Three-dimensional Electron Microscopy Research Articles

Program VUE: analysing distributions of cryo-EM projections using uniform spherical grids

Three-dimensional cryo electron microscopy reconstructions are obtained by extracting information from a large number of projections of the object. These projections correspond to different `views' or `orientations', i.e. directions in which these projections show the reconstructed object. Uneven distribution of these views and the presence of dominating preferred orientations may distort the reconstructed spatial images. This work describes the program VUE (views on uniform grids for cryo electron microscopy), designed to study such distributions. Its algorithms, based on uniform virtual grids on a sphere, allow an easy calculation and accurate quantitative analysis of the frequency distribution of the views. The key computational element is the Lambert azimuthal equal-area projection of a spherical uniform grid onto a disc. This projection keeps the surface area constant and represents the frequency distribution with no visual bias. Since it has multiple tunable parameters, the program is easily adaptable to individual needs, and to the features of a particular project or of the figure to be produced. It can help identify problems related to an uneven distribution of views. Optionally, it can modify the list of projections, distributing the views more uniformly. The program can also be used as a teaching tool.

Dynamic Behavior of Twisted UHMWPE Yarns

Twisting is an effective method to reduce defects during fabric weaving and improve the impact resistance of the armor material. This study examined the dynamic behavior of twisted ultra-high molecular weight polyethylene (UHMWPE) yarns. Material characterization was conducted using three-dimensional X-ray tomography and scanning electron microscopy (SEM). Dynamic mechanical experiments were carried out on the twisted yarns using a Kolsky bar, with the yarn's failure process captured in real time through high-speed imaging. Additionally, the digital imaging correlation (DIC) technique was used to calibrate the strain measured by the Kolsky bar. Post-fracture analysis was performed using SEM. The effects of twisting extent, strain rate, and gauge length on the dynamic behavior of twisted yarns were studied. It was observed that all twisted yarns underwent elongation, shrinkage, fiber breakage, untwisting, and yarn rupture. Furthermore, as the twisting degree and strain rate increased, and the gauge length decreased, the fracture area became more concentrated. The tensile strength peaked at a twisting degree of 250 t/m and decreased thereafter. Moreover, twisting was found to alter the sensitivity of the yarns' stress–strain curves and tensile properties to the strain rate and gauge length.

RoboEM: automated 3D flight tracing for synaptic-resolution connectomics

Mapping neuronal networks from three-dimensional electron microscopy (3D-EM) data still poses substantial reconstruction challenges, in particular for thin axons. Currently available automated image segmentation methods require manual proofreading for many types of connectomic analysis. Here we introduce RoboEM, an artificial intelligence-based self-steering 3D ‘flight’ system trained to navigate along neurites using only 3D-EM data as input. Applied to 3D-EM data from mouse and human cortex, RoboEM substantially improves automated state-of-the-art segmentations and can replace manual proofreading for more complex connectomic analysis problems, yielding computational annotation cost for cortical connectomes about 400-fold lower than the cost of manual error correction.

Multiscale observation of nano-oxide particles in oxide-dispersion-strengthened Cu alloys

Cu alloys with high durability and high thermal and electric conductivities are the key to achieving a more energetically efficient and eco-friendly future. Noble Zr-added oxide-dispersion-strengthened Cu alloys (ODS-Cu) with high strengths are promising materials for meeting all future service requirements. In this study, three-dimensional atom probe (3D-AP) and transmission electron microscopy (TEM) revealed that the majority of nano-oxide particles in the Zr-added ODS-Cu were novel yttria-stabilized zirconia (YSZ) particles with a diameter of less than 4 nm, which were completely different from Y2Zr2O7 and Y4Zr3O12 observed in other Zr-added ODS alloys. Multiscale observations including small-angle X-ray scattering (SAXS), 3D-AP, and TEM were performed to clarify the morphology of the oxide particles in the Zr-added ODS-Cu. Further strength enhancement through microstructural homogenization and specific incremental strength values were proposed.

Motion of VAPB molecules reveals ER-mitochondria contact site subdomains.

