherent specimens are typically cultured directly on cryo-EM grids, while suspended specimens are applied to grids similarly to isolated biomolecules. The choice of vitrification method depends on sample type and thickness. Plunge freezing is suitable for specimens thinner than ~10 μm, although cryoprotectants are often required for samples exceeding ~5 μm [8]. For thicker specimens, such as organismal samples or tissues, high-pressure freezing is necessary to ensure effective vitrification and structural preservation [9]. These specimens can be vitrified in various carriers and are processed accordingly [8].

For imaging via modern transmission electron microscopy (TEM), samples must be thinner than 500 nm to achieve electron transparency. As many cellular and organismal samples exceed this limit in dense regions, such as near nuclei, imaging is often limited to thinner peripheral areas or requires methods to create electron-transparent windows.

Sample thinning

Cells and tissues can sometimes be rendered electron-transparent using mild purification methods—such as unroofing or cell permeabilization—although these approaches risk compromising sample integrity. To thin samples without chemical perturbation, cryogenic sectioning was initially developed, enabling cryo-EM of vitreous sections (CEMOVIS) [10,11]. In this technique, an ultramicrotome equipped with a cold diamond knife is used to slice ultrathin sections, which are then transferred onto cryo-EM grids for imaging. Although CEMOVIS has facilitated the visualization of large specimens, including tissues and multicellular organisms, artifacts induced by section compression frequently limit its utility.

In recent years, cryo-FIB milling has gained attraction for sample thinning [3]. This method employs ion beam–directed ablation to generate lamellae thinner than 300 nm and can be tailored for both plunge-frozen and high-pressure frozen sample types [12]. Despite its considerable utility, cryo-FIB milling is currently hindered by efficiency constraints due to time-consuming procedures. However, the adoption of plasma ion sources and automated pipelines are expected to significantly enhance its throughput [13]. Moreover, hybrid approaches that combine sectioning and cryo-FIB milling are now emerging [14]. By leveraging the strengths of both methods, these integrated strategies promise to expand the versatility of sample preparation, thereby enabling high-resolution imaging across a broader spectrum of biological specimens.

Feature localization

As the field advances to address increasingly complex questions in cellular and organismal biology, there is an increasing demand for spatiotemporal precision in cryo-EM and cryo-ET. Immunolabeling and the application of electron dense probes have long served as strategies to enable the in situ localization of cellular events and biomolecules [15,16]. However, the integration of fluorescent markers with cryo-imaging has led to correlative light and cryo-electron microscopy (cryo-CLEM) as a powerful means to enhance spatial and molecular precision [4,17].

In a typical cryo-CLEM workflow, vitrified samples are first imaged using cryogenic light (fluorescence) microscopy (cryo-LM) to identify features of interest. These fluorescence images are then aligned with scanning or transmission electron microscopy images via coordinate transformation, allowing seamless cross–modality correlation [18]. For events located in the thin sample areas, fluorescence guides area selection for high-resolution imaging. For events in thick regions, it guides cryo-sectioning or lamella preparation via FIB milling, and subsequent targeted imaging.

Although cryo-CLEM significantly enhances feature localization, the resolution attainable with cryo-LM remains lower than that of conventional, non-cryogenic systems. The adaptation of super-resolution fluorescence techniques—such as stimulated emission depletion (STED), structured illumination microscopy (SIM), photoactivatable localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM)—to cryogenic conditions has introduced single-molecule sensitivity and specificity [19]. However, workflows involving multiple instrument transfers increase the risk of sample contamination and devitrification due to exposure outside of vacuum. As a practical solution, integrated cryogenic instruments have been developed that incorporate fluorescence imaging directly within FIB chambers [20]. While the resolution of commercially available integrated setups is currently lower than that of stand-alone cryo-LM systems, they enable direct signal verification on lamellae, thereby improving the reliability of downstream analyses.

Imaging cellular and organismal samples

Cryo-EM and cryo-ET are both powerful techniques for structural studies of cellular and organismal samples, with the choice of method depending on the research objectives. These methods differ fundamentally in data acquisition strategies and the type of information they provide.

Cryo-EM captures single projection images at fixed sample stage angles, producing two-dimensional (2D) representations of the specimen. This approach underlies single-particle analysis (SPA), where individual projections provide randomly oriented views of purified biomolecules. Through computational alignment and averaging of these images, high-resolution three-dimensional (3D) structures can be reconstructed [21].

In contrast, cryo-ET acquires a series of projection images across a defined tilt range of the specimen, which are then computationally reconstructed into 3D volumes or tomograms [22,23]. To analyse recurring structural patterns within these tomograms, coordinates are selected using either template-based or template-free approaches [24,25]. This is followed by subvolume or subtomogram averaging (STA), which aligns and averages the extracted subvolumes to generate 3D reconstructions [26,27]. Consequently, cryo-ET enables the in situ visualization of macromolecular assemblies and cellular ultrastructure, preserving their native spatial and contextual organization.

While technological advancements have markedly improved the quality of cryo-EM data—enabling the visualization of cellular features previously beyond detection—the resolution achievable through STA continues to lag behind that attained via SPA. This limitation is largely due to the intrinsically low signal-to-noise ratio of cryo-ET data, which results from the densely packed cellular environment and the necessity of distributing the electron dose over a wide tilt range.

To mitigate these constraints, a range of hybrid data acquisition and processing strategies has emerged to maximize structural information [28–30]. In the cellular context, a notable development is hybrid single-particle tomography, in which a high-dose, untilted image is first acquired, followed by a tilt series of the same field of view [28,29]. This facilitates the determination of 3D structures, while preserving information on particle orientations and positions. Other approaches, such as 2D template matching, involve comparing projections of known structures against untilted images of thin cellular specimens, enabling the identification of macromolecular complexes without requiring tilt-series acquisition [31,32]. By combining the high-resolution potential of SPA with the spatial contextualization afforded by cryo-ET, these hybrid cryo-EM approaches offer a powerful framework for in situ structural biology—providing the possibility of achieving resolutions that surpass those attainable by conventional STA alone.

Parallel advances in image processing—particularly the integration of deep learning for structural pattern recognition, denoising, classification, and segmentation—have significantly enhanced the analytical capabilities of cryo-ET. A detailed discussion of these developments lies beyond the scope of the present review; readers are instead referred to the following comprehensive review for further information [6].

Applications of Cryo-EM and Cryo-ET

Subcellular architecture

Cryo-imaging techniques are essential for elucidating subcellular architecture and characterizing ultrastructural features across diverse biological contexts. Given the complexity of cellular interiors, research has traditionally focused on readily identifiable structures such as membranes, cytoskeletal elements, vesicles, and internalized or isolated pathogens. By segmenting these features, researchers have quantified key biophysical parameters, including membrane curvature, inter-feature distances, and filament persistence lengths [33–38]. These measurements have substantially refined our understanding of organelle ultrastructure, inter-organelle interactions, membrane dynamics, and overall subcellular morphology.

In recent years, cryo-ET has provided unprecedented ultrastructural insights into viral infections, illustrating key processes such as viral entry [39,40], nuclear import [41,42], the formation of replication organelles [43], and the hijacking of autophagy [44]. It has also elucidated infection strategies that pathogens adopt to subvert host cellular machinery [45]. Beyond viral pathogenesis, these investigations have been instrumental in uncovering biomolecular condensates across diverse cellular contexts [46,47], underscoring their fundamental role in cellular organization and function. Additionally, they have provided critical insights into inflammasome signaling by capturing NLRP3-activated ASC complexes [48], their associated mitochondrial dynamics, and inflammation-associated condensate formation at the microtubule-organizing centre [49].

Further, ultrastructural elucidations have advanced our understanding of protein quality control [50] and autophagy, shedding light on how cells orchestrate the degradation and recycling of damaged proteins, organelles, and pathogens [51–53]. These findings have significant implications for cellular stress responses and neurodegenerative conditions.

While cryo-ET investigations of neurons have been reported previously, recent studies represent extensions of this earlier work, continuing to characterize synaptic components such as vesicles and scaffold proteins—key elements for neurotransmission and synaptic plasticity [54,55]. In contrast, major recent advancements have been made at the tissue level, where cryo-ET has provided detailed insights into native hippocampal glutamatergic synapses in transgenic mouse brains [56], as well as β-amyloid and tau pathology in postmortem Alzheimer’s disease brains [57].

Across biological kingdoms, cryo-imaging has revealed fundamental structural adaptations that drive cellular function and evolution. For instance, it has elucidated the complex cellular architecture of the Asgard archaeon [58], demonstrated how ferrosome organelles mitigate nutritional immunity [59], and uncovered a protein-shell-mediated mechanism for CO2 fixation in diatom pyrenoids [60]. Collectively, these findings provide a structural framework for understanding cellular adaptations from an evolutionary perspective.

Macromolecules in cellular contexts

Imaging of cellular and organismal samples enables the structural characterization of macromolecular assemblies within their native contexts. While these methods are most effective when applied to large, abundant, high-contrast, or symmetric structures, they have significantly advanced our understanding of cellular machinery by providing unique insights into spatial organization, dynamics, and molecular mechanisms.

