In structural cellular biology applications, cryo-EM has two main focuses. One is SPA, a method routinely used to determine the 3D structures of macromolecular complexes with sub-nanometer resolution without the need of crystals or intrinsic symmetries.^[12] SPA is an averaging technique, which requires a large number of structurally identical protein complexes of interest, called “single particles”, to be frozen, imaged, aligned, and averaged. Recent advances in detector devices^[15–17] and software algorithms^[16,18–23] enable researchers to study structures of these architectures at atomic resolution.^[24–28] The compositional and conformational heterogeneity of the protein complexes of interest is a challenging problem, which needs to be addressed in order to achieve a sharp electron density map, upon which an atomic model can be automatically built.^[29]

Although SPA is a powerful technique, it has limitations. For example, structurally unique or pleiomorphic objects such as organelles, intact cells, and multi-cell systems are not accessible by SPA. Extracting high-fidelity structural information of certain supramolecular assemblies in their native, physiological, unperturbed, crowding cellular, or organellar environment is also challenging to SPA. Furthermore, as certain crucial cellular processes are carried out by molecular actors, which are only fully functional in transiently formed assemblies in the presence of their crowded cellular environment, a genuine understanding of their structural mechanism requires the acquisition of their molecular architecture in situ. Furthermore, several of these functional multi-component machineries are either challenging to purify^[30] or difficult to reconstitute in vitro; therefore, other methods are needed.

The other focus of cryo-EM is cryo-electron tomography (cryo-ET),^[31–33] which is a method with the potential to tackle these intriguing scientific problems. Cryo-ET enables dissecting faithfully preserved molecular mechanisms directly in frozen-hydrated cells. It allows direct visualization of unperturbed 3D structures of protein assemblies, organelles, cellular fragments, and multi-cellular systems with varying molecular detail in situ.^[14,34] Tomographic reconstruction, often followed by sub-tomogram averaging, has the capacity to achieve sub-nanometer resolution;^[35,36] the pipelines of the promising technique are reviewed in the following sections. Besides SPA and cryo-ET, there are other commonly used TEM approaches, such as electron crystallography,^[37,38] helical reconstruction,^[39,40] and icosahedral symmetry reconstruction.^[41] The different approaches of TEM are outlined in Table 1.

Table 1.

Table 1.

Table 1. Typical Approaches of TEM. . Single particle reconstruction Electron crystallography Helical reconstruction Icosahedral symmetry Electron tomography Symmetry any symmetry 2D crystals helixes icosahedral any symmetry Representative Ribosome (2.9 Å, 2015) Bacteriorho-dopsin (3 Å, 1999) TMV (4.5 Å, 2007) microtubule (3.5 Å, 2015) Capsid of bacteriophage Sf6 virion (2.9 Å, 2017) Immature HIV-1 CA-SP1 (3.9 Å, 2016) Ryanodine receptor (3.8 Å, 2015) aquaporin-1 (3.8 Å, 2000) actin (8 Å, 2013) Aquareovirus (3.3 A, 2010) immature HIV-1 CA-SP1 with novaCTF (3.4 Å, 2017) GroEL (3.5 Å, 2017) MAPEG proteins (2005) acetylcholine receptor (4 Å, 2003) flagella (4 Å, 2003) tulane virus (2.6 Å, 2016) salmonella type III secretion machine (17 Å, 2017)

Table 1. Typical Approaches of TEM. .

Molecules in cells rarely act alone; they interact with each other, stably or transiently, to form large functional modules.^[42,43] A thorough study of the spatial relationships of these intricate ensembles and complicated macromolecular machines would expand our knowledge of the mechanisms underlying their specialized cellular tasks.^[44] To achieve this essential in situ structural information, instead of 2D, a reliable 3D structural signature of these functional cellular content is absolutely necessary.^[45] Cryo-ET, developed by combining tomography with cryo-EM, provides faithful 3D structural blueprints for these cellular components (Fig. 1).^[46]

Fig. 1.

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Fig. 1. (color online) Basic pipelines of cryo-ET. Basic pipeline for cryo-ET. (a) The sample stages are usually tilted from −60° to +60°, (b) allowing projection images at varying angles to be recorded. (c) These 2D projection images are then reconstructed computationally to generate a 3D volumetric structure known as a “tomogram”. Structural features of interest in the tomogram are segmented either manually or automatically by a specialized visualization software, to create 3D annotations, which can then be used for both visualization and statistical analysis. (d) Repetitive structures in a tomogram can be extracted as “sub-tomograms” for furthering averaging. Modified with permission from Galaz-Montoya et al., 2017.