To coordinate cellular physiology, eukaryotic cells rely on the rapid exchange of molecules at specialized organelle-organelle contact sites1,2. Endoplasmic reticulum-mitochondrial contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signalling molecules, lipids and metabolites3,4. ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle5,6. However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation7,8, a clear understanding of their nanoscale organization and regulation is still lacking. Here we combine three-dimensional electron microscopy with high-speed molecular tracking of a model organelle tether, Vesicle-associated membrane protein (VAMP)-associated protein B (VAPB), to map the structure and diffusion landscape of ERMCSs. We uncovered dynamic subdomains within VAPB contact sites that correlate with ER membrane curvature and undergo rapid remodelling. We show that VAPB molecules enter and leave ERMCSs within seconds, despite the contact site itself remaining stable over much longer time scales. This metastability allows ERMCSs to remodel with changes in the physiological environment to accommodate metabolic needs of the cell. An amyotrophic lateral sclerosis-associated mutation in VAPB perturbs these subdomains, likely impairing their remodelling capacity and resulting in impaired interorganelle communication. These results establish high-speed single-molecule imaging as a new tool for mapping the structure of contact site interfaces and reveal that the diffusion landscape of VAPB at contact sites is a crucial component of ERMCS homeostasis.

The effects of axonal beading and undulation on axonal diameter estimation from diffusion MRI: Insights from simulations in human axons segmented from three-dimensional electron microscopy.

The increasing availability of high-performance gradient systems in human MRI scanners has generated great interest in diffusion microstructural imaging applications such as axonal diameter mapping. Practically, sensitivity to axon diameter in diffusion MRI is attained at strong diffusion weightings , where the deviation from the expected scaling in white matter yields a finite transverse diffusivity, which is then translated into an axon diameter estimate. While axons are usually modeled as perfectly straight, impermeable cylinders, local variations in diameter (caliber variation or beading) and direction (undulation) are known to influence axonal diameter estimates and have been observed in microscopy data of human axons. In this study, we performed Monte Carlo simulations of diffusion in axons reconstructed from three-dimensional electron microscopy of a human temporal lobe specimen using simulated sequence parameters matched to the maximal gradient strength of the next-generation Connectome 2.0 human MRI scanner ( 500 mT/m). We show that axon diameter estimation is accurate for nonbeaded, nonundulating fibers; however, in fibers with caliber variations and undulations, the axon diameter is heavily underestimated due to caliber variations, and this effect overshadows the known overestimation of the axon diameter due to undulations. This unexpected underestimation may originate from variations in the coarse-grained axial diffusivity due to caliber variations. Given that increased axonal beading and undulations have been observed in pathological tissues, such as traumatic brain injury and ischemia, the interpretation of axon diameter alterations in pathology may be significantly confounded.

Acoustic emission and splitting surface roughness of sandstone in a Brazilian splitting test under the influence of water saturation

Water weakens rock masses, which induces a series of engineering geological hazards. In this study, Brazilian splitting and acoustic emission (AE) experiments were conducted on sandstone with different saturation levels (0%, 25%, 50%, 75%, and 100%), and the effect of saturation on the AE and fracture characteristics was investigated. Meanwhile, nuclear magnetic resonance (NMR) test was used to analyze the water absorption and distribution characteristics of the samples with different saturation. Three-dimensional (3D) morphological scanning and scanning electron microscopy (SEM) were performed to analyze the roughness of the splitting surface. The tensile strength of rocks decreased from 3.05 MPa to 0.98 MPa with a moisture content from dry to saturated conditions, which obeyed an exponential function. The hardness of the sandstone decreased from dry to saturated by 56.6%, whereas the plasticity of the high-saturation sandstone increased. The AE counts and cumulative energy distribution decreased from 3880 and 39,550 to 1248 and 476, respectively, from dry to saturated conditions. Based on the Ib value, the rock damage process can be divided into three stages: fluctuation, stabilization, and decline. As the saturation increased, the initial Ib value decreased from 0.1047 to 0.0755. The development of large fractures increased during the splitting of the highly saturated rocks. With increasing saturation, the fracture surface joint roughness coefficient (JRC) and fractal dimension increased. The mean JRC increased from 5.98 to 13.6025. The unsaturated JRC showed an obvious dispersion compared with the dry and saturated sandstones. Combined with micro-macro characterization, it was found that an increase in saturation caused the rock to generate a weak structure. The damage gradually evolved from transgranular to intergranular fractures, leading to the macroscopic performance of rougher splitting surfaces and lower AE.