Historically, ribosomes have been among the most visually recognizable targets for in situ structure determination, offering invaluable insights into translation. These studies have enabled the visualization of translation dynamics under various conditions, including the effects of cancer drugs [61], ribosome collision stress [62], and chloramphenicol exposure [63].

Recent in situ analyses have expanded to other essential complexes, such as the chaperonins GroEL–GroES in bacteria [64] and TRiC (CCT) in human cells [65]. By defining their spatial organization and conformational states, these studies have provided valuable insights into protein folding mechanisms. Structural features of large assemblies, such as the nuclear pore complex basket [66] and cardiac myosin filaments [67], have also been elucidated. Additionally, cryo-ET has facilitated the visualization of centrosomal organization in Caenorhabditis elegans embryos [68] and identified tubulin chaperones as a distinct subset of microtubule luminal particles, suggesting a role in neuronal microtubule maintenance [69].

Viral pathogenesis has been a major area of advancement. In situ structural studies have provided mechanistic insights into SARS-CoV-2 infection [70], vaccinia virus maturation [71], rotavirus assembly [72], herpesvirus nuclear egress [73], and RNA genome packaging in bluetongue virus [74]. Native structures of the ChAdOx1 nCoV-19 vaccine product have also been resolved on the surface of transfected cells [75]. Further breakthroughs include the visualization of Ebola virus nucleocapsid assembly [76], viral factory maturation and dispersion [77], and copia virus-like particles in Drosophila ovarian cells and egg chambers [78].

These imaging approaches have also enhanced our understanding of host–pathogen interactions and immune defence. For example, they have clarified bacterial predation by ixotrophy [79], revealed the detailed architecture of actin networks in apicomplexans and polar tube dynamics in microsporidia [80], and unveiled the molecular organization of the human GBP1 defence complex [81].

Beyond pathogenesis, structural studies have illuminated carbon fixation strategies. The spatial arrangement of Rubisco within α- and β-carboxysomes [82–84], as well as within the pyrenoid of Chlamydomonas reinhardtii [85], has shed light on CO2-concentrating mechanisms in cyanobacteria and eukaryotic chloroplasts.

Furthermore, advances in imaging technologies and data quality have led to significant breakthroughs in resolving membrane protein complexes, such as SARS-CoV-2 vaccine spikes [75], the synaptic V-ATPase–synaptophysin complex [86], mitochondrial respiratory chain assemblies [87,88], bacterial chemoreceptor signalling arrays [89], the particulate methane monooxygenase [90], and the human prohibitin complex [91]. Notably, it is now also possible to resolve macromolecular assemblies within the relatively low-contrast environment of the nucleus. For example, this has enabled the visualization of chromatin fibers and nucleosome structures, opening new avenues for investigating three-dimensional genome organization and its regulation [92].

Concluding remarks

Despite significant advances in cryo-imaging over the past decade, its routine application remains hindered by several challenges. Sample preparation, target identification, and data interpretation continue to pose major obstacles. Although cryo-FIB milling has facilitated sample thinning, further refinement of vitrification and milling protocols is needed to accommodate the heterogeneity, morphology, and complexity of biological specimens, as well as to enhance throughput. Additionally, many intracellular structures remain uncharacterized, with densely packed cytoplasm and featureless nuclei contributing to poor signal-to-noise ratios and complicating structural interpretation. While cryo-CLEM supports targeting, its effectiveness is limited by low resolution, reliance on fluorescent labelling, and labour–intensive correlation workflows.

Looking forward, hybrid strategies such as integrating cryo-EM/ETwith volume EM offer exciting possibilities for bridging molecular detail with broader cellular context [93]. Artificial intelligence, though currently applied in limited ways—primarily in denoising and segmentation—holds substantial promise for the field [94,95]. As AI technologies advance, especially in areas like real-time targeting, automated acquisition, and biologically meaningful interpretation, they may drive paradigm-shifting changes in cryo-EM/ET. Tools such as SPACE tomo [96] and the emergence of annotated tomogram databases [97] mark important steps toward high-resolution cellular mapping and visual proteomics.

Acknowledgements

This work was supported by the Wellcome Trust Investigator Award (206422/Z/17/Z), the Wellcome Discovery Award (311427/Z/24/Z), the BBSRC grant (BB/S003339/1), the ERC AdG grant (101021133), and the NIH grants (AI184080, AI170791-7522).

Footnotes

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

No data was used for the research described in the article.

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Papers of particular interest, published within the period of review, have been highlighted as:

* of special interest

* * of outstanding interest

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Data Availability Statement

No data was used for the research described in the article.

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