2.2.3. Sub-tomogram averaging and segmentation

The tomogram can be further analyzed in several ways. Macromolecular assemblies in multiple copies in a tomogram, such as phages at a certain stage in a bacterium, can be identified visually and then extracted into smaller volumes known as sub-tomograms.^[78] Sub-tomograms can be aligned and averaged to produce 3D structures with medium-to-high resolution (Fig. 2). This methodology is called sub-tomogram averaging^[79] and has increasingly been adopted for in situ observation of molecular architectures of macromolecular assemblies with multiple copies in the cell, such as macromolecular complexes, phages, ribosomes.^[80] Florian K M Schur et al. have determined the structure of HIV-1 capsid-SP1 to 3.9 Å using this method.^[81] Sub-tomogram averaging is widely used to solve structures of protein machines in vivo, often as a complementary method for the cryo-EM SPA technology. It is particularly useful when the studied complex is superimposed in projection by confounding densities and rendering the 2D classification in SPA is very difficult.

Fig. 2.

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Fig. 2. (color online) Overview of sub-tomogram averaging. Sub-tomograms are extracted from the tomogram. Each sub-tomogram contains a “repeating particle” of interest. Sub-tomograms are rotationally and translationally aligned against a reference. Then, the aligned sub-tomograms are averaged to generate a new reference. Then, the new reference is used for alignment of the sub-tomograms again. This procedure is repeated until the reference stabilizes. Modified with permission from Briggs et al., 2013.

For further non-uniform cellular features of interest, the segmentation method can be applied. Segmentation is performed either manually with specialized visualization packages such as Amira/Avizo^[82] or can be achieved automatically with algorithms such as the template-based matching developed by Wolfgang Baumeister’s group^[83] or convolutional neural-network (CNN) based tools implemented in EMAN 2.2,^[84] to produce a multicolor volume for purposes of better visualization. Informative movies clearly illustrating the spatial relationship of each features of interest can be created with segmented data, clarifying the observed morphology and therefore facilitating the derivation of the hypothesis.^[85] In certain cases, quantitative statistical analysis can be applied on the segmented tomograms to understand the biological/clinical problems in a more specialized way.^[86]

When integrated with other techniques, cryo-ET has the potential of mechanistically understanding cellular structural biology across wide size and time scales, sometimes in a more dynamic way.^[14] Although cryo-ET can provide structural information on the actual mechanisms of molecular assemblies in situ at molecular resolution, atomic details are often missing. This information gap can sometimes be partially complemented by atomic details of those mechanisms in vitro, from scientific tools like x-ray crystallography, nuclear magnetic resonance (NMR), and cryo-EM SPA. The integration of cryo-ET with these high-resolution in vitro techniques can generate structural information in a more native cellular context, closer to the empirical evidence.^[14] Cryo-ET also suffers from shortcomings, such as its smaller field of view, which can be complemented by fluorescent microscopy.^[87] Correlative light and electron microscopy (CLEM)^[88] is often used to pinpoint areas of interest in a cell on the EM grid before loading the sample for cryo-ET visualization, which gives the researcher structural details at a much higher resolution compared to that of light microscopy. Another challenge for cryo-ET is the thickness of the large eukaryotes. This problem can be solved by an essential integrative technique for cryo-ET: the cryo-focused ion beam (cryo-FIB) milling.^[89–91] Cryo-FIB has been shown to reproducibly yield homogenously thin, distortion-free, vitreous sections which are suitable for cryo-ET study.

Therefore, when combined with integrative approaches, cryo-ET has the potential to investigate structural biophysics in the cellular context. Consequently, cryo-ET is capable of bridging the gap between structural biophysics and cell biology.