Electrochemical performance of lithium-ion batteries with two-layer gradient electrode architectures

Thick electrodes whose active materials have high areal density may improve the energy densities of lithium-ion batteries. However, the weakened rate abilities and cycle lifetimes of such electrodes significantly limit their practical applications. In this study, a modified two-dimensional model was built to evaluate the influence of the electrode structure on the lithiation process. Gradient electrodes with different particle sizes along the thickness direction are designed and fabricated via a two-layer coating process. The gradient design in the thickness direction is confirmed by three-dimensional digital microscopy, energy-dispersive spectroscopy, X-ray diffraction, and scanning electron microscopy. Galvanostatic charge/discharge cycling tests and electrochemical impedance spectroscopy are performed to investigate the electrochemical properties of the fabricated gradient electrodes. The simulation results indicate that the gradient electrode structure can enhance the utilization of electrode particles on the current collector side and alleviate the nonuniformity of the solid-phase Li concentration along the thickness direction. High electrochemical performance and long-term cycling stability of the designed gradient electrodes are verified by electrochemical test. The gradient structure fabricated via multi-layer coating processes can be used to improve the electrochemical performance of thick electrodes.

EMDB-the Electron Microscopy Data Bank.

The Electron Microscopy Data Bank (EMDB) is the global public archive of three-dimensional electron microscopy (3DEM) maps of biological specimens derived from transmission electron microscopy experiments. As of 2021, EMDB is managed by the Worldwide Protein Data Bank consortium (wwPDB; wwpdb.org) as a wwPDB Core Archive, and the EMDB team is a core member of the consortium. Today, EMDB houses over 30000 entries with maps containing macromolecules, complexes, viruses, organelles and cells. Herein, we provide an overview of the rapidly growing EMDB archive, including its current holdings, recent updates, and future plans.

Cell surface plasticity in response to shape change in the whole organism

Plasma membrane rupture can result in catastrophic cell death. The skeletal muscle fiber plasma membrane, the sarcolemma, provides an extreme example of a membrane subject to mechanical stress since these cells specifically evolved to generate contraction and movement. A quantitative model correlating ultrastructural remodeling of surface architecture with tissue changes invivo is required to understand how membrane domains contribute to the shape changes associated with tissue deformation in whole animals. We and others have shown that loss of caveolae, small invaginations of the plasma membrane particularly prevalent in the muscle sarcolemma, renders the plasma membrane more susceptible to rupture during stretch.1,2,3 While it is thought that caveolae are able to flatten and be absorbed into the bulk membrane to buffer local membrane expansion, a direct demonstration of this model invivo has been unachievable since it would require measurement of caveolae at the nanoscale combined with detailed whole-animal morphometrics under conditions of perturbation. Here, we describe the development and application of the "active trapping model" where embryonic zebrafish are immobilized in a curved state that mimics natural body axis curvature during an escape response. The model is amenable to multiscale, multimodal imaging including high-resolution whole-animal three-dimensional quantitative electron microscopy. Using the active trapping model, we demonstrate the essential role of caveolae in maintaining sarcolemmal integrity and quantify the specific contribution of caveolar-derived membrane to surface expansion. We show that caveolae directly contribute to an increase in plasma membrane surface area under physiologically relevant membrane deformation conditions.

SV2B defines a subpopulation of synaptic vesicles.

Synaptic vesicles can undergo several modes of exocytosis, endocytosis, and trafficking within individual synapses, and their fates may be linked to differences in the vesicular protein composition. Here, we mapped the intrasynaptic distribution of the synaptic vesicle proteins SV2B and SV2A in glutamatergic synapses of the hippocampus using three-dimensional electron microscopy. SV2B is almost completely absent from both docked vesicles and a distinct cluster of vesicles found near the active zone. In contrast, SV2A was found in all domains of the synapse and was slightly enriched near the active zone. SV2B and SV2A were found on the membrane in the peri-active zone, suggesting recycling from both clusters of vesicles. SV2B knockout mice displayed an increased seizure induction threshold only in a model employing high-frequency stimulation. Our data show that glutamatergic synapses generate molecularly distinct populations of synaptic vesicles and are able to maintain them at steep spatial gradients. The almost complete absence of SV2B from vesicles at the active zone of wildtype mice may explain why SV2A has been found to be more important for vesicle release.

