Three-dimensional Electron Microscopy Research Articles

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

In situ electron tomography for the thermally activated solid reaction of anaerobic nanoparticles.

The nanoscale characterization of thermally activated solid reactions plays a pivotal role in products manufactured by nanotechnology. Recently, in situ observation in transmission electron microscopy combined with electron tomography, namely four-dimensional observation for heat treatment of nanomaterials, has attracted great interest. However, because most nanomaterials are highly reactive, i.e., oxidation during transfer and electron beam irradiation would likely cause fatal artefacts; it is challenging to perform the artifact-free four-dimensional observation. Herein, we demonstrate our development of a novel in situ three-dimensional electron microscopy technique for thermally activated solid-state reaction processes in nanoparticles (NPs). The sintering behaviour of Cu NPs was successfully visualized and analyzed in four-dimensional space-time. An advanced image processing protocol and a newly designed state-of-the-art MEMS-based heating holder enable the implementation of considerably low electron dose imaging and prevent air exposure, which is of central importance in this type of observation. The total amount of electron dose for a single set of tilt-series images was reduced to 250 e- nm-2, which is the lowest level for inorganic materials electron tomography experiments. This study evaluated the sintering behaviour of Cu NPs in terms of variations in neck growth and particle distance. A negative correlation between the two parameters is shown, except for the particle pair bound by neighbouring NPs. The nanoscale characteristic sintering behavior of neck growth was also captured in this study.

Three-dimensional analysis of membrane structures associated with tomato spotted wilt virus infection.

To study viral infection, the direct structural visualization of the viral life cycle consisting of virus attachment, entry, replication, assemblyand transport is essential. Although conventional electron microscopy (EM) has been extremely helpful in the investigation of virus-host cell interactions, three-dimensional (3D) EM not only provides important information at the nanometer resolution, but can also create 3D maps of large volumes, even entire virus-infected cells. Here, we determined the ultrastructural details of tomato spotted wilt virus (TSWV)-infected plant cells using focused ion beam scanning EM (FIB-SEM). The viral morphogenesis and dynamic transformation of paired parallel membranes (PPMs) were analyzed. The endoplasmic reticulum (ER) membrane network consisting of tubules and sheets was related to viral intracellular trafficking and virion storage. Abundant lipid-like bodies, clustering mitochondria, cell membrane tubules, and myelin-like bodies were likely associated with viral infection. Additionally, connecting structures between neighboring cells were found only in infected plant tissues and showed the characteristics of tubular structure. These novel connections that formed continuously in the cell wall or were wrapped by the cell membranes of neighboring cells appeared frequently in the large-scale 3D model, suggesting additional strategies for viral trafficking that were difficult to distinguish using conventional EM.

Surface texture and heat treatment on the friction performance of cam tappet experimental and fluid-solid coupling numerical study

In order to improve tribological properties of tappets made of GCr15 bearing steel, a combination of martensitic heat treatment and surface texture was applied to the GCr15 bearing steel. The texture was designed based on a fluid-solid coupling simulation performed using COMSOl, and effectiveness of the designed texture was verified by performed wear tests using a pin-disk line contact rotary motion tester under oil lubrication condition. The worn surface morphology and wear mechanism for the friction pair were analyzed with three-dimensional scanning microscopy and scanning electron microscopy to reveal the antifriction behavior and wear resistance. Results of the study show that the heat treatment increased the surface hardness and significantly reduced wear of the GCr15 bearing steel. The designed surface texture helped reduce the contact area, store tiny wear debris, and increase the oil film pressure, thus improving the tribological performance. However, the texture edge was prone to severe plastic deformation due to the stress concentration and lowered lattice confinement. The martensitic heat treatment involving tempering effectively reduced plastic flow at the edge of texture. The combination of texture and the martensitic heat treatment considerably improved the tribological properties of GCr15 steel.

Igneous: Distributed dense 3D segmentation meshing, neuron skeletonization, and hierarchical downsampling.

Three-dimensional electron microscopy images of brain tissue and their dense segmentations are now petascale and growing. These volumes require the mass production of dense segmentation-derived neuron skeletons, multi-resolution meshes, image hierarchies (for both modalities) for visualization and analysis, and tools to manage the large amount of data. However, open tools for large-scale meshing, skeletonization, and data management have been missing. Igneous is a Python-based distributed computing framework that enables economical meshing, skeletonization, image hierarchy creation, and data management using cloud or cluster computing that has been proven to scale horizontally. We sketch Igneous's computing framework, show how to use it, and characterize its performance and data storage.

A Cristae-Like Microcompartment in Desulfobacterota.