Followed by sub-tomogram averaging, Cryo-ET has widely been adopted to study structural details of macromolecular assemblies.^[80] For example, it has been used to obtain the first 3D structure of the flagellar motor of leptospira, with novel features in the flagellar C ring, export apparatus, and stator.^[92] Cryo-ET has also been applied to understand the architecture of the flagellar motor, which is responsible for bacterial motility, revealing its functional mechanism from a structural point of view.^[93] It has been adopted to reveal the organization of microtubule and actin filaments^[94] to substantial detail in both eukaryotic and prokaryotic cells. It is crucial in understanding the nuclear pore complex (NPC) (Fig. 3) and the underlying nuclear lamina.^[95]

Fig. 3.

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Fig. 3. (color online) TEM Study of nuclear pore complex (NPC). (a) Callan and Tomlin isolated the nuclear membrane of the oocyte of Xenopus laevis, and reported orderly array pores in the outer layer by TEM for the first time in 1950. (b) Using the ultra-thin sectioning technique, Bahr and Beermann reported on the nuclear membrane of salivary glands. (c) Faberge isolated the nuclei of Oocytes of the new Taricha granulosa, and directly demonstrated the eight-fold symmetry of the nuclear pores. (d) Akey and Radermacher isolated the Oocytes of Xenopus, and solved the 3D structure of NPC by Cryo-EM in 1993, with the resolution of 25 Å. (e) Composite structure of the human NPC acquired by docking all available single or subcomplex crystal structures into the cryo-ET map of the intact human NPC (EMD-3103).

Another example should be mentioned is ribosome. Although its purified structure had been extensively studied by cryo-EM SPA,^[96–99] little was known about its dynamics and in situ structure. Robert Englmeier et al. discovered that mitoribosomes are organized in clusters in the inner membrane of the mitochondria, and they also identified the region, which mediates the contact of ribosome with the inner membrane by using sub-tomogram averaging.^[100] In the future, more and more attempts are expected to be made to decipher the detailed mechanisms and functions underlying the in situ structural architecture of the ribosome using cryo-ET and sub-tomogram averaging.

In certain cases cryo-ET is capable of discovering new phenomena, indicated by structures, which were directly visualized for the first time by it. Two examples are the architecture of the ring formed by tubulin homologue FtsZ in bacterial cell division^[101] and bacterial compartmentalization structures created by protein diffusion barriers.^[102]

In addition to high-resolution structures obtained from averaging, pleomorphic structures are also extensively studied by cryo-ET, often followed by other integrative methods such as CLEM or cryo-FIB. For example, research on human platelet structural variation with cryo-ET (Fig. 4) indicates the possibility of using cryo-ET as a clinical diagnostic tool.^[86] Furthermore, an in situ structure of Chlamydomonas chloroplast is visualized using cryo-ET, in combination with cryo-FIB,^[103] and cryo-ET, together with CLEM, are used to discover a novel member of the bacterial cytoskeleton.^[104]

Fig. 4.

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Fig. 4. (color online) Cryo-ET has potential as a clinical diagnostic technique on a single cell level. Platelets from control subjects and patients with cancer, annotated tomogram. (a) Three randomly selected annotated platelets from healthy donors. All of them have an intact marginal band of microtubules (blue) enclosing most of their granules (α pink, dense green) and mitochondria (red) inside. The plasma membrane is gray and the low-contrast vacuole-like (LCV) feature is yellow. (b) Three randomly selected annotated platelets from patients with benign masses. Their structures are similar to those shown in panel (a). (c) Three randomly selected annotated platelets from patients with invasive ovarian cancer. Their morphologies appear to be more heterogeneous compared with the six platelets in the other two panels. They seem to have more LCV, fewer and shorter microtubule filaments, and more mitochondria. Modified with permission from Wang et al., 2015.

Previously, charge-coupled detectors (CCDs) were used in cameras for image acquisition for cryo-EM and cryo-ET.^[105] Electrons are not directly detected in a CCD camera, but they are converted to photons when passing through a scintillator. Following this, a fiber-optic bundle transmits the photons and converts them to charges before detection on the sensor. Although faster than the film as a recording device, the conversions not only degrade the high-frequency information, but also inevitably append noise to the image, reducing the chance of retrieving high-resolution signal significantly.^[11] The introduction of direct detector device (DDD) cameras^[106] revolutionized both cryo-EM and cryo-ET.

As its name suggests, DDD cameras have a complementary metal-oxide semiconductor (CMOS) chip enabling direct detection of electrons.^[107] These cameras exhibit a significant improvement in detective quantum efficiency (DQE) compared to CCDs.^[108] DDDs also have a high frame rate, enabling fine-tuning of the acquired data if motion correction is performed. Motion correction is crucial for achieving atomic resolution in cryo-EM studies as it significantly reduces the beam-induced movement of the sample during exposure.^[16] Among the commercially available DDD cameras, the Gatan K2 Summit is specifically designed for counting individual electrons. Counting further improves DQE, eliminates noise, and reduces the artifacts due to the point spread function (PSF) effect from each incident electron hitting the camera.^[109] With the application of DDDs, both cryo-EM and cryo-ET have experienced a so-called “resolution revolution”.

Although the “resolution revolution”^[110] significantly enhanced the performance of both cryo-EM and cryo-ET, both techniques still suffer from the fundamental dose limit problem. This is further exacerbated by the low contrast of the cryo-ET images of frozen hydrated biological samples predominantly comprising of light elements. As phase contrast, not amplitude contrast is the dominant form of contrast in the cryo-ET pictures, enhancing the phase contrast of the cryo-ET images is crucial for retrieving structural information from biological specimens. Traditionally, the phase contrast in a typical cryo-ET imaging session is generated by defocusing the objective lens.^[111] However, images produced using this method, especially those taken under high under-focus conditions, suffer from a dramatic degradation of high-frequency signal; therefore, a novel methodology is required.

The idea of phase plate was theoretically proposed during the early application of cryo-EM, and phase contrast can be generated over a wide range of spatial frequencies without the need of defocusing.^[112] Phase plate has been adopted in cryo-ET studies and it enables the visualization of unprecedented ultrastructural details in a cellular environment. The application of phase plate to cryo-ET begin with the use of the thin film Zernike phase plate (ZPP),^[113] which comprises a thin carbon film with a tiny hole at the center. Dai et al. successfully used ZPP to investigate the maturation process of cyanophage Syn5 inside Synechococcus host cells.^[110,111] Although high quality results can be achieved, ZPP has practical limitations. The first problem with ZPP is its short lifespan: the performance of ZPP degrades quickly and therefore it needs to be replaced frequently. A further problem is that ZPP cannot be used in automated data acquisition sessions, as the hole of the ZPP on the beam path needs to be centered manually. These problems lead to low efficiency in ZPP tomographic data collection; therefore, restricting its range of application.

Another type of phase plate, the Volta phase plate (VPP), which solved most of the practical issues with ZPP, was proposed by Radostin Danev.^[114] The design of the VPP is very similar to that of the ZPP, but without the central hole. When being irradiated by an electron beam, the heated carbon film displays a negative “Volta potential” which, when used constructively, generates a phase shift with the same sign and magnitude to that of a ZPP. Experimental evidence suggests that beam-induced surface modifications are essential in the process resulting in the “Volta potential”. Contrary to the ZPP, the VPP has a relatively longer usable life. Furthermore, images taken with the VPP do not show fringing artifacts commonly seen in images taken with the ZPP. Moreover, as the phase shift is self-created by the electron beam, a VPP can be easily implemented in the automated tomographic image acquisition schemes. Owing to these improvements over the ZPP, the VPP has been widely used in cryo-ET studies in several systems following its recent introduction. Asano et al. studied the spatial distribution, state of assembly, and conformational states of 26S proteasome in neurons using VPP cryo-ET, proving the potential of cryo-ET to reveal structural information in situ.^[115] Mahamid et al. observed the meshwork of nuclear lamina in situ for the first time with this approach.^[116] Englmeier R et al. studied ribosomes with VPP cryo-ET. In their work, human ribosomes tethering to the inner membrane of mitochondria could not be identified clearly in the tomograms for later classification and for sub-tomogram averaging procedures without the application of VPP.^[100] The performance of the VPP was also tested in combination with other techniques. For instance, Fan X et al. demonstrated the feasibility of combining the VPP with spherical aberration (Cs) correction for high-resolution imaging at both over and under-focus conditions.^[117] VPP cryo-ET is powerful, but still suffers from practical issues, such as the electrostatic charging of the specimen^[118] and challenges for maintaining accurate focus throughout the tilt series.^[118,119]

A further important limitation of the cryo-ET is the accessible thickness of the specimen; thus, the cryo-ET approach is not suitable for most of the eukaryotic cells with thickness over 1 μm. Therefore, the thickness problem needs to be addressed.

Several solutions have been suggested to address this problem. One approach is to mechanically cut the biological specimens to sufficiently thin sections with a diamond knife.^[120] This approach is known as cryo-electron microscopy of vitreous section, or CEMOVIS, for short.^[121,122] So far, different types of specimens, such as mitochondria, human skin, microtubules, bacteria, and bacterial spores, etc., have been studied with this method.^[123–127] However, this technique does have limitations. The most severe one is the compression along the cutting direction, which almost inevitably distorts the observed structure to a certain degree,^[128] significantly complicating the accurate scientific interpretation of the data. Because of this problem, another approach, the cryo-focused ion beam (cryo-FIB) milling has emerged as an alternative.^[129–132]

In cryo-FIB milling, the regions of interest within the sample are abraded layer-by-layer by a beam of accelerated ions from both above and below, and consequently, lamellas with thickness of approximately 200 nm, suitable for cryo-ET analysis, are obtained.^[133] Cryo-FIB milling significantly expands the boundaries of cryo-ET, rendering almost any interior regions of interest in any types of cells suitable for cryo-ET study.^[89] As a result, cryo-FIB milling, in combination with cryo-ET, has been increasingly applied in dissecting interesting features, reactions, and processes in cells or simple organisms. In the following, a few examples are provided. Jasnin et al. studied the effect of reorganization of actin filaments on the ability of Dictyostelium discoideum to adapt to the environment.^[94] Hagen and colleagues discovered the mechanism underlying the herpesvirus capsid budding from the nuclear envelope.^[134] A research conducted by Wolfgang Baumeister’s group showed the native architecture of Chlamydomonas chloroplast in situ.^[103] These significant achievements by cryo-FIB milling demonstrate the feasibility of this technique used together with cryo-ET to prepare and study thin, distortion-free lamellae of frozen hydrated biological materials in situ.^[135]

Correlative light and electron microscopy (CLEM) has become widely used in structural studies of cells and tissues. Cryo-CLEM combines spatiotemporal information from dynamic fluorescent microscopy with high-resolution in situ structural architecture from cryo-ET.^[88,90,136] As previously shown, cryo-ET alone has several limitations. Sample thickness is a major issue, which can be solved by combing cryo-ET with cryo-FIB.^[137] Its inability to unambiguously locate a particular reaction or process within the crowded cellular environment, partly due to the low contrast, low signal-to-noise ratio (SNR), and low copy number of target molecules of cryo-ET, is another limitation which restricts its applicability. Moreover, cryo-ET typically has a relatively smaller field of view compared to light microscopy, which further limits its potential of locating specific areas of interest in large cells. CLEM, which undergoes many technical improvements recently,^[88] can be adopted as a complementary approach to cryo-ET, because it can accurately label and target specific complexes or events using fluorescent dye. This integrative method has been applied to study cell division in vitrified Streptomyces bacteria.^[138] It was also applied to study virus infected or transfected mammalian cells.^[139] Therefore, CLEM together with cryo-ET, has the potential of bridging the gap between cellular biology and structural biophysics.

Observing macromolecular complexes in situ is a significant accomplishment, but currently achieving near-atomic resolution routinely is very challenging for sub-tomogram averaging. One major problem is the difficulty to accurately perform contrast transfer function (CTF) correction for the tomographic data. As CTF is an oscillating sine function depending on the defocus parameter, determining the accurate defocus is essential. It should be noted that there is a defocus gradient across the tilting angle within each tilt series collected. Therefore, each tilting projection needs to be CTF-corrected multiple times. This is commonly performed by a strip-based correction algorithm. Moreover, thick specimens, such as cells, have multiple features of interests reside in different Z-heights within the sample. A recent publication by Turonova et al. introduced a NovaCTF-correction algorithm, where both the CTF gradient across the tilting angles and the CTF gradient due to the sample thickness are considered.^[36] They achieved a result with 3.4-Å resolution with sub-tomogram averaging using this correction method. Galaz-Montoya et al. also demonstrated their CTF-correction method, which can routinely yield sub-tomogram averages close to 4/5 Nyquist frequency of the detector under their experimental conditions.^[140] Performing accurate CTF correction for tomographic data has been gradually becoming a routine in the cryo-ET field.