Multiscale head anatomy of Megaphragma (Hymenoptera: Trichogrammatidae)

Methods of three-dimensional electron microscopy have been actively developed recently and open up great opportunities for morphological work. This approach is especially useful for studying microinsects, since it is possible to obtain complete series of high-resolution sections of a whole insect. Studies on the genus Megaphragma are especially important, since the unique phenomenon of lysis of most of the neuron nuclei was discovered in species of this genus. In this study we reveal the anatomical structure of the head of Megaphragma viggianii at all levels from organs to subcellular structures. Despite the miniature size of the body, most of the organ systems of M. viggianii retain the structural plan and complexity of organization at all levels. The set of muscles and the well-developed stomatogastric nervous system of this species correspond to those of larger insects, and there is also a well-developed tracheal system in the head of this species. Reconstructions of the head of M. viggianii at the cellular and subcellular levels were obtained, and of volumetric data were analyzed. A total of 689 nucleated cells of the head were reconstructed. The ultrastructure of M. viggianii is surprisingly complex, and the evolutionary benefits of such complexity are probably among the factors limiting the further miniaturization of parasitoid wasps.

Enhancement of Arc Erosion Resistance in AgCuO Electrical Contact Materials through Rare Earth Element Doping: First-Principles and Experimental Studies.

To investigate the stability and electrical and physical properties of undoped CuO and CuO doped with rare earth elements, electronic structures and elastic constants were calculated using first-principles density functional theory. Additionally, experimental verification was carried out on AgCuO and AgCuO-X (La, Ce, Y) electrical contacts, which were prepared using sol-gel and powder metallurgy methods. The contacts were tested under an 18 V/15 A DC resistive load using the JF04D contact material testing system. Arc parameters were analyzed, and three-dimensional surface profilometry and scanning electron microscopy were used to study the altered erosion morphology of the electrically contacted materials; moreover, the potential mechanisms behind their arc erosion behavior were investigated in depth. The results demonstrate that the doping of rare earth elements can improve the electrical conductivity and physical properties of the contacts, optimize the arc parameters, and enhance their resistance to arc erosion. Notably, AgCuO-Ce exhibited the highest electrical conductivity and the least amount of material transfer; moreover, it had excellent arc time and energy parameters, resulting in the best resistance to arc erosion. This study provides a theoretical basis for the screening of doping elements to enhance the performance of AgCuO contact materials and offers new ideas and scientific references for this field.

Chronic hyperglycemia reduces the expression of intercellular adhesion molecules and increases intercellular hyperpermeability in the periodontal epithelium.

Hyperglycemia in diabetes is closely associated with periodontal disease progression. This study aimed to investigate the effect of hyperglycemia on the barrier function of gingival epithelial cells as a cause of hyperglycemia-exacerbated periodontitis in diabetes mellitus. The abnormal expression of adhesion molecules in gingival epithelium in diabetes was compared between db/db and control mice. To study the effects of hyperglycemia on interepithelial cell permeability, the mRNA and protein expressions of adhesion molecules were investigated using a human gingival epithelial cell line (epi 4 cells) in the presence of either 5.5 mM glucose (NG) or 30 mM glucose (HG). Immunocytochemical and histological analyses were performed. We also studied HG-related intracellular signaling to assess abnormal adhesion molecule expression in the cultured epi 4 cells. The results of the proteomic analysis implied the abnormal regulation of cell-cell adhesion, and mRNA and protein expression assessments revealed the significant downregulation of Claudin1 expression in the gingival tissues of db/db mice (p < .05 vs control). Similarly, the mRNA and protein expressions of adhesion molecules were lower in epi 4 cells cultured under HG conditions than in those cultured under NG conditions (p < .05). Three-dimensional culture and transmission electron microscopy revealed reduced thickness of the epithelial cell layers with no flattened apical cells and heterogeneously arranged intercellular spaces among adjacent epi 4 cells under the HG. These results were consistent with the increased permeability of epi 4 cells under the HG relative to that of cells under the NG. This abnormal expression of intercellular adhesion molecules under the HG was related to the increased expression of receptors for advanced glycation end products (AGEs) and oxidative stress relative to that seen under the NG, along with stimulation of ERK1/2 phosphorylation in epi 4 cells. High glucose-induced impairment of intercellular adhesion molecule expression in gingival epithelial cells was related to the intercellular permeability of gingival cells, representing a possible link to hyperglycemia-related AGE signaling, oxidative stress, and ERK1/2 activation.

Formation and three-dimensional architecture of Leishmania adhesion in the sand fly vector.

Attachment to a substrate to maintain position in a specific ecological niche is a common strategy across biology, especially for eukaryotic parasites. During development in the sand fly vector, the eukaryotic parasite Leishmania adheres to the stomodeal valve, as the specialised haptomonad form. Dissection of haptomonad adhesion is a critical step for understanding the complete life cycle of Leishmania. Nevertheless, haptomonad studies are limited, as this is a technically challenging life cycle form to investigate. Here, we have combined three-dimensional electron microscopy approaches, including serial block face scanning electron microscopy (SBFSEM) and serial tomography to dissect the organisation and architecture of haptomonads in the sand fly. We showed that the attachment plaque contains distinct structural elements. Using time-lapse light microscopy of in vitro haptomonad-like cells, we identified five stages of haptomonad-like cell differentiation, and showed that calcium is necessary for Leishmania adhesion to the surface in vitro. This study provides the structural and regulatory foundations of Leishmania adhesion,which are critical for a holistic understanding of the Leishmania life cycle.

Atomic-Scale Corrugations in Crystalline Polypeptoid Nanosheets Revealed by Three-Dimensional Cryogenic Electron Microscopy.

Amphiphilic molecules that can crystallize often form molecularly thin nanosheets in aqueous solutions. The possibility of atomic-scale corrugations in these structures has not yet been recognized. We have studied the self-assembly of amphiphilic polypeptoids, a family of bio-inspired polymers that can self-assemble into various crystalline nanostructures. Atomic-scale structure of the crystals in these systems has been inferred using both X-ray diffraction and electron microscopy. Here we use cryogenic electron microscopy to determine the in-plane and out-of-plane structures of a crystalline nanosheet. Data were collected as a function of tilt angle and analyzed using a hybrid single-particle crystallographic approach. The analysis reveals that adjacent rows of peptoid chains, which are separated by 4.5 Å in the plane of the nanosheet, are offset by 6 Å in the direction perpendicular to the plane of the nanosheet. These atomic-scale corrugations lead to a doubling of the unit cell dimension from 4.5 to 9 Å. Our work provides an alternative interpretation for the observed Å X-ray diffraction peak often reported in polypeptoid crystals.

Secretion Bias of Lamellar Granules Revealed by Three-Dimensional Electron Microscopy

Secretion Bias of Lamellar Granules Revealed by Three-Dimensional Electron Microscopy

Spatial mapping of mitochondrial networks and bioenergetics in lung cancer

Mitochondria are critical to the governance of metabolism and bioenergetics in cancer cells1. The mitochondria form highly organized networks, in which their outer and inner membrane structures define their bioenergetic capacity2,3. However, in vivo studies delineating the relationship between the structural organization of mitochondrial networks and their bioenergetic activity have been limited. Here we present an in vivo structural and functional analysis of mitochondrial networks and bioenergetic phenotypes in non-small cell lung cancer (NSCLC) using an integrated platform consisting of positron emission tomography imaging, respirometry and three-dimensional scanning block-face electron microscopy. The diverse bioenergetic phenotypes and metabolic dependencies we identified in NSCLC tumours align with distinct structural organization of mitochondrial networks present. Further, we discovered that mitochondrial networks are organized into distinct compartments within tumour cells. In tumours with high rates of oxidative phosphorylation (OXPHOSHI) and fatty acid oxidation, we identified peri-droplet mitochondrial networks wherein mitochondria contact and surround lipid droplets. By contrast, we discovered that in tumours with low rates of OXPHOS (OXPHOSLO), high glucose flux regulated perinuclear localization of mitochondria, structural remodelling of cristae and mitochondrial respiratory capacity. Our findings suggest that in NSCLC, mitochondrial networks are compartmentalized into distinct subpopulations that govern the bioenergetic capacity of tumours.

Quantitative Measurement of Brain Extracellular Space with Three-Dimensional Electron Microscopy Imaging

Quantitative Measurement of Brain Extracellular Space with Three-Dimensional Electron Microscopy Imaging

Three-dimensional electron microscopy by STEM depth sectioning

Three-dimensional electron microscopy by STEM depth sectioning