Some Alphaproteobacteria contain intracytoplasmic membranes (ICMs) and proteins homologous to those responsible for the mitochondrial cristae, an observation which has given rise to the hypothesis that the Alphaproteobacteria endosymbiont had already evolved cristae-like structures and functions. However, our knowledge of microbial fine structure is still limited, leaving open the possibility of structurally homologous ICMs outside the Alphaproteobacteria. Here, we report on the detailed characterization of lamellar cristae-like ICMs in environmental sulfate-reducing Desulfobacterota that form syntrophic partnerships with anaerobic methane-oxidizing (ANME) archaea. These structures are junction-bound to the cytoplasmic membrane and resemble the form seen in the lamellar cristae of opisthokont mitochondria. Extending these observations, we also characterized similar structures in Desulfovibrio carbinolicus, a close relative of the magnetotactic D. magneticus, which does not contain magnetosomes. Despite a remarkable structural similarity, the key proteins involved in cristae formation have not yet been identified in Desulfobacterota, suggesting that an analogous, but not a homologous, protein organization system developed during the evolution of some members of Desulfobacterota. IMPORTANCE Working with anaerobic consortia of methane oxidizing ANME archaea and their sulfate-reducing bacterial partners recovered from deep sea sediments and with the related sulfate-reducing bacterial isolate D. carbinolicus, we discovered that their intracytoplasmic membranes (ICMs) appear remarkably similar to lamellar cristae. Three-dimensional electron microscopy allowed for the novel analysis of the nanoscale attachment of ICMs to the cytoplasmic membrane, and these ICMs are structurally nearly identical to the crista junction architecture seen in metazoan mitochondria. However, the core junction-forming proteins must be different. The outer membrane vesicles were observed to bud from syntrophic Desulfobacterota, and darkly stained granules were prominent in both Desulfobacterota and D. carbinolicus. These findings expand the taxonomic breadth of ICM-producing microorganisms and add to our understanding of three-dimensional microbial fine structure in environmental microorganisms.

Performance Improvement of Lithium Metal Anode All-Solid-State Batteries By High-Speed Blowing of Abrasive Grains

All-solid-state lithium metal batteries (ASSLiMBs) are one of the candidates for a next-generation automotive battery. In particular, oxide-based SEs have relatively high electrochemical stability against lithium metal. Thus, ASSLiMB with oxide SE is expected to be long life and high energy density battery.Poor contact between the lithium metal and oxide SE is an issue to realize ASSLiMB with oxide SE. This poor contact causes high overvoltage at the SE-Li interface, and lithium dendrite grows into the SE, and it reaches to the cathode, then causing a short circuit at high-speed charging that exceeds critical current density (CCD). Several methods (e.g., thin-film insertion method, interface structure modification methods) are proposed to suppress the interface resistance and enhance the CCD; however, more low interface resistance and high CCD is required to achieve high-speed charging by ASSLiMB with oxide SE.In this study, we propose a thin surface structure control method by shot peening (S.P.) to suppress the interface resistance and increase the CCD of ASSLiMB with oxide SE. S.P. is a surface treatment method by blowing abrasive grains with high-pressure air. S.P. has the effect of imparting an uneven surface and compressive residual stresses to the surface. The uneven shape is expected to increase the interfacial stress and improve the contact property as the SE bites into the Li. The compressive residual stresses suppress crack initiation on the SE surface. It has been reported that Li dendrites grow with cracks, and suppression of cracks is expected to improve CCD . The S.P. has above two effects that can improve the lithium metal anode performance, so, we investigated the influence of the S.P. on the lithium metal anode performance. We also investigated the influence of the grain size on the electrode performance because the size of the surface roughness and the compressive residual stresses can be changed by the change of the grain size used in S.P.Pellets of oxide SE (Li6.25Ga0.25La3Zr2O12) synthesized by the solid phase reaction method were processed by S.P. with abrasive grain of Al2O3 powder. After the S.P., the surface of the pellet is coated with gold by gold sputtering. Three experiments with three abrasive gain sizes (50 µm,100 µm,300 µm) and the SE without S.P. were carried out to check the influence of the S.P.. Li metal was attached to both sides of the SE layer to construct a symmetric cell, and CCD measurements and electrochemical impedance spectroscopy measurements were performed. Surface characteristics were measured by surface three-dimensional scanning electron microscopy (surface 3DSEM) and indentation.Fig. 1 shows the CCD and interfaces resistance without S.P. and S.P. using each abrasive grain. As shown in the figure, higher CCD and low interface resistance are achieved for the samples with S.P. with 50 µm, and 100 µm grain size than non S.P. processed samples. On the other hand, the CCD of S.P. with 300 µm grain is almost the same as the non S.P. processed samples and the interface resistance is high. These results indicate that the S.P. can improve electrode performance, and there is an optimum S.P. grain size. From indentation, the fracture toughness of S.P. processed samples were higher than a sample without S.P., and the fracture toughness increased with increasing in grain diameter. This increase in fracture toughness may have suppressed the growth of Li dendrites into the SