On geometric artifacts in cryo electron tomography

Cryogenic single-particle photoluminescence (PL) spectroscopy has been used with great success to directly observe the heterogeneous photophysical states present in a population of luminescent particles. Cryogenic electron tomography provides complementary nanometer scale structural information to PL spectroscopy, but the two techniques have not been correlated due to technical challenges. Here, we present a method for correlating single-particle information from these two powerful microscopy modalities. We simultaneously observe PL brightness, emission spectrum, and in-plane excitation dipole orientation of CdSSe/ZnS quantum dots suspended in vitreous ice. Stable and fluctuating emitters were observed, as well as a surprising splitting of the PL spectrum into two bands with an average energy separation of 80 meV. In some cases, the onset of the splitting corresponded to changes in the in-plane excitation dipole orientation. These dynamics were assigned to structures of individual quantum dots and the excitation dipoles were visualized in the context of structural features.

In the recent years, cryo-electron microscopy (cryo-EM) single-particle analysis (SPA) has greatly benefited from a multitude of technological advances cumulating in breaking the atomic-resolution barrier for biological macromolecules. Since then, structural insights by cryo-EM have led to driving rational design of new vaccines and therapeutics due to high throughput and high-resolution screening and data collection technologies. Additionally, cryo-electron tomography (cryo-ET) has become an indispensable tool for structural cell biology. In this presentation we elaborate on the latest Thermo Fisher Scientific innovations for 300kV cryo electron microscopy for SPA and cryo-ET to advance more workflow efficiency and performance. Our ongoing mission is to further improve the connectivity between the cryo-FIB and cryo-TEM for an even more streamlined tomography workflow that is safer, faster and cleaner. Data acquisition and processing can be a significant cryo-ET bottleneck. Our integrated solution, combining Thermo Scientific Tomography 5 and Tomo Live, automates pre-acquisition tasks, enhances productivity with predictive tracking and enables rapid quality assessment. Robust and optimized motion correction helps produce crisp tomograms and image quality needed for sub-tomogram averaging. Results are displayed with new quality metrics on a web platform, making the workflow user-friendly and facilitating structural biology advancements.

This review of the development and current status of electron tomography deals mainly with the mathematical and algorithmic aspects. After a very brief description of the role of electron tomography in structural biology, we turn our attention to the derivation of the forward operator. Starting from the Schrödinger equation, the electron–specimen interaction is modelled as a diffraction tomography problem and the picture is completed by adding a description of the optical system of the transmission electron microscope. The first-order Born approximation enables one to explicitly express the intensity for any finite wavenumber in terms of the propagation operator acting on the specimen convolved with a point spread function, derived from the optics in the transmission electron microscope. Next, we focus on the difficulties that cause the reconstruction problem to be quite challenging. Special emphasis is put on explaining the extremely low signal-to-noise ratio in the data combined with the incomplete data problems, which lead to severe ill-posedness. The next step is to derive the standard phase contrast model used in the electron tomography community. The above-mentioned expression for the intensity generalizes the standard phase contrast model which can be obtained by replacing the propagation operator by its high-energy limit, the x-ray transform, as the wavenumber tends to infinity. The importance of more carefully including the wave nature of the electron–specimen interaction is supported by performing an asymptotic analysis of the intensity as the wavenumber tends to infinity. Next we provide an overview of the various reconstruction methods that have been employed in electron tomography and we conclude by mentioning a number of open problems. Besides providing an introduction to electron tomography written in the ‘language of inverse problems’, the authors hope to raise interest among experts in integral geometry and regularization theory for the mathematical and algorithmic difficulties that are encountered in electron tomography.

The type three secretion system (T3SS) is a bacterial weapon found in many Gram-negative bacteria. It consists out of a needle like structure called injectisome, which enables bacteria to inject effector proteins into eukaryotic cells. The injectisome is a very complex molecular machine, inserted in the inner and outer bacteria membrane, passing the periplasm with the peptidoglycan layer. We used an approach with fluorescent hybrid proteins to study the assembly order of the injectisome in Yersinia enterocolitica. First the outer membrane protein YscC was genetically fused to the red fluorescent protein mCherry. This construct was able to build fluorescent foci in the bacterial membrane by itself, without any other components of the T3SS. Then we engineered fusions between the green fluorescent protein EGFP and the inner membrane protein YscD, the putative C-ring protein YscQ or the ATPase YscN. All three constructs showed fluorescent foci at the bacterial membrane. Comparison of the different EGFP constructs with the YscC-mCherry construct in double mutants showed that the proteins co-localize. Thus we considered the foci to be a read out for injectisome assembly. By combining the EGFP constructs with different deletion mutants we found that the assembly occurs from the outside to the inside. Starting with the outer membrane protein YscC to the inner membrane. Then the ATPase and C-ring assemble together and finally the needle is formed. 
\nDifferent single particle structures of T3SS, needle complexes, purified from Shigella flexneri and Salmonella enterica are available. But in the course of purification the inner membrane export apparatus, the ATPase complex and the C-ring were lost. As well no such complex has been purified from Y. enterocolitica. Thus we investigated the Y. enterocolitica injectisomes in situ by cryo electron tomography (cryo-ET). Unfortunately, the 1-?m diameter of Y. enterocolitica is too large to obtain optimal resolution with cryo-ET. Thus we engineered a minD mutant that forms so called minicells, due to asymmetric septum placement. We collected tomograms of particles from wild type and minicells and constructed an average structure with a resolution of 3.7 nm. In addition the 6 nm resolution in situ structure of the injectisome of S. flexneri was made. We saw significant stretching of the in situ structure compared to the isolated particles. Moreover we saw flexibility of the basal body. We can only speculate that such flexibility might increase the stability of the structure and protect it of mechanical forces. In addition for the first time we could visualize a mass in cytoplasm just below the middle of the injectisome. Due to homology to the flagellum we can assume, that this is the ATPase. But to conclusively assign proteins to masses seen in the in situ structure we would need to label them.
\nA question mark in the Y. enterocolitica injectisome assembly is, how the structure can pass the peptidoglycan layer. The flagellum, which is evolutionary closely related to the injectisome, as well as other types of injectisomes have a muramidase or more specifically lytic transglycosylase (LT) encoded within their loci. No gene encoding for a LT, can be found on the pYV plasmid that encodes otherwise for the entire T3SS. Thus we tested several genomic LT for their involvement in the assembly of the T3SS. It is possible that the injectisome assembles through temporary gaps generated during the synthesis of new peptidoglycan strands. This theory is very intriguing, as the pattern of the fluorescent foci resembled closely the arrangement of the bacterial cytoskeleton protein MreB, which is assumed to be involved in placing the peptidoglycan synthesis machinery. Comparing the localization of MreB and the injectisome showed that both constructs seem to be arranged on two different helical paths. This convinced us that injectisome arrangement is not stochastic but rather controlled. To find the underlying structure responsible for this arrangement, we compared the localization of other proteins with similar arrangement as MreB. In Bacillus subtilis different membrane compositions were shown to be helically distributed. Unfortunately staining of the inner membrane is difficult in Gram-negative bacteria. Therefore we compared injectisome location with a potential bacterial lipid raft marker, which showed injectisomes do not insert into the lipid rafts. 
\nThus although our knowledge about the assembly, the structure and the function of the T3SS is improving enormously, the question of how injectisomes are localized remains to be answered.
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Cryogenic electron tomography (cryoET) allows visualization of cellular structures in situ. However, anisotropic resolution arising from the intrinsic “missing-wedge” problem has presented major challenges in visualization and interpretation of tomograms. Here, we have developed IsoNet, a deep learning-based software package that iteratively reconstructs the missing-wedge information and increases signal-to-noise ratio, using the knowledge learned from raw tomograms. Without the need for sub-tomogram averaging, IsoNet generates tomograms with significantly reduced resolution anisotropy. Applications of IsoNet to three representative types of cryoET data demonstrate greatly improved structural interpretability: resolving lattice defects in immature HIV particles, establishing architecture of the paraflagellar rod in Eukaryotic flagella, and identifying heptagon-containing clathrin cages inside a neuronal synapse of cultured cells. Therefore, by overcoming two fundamental limitations of cryoET, IsoNet enables functional interpretation of cellular tomograms without sub-tomogram averaging. Its application to high-resolution cellular tomograms should also help identify differently oriented complexes of the same kind for sub-tomogram averaging.

Resolving structural heterogeneity in situ through cryogenic electron tomography

Electron tomography (ET) offers a unique capacity to image biological structures in situ. However, the resolution of ET reconstructed tomograms is not comparable to that of the single-particle cryo-EM. If many copies of the object of interest are present in the tomograms, their structures can be reconstructed in the tomogram, picked, aligned and averaged to increase the signal-to-noise ratio and improve the resolution, which is known as the subtomogram averaging. To date, the resolution improvement of the subtomogram averaging is still limited because each reconstructed subtomogram is of low reconstruction quality due to the missing wedge issue. In this article, we propose a novel computational model, the constrained reconstruction model (CRM), to better recover the information from the multiple subtomograms and compensate for the missing wedge issue in each of them. CRM is supposed to produce a refined reconstruction in the final turn of subtomogram averaging after alignment, instead of directly taking the average. We first formulate the averaging method and our CRM as linear systems, and prove that the solution space of CRM is no larger, and in practice much smaller, than that of the averaging method. We then propose a sparse Kaczmarz algorithm to solve the formulated CRM, and further extend the solution to the simultaneous algebraic reconstruction technique (SART). Experimental results demonstrate that CRM can significantly alleviate the missing wedge issue and improve the final reconstruction quality. In addition, our model is robust to the number of images in each tilt series, the tilt range and the noise level. The codes of CRM-SIRT and CRM-SART are available at https://github.com/icthrm/CRM. Supplementary data are available at Bioinformatics online.

Progressive Stochastic Reconstruction Technique (PSRT) for cryo electron tomography.

Structural features of the salivary gland hypertrophy virus of the tsetse fly revealed by cryo-electron microscopy and tomography

The demand for ever more complex nanostructures in materials and soft matter nanoscience also requires sophisticated characterization tools for reliable visualization and interpretation of internal morphological features. Here, we address both aspects and present synthetic concepts for the compartmentalization of nanoparticle peripheries as well as their in situ tomographic characterization. We first form negatively charged spherical multicompartment micelles from ampholytic triblock terpolymers in aqueous media, followed by interpolyelectrolyte complex (IPEC) formation of the anionic corona with bis-hydrophilic cationic/neutral diblock copolymers. At a 1:1 stoichiometric ratio of anionic and cationic charges, the so-formed IPECs are charge neutral and thus phase separate from solution (water). The high chain density of the ionic grafts provides steric stabilization through the neutral PEO corona of the grafted diblock copolymer and suppresses collapse of the IPEC; instead, the dense grafting results in defined nanodomains oriented perpendicular to the micellar core. We analyze the 3D arrangements of the complex and purely organic compartments, in situ, by means of cryogenic transmission electron microscopy (cryo-TEM) and tomography (cryo-ET). We study the effect of block lengths of the cationic and nonionic block on IPEC morphology, and while 2D cryo-TEM projections suggest similar morphologies, cryo-ET and computational 3D reconstruction reveal otherwise hidden structural features, e.g., planar IPEC brushes emanating from the micellar core.

Force generation by actin assembly shapes cellular membranes. An experimentally constrained multiscale model shows that a minimal branched actin network is sufficient to internalize endocytic pits against membrane tension. Around 200 activated Arp2/3 complexes are required for robust internalization. A newly developed molecule-counting method determined that ~200 Arp2/3 complexes assemble at sites of clathrin-mediated endocytosis in human cells. Simulations predict that actin self-organizes into a radial branched array with growing ends oriented toward the base of the pit. Long actin filaments bend between attachment sites in the coat and the base of the pit. Elastic energy stored in bent filaments, whose presence was confirmed by cryo-electron tomography, contributes to endocytic internalization. Elevated membrane tension directs more growing filaments toward the base of the pit, increasing actin nucleation and bending for increased force production. Thus, spatially constrained actin filament assembly utilizes an adaptive mechanism enabling endocytosis under varying physical constraints.

Single particle and molecular assembly analysis of polyribosomes by single- and double-tilt cryo electron tomography

Transmission electron microscopy can be used for the characterisation of a wide range of thin specimens, but soft matter and aqueous samples such as gels, nanoparticle dispersions, and emulsions will dry out and collapse under the microscope vacuum, therefore losing information on their native state and ultimately limiting the understanding of the sample. This study examines commonly used techniques in transmission electron microscopy when applied to the characterisation of cryogenically frozen Pickering emulsion samples. Oil-in-water Pickering emulsions stabilised by 3 to 5nm platinum nanoparticles were cryogenically frozen by plunge-freezing into liquid ethane to retain the native structure of the system without inducing crystallisation of the droplet oil cores. A comparison between the droplet morphology following different sample preparation methods has confirmed the effectiveness of using plunge-freezing to prepare these samples. Scanning transmission electron microscopy imaging showed that dry droplets collapse under the microscope vacuum, changing their shape and size (average apparent diameter: ∼342nm) compared to frozen samples (average diameter: ∼183nm). Cryogenic electron tomography was used to collect additional information of the 3D shape and size of the emulsion droplets, and the position of the stabilising nanoparticles relative to the droplet surface. Cryogenic energy dispersive X-ray and electron energy loss spectroscopy were used to successfully obtain elemental data and generate elemental maps to identify the stabilising nanoparticles and the oil phase. Elemental maps generated from spectral data were used in conjunction with electron tomography to obtain 3D information of the oil phase in the emulsion droplets. Beam-induced damage to the ice was the largest limiting factor to the sample characterisation, limiting the effective imaging resolution and signal-to-noise ratio, though careful consideration of the imaging parameters used allowed for the characterisation of the samples presented in this study. Ultimately this study shows that cryo-methods are effective for the representative characterisation of Pickering emulsions.

The structures of a series of 3D-SBA-15 materials have been systematically studied by X-ray diffraction (XRD), nitrogen sorption, and electron tomography (ET) techniques. All the 3D-SBA-15 materials possess an ordered 2D hexagonal mesostructure with similar cell parameters, while the pore size measured from nitrogen sorption increases with the hydrothermal treatment temperature, in some cases even larger than the cell parameter calculated from X-ray diffraction. Taking advantage of the ET technique, it is shown that there exist two types of pores in 3D-SBA-15 materials: one is the ordered channels associated with the hexagonal symmetry, and the other is locally disordered and randomly distributed merged pores. During the hydrothermal treatment events at various temperatures, the size of ordered channels changes little, while the volume fraction and the overall size of the latter type of pores increase and eventually become dominant. Thus, the attribution of the pore sizes obtained from nitrogen sorption analysis to which types of pores is synthesis condition dependent and ET is an important tool to answer this question.

Cryogenic-electron tomography (cryo-ET) has provided an unprecedented glimpse into the nanoscale architecture of cells by combining cryogenic preservation of biological structures with electron tomography. Micropatterning of extracellular matrix proteins is increasingly used as a method to prepare adherent cell types for cryo-ET as it promotes optimal positioning of cells and subcellular regions of interest for vitrification, cryo-focused ion beam (cryo-FIB) milling, and data acquisition. Here we demonstrate a micropatterning workflow for capturing minimally adherent cell types, human T-cells and Jurkat cells, for cryo-FIB and cryo-ET. Our affinity capture system facilitated the nanoscale imaging of Jurkat cells, revealing extracellular filamentous structures. It improved workflow efficiency by consistently producing grids with a sufficient number of well positioned cells for an entire cryo-FIB session. Affinity capture can be extended to facilitate high resolution imaging of other adherent and non-adherent cell types with cryo-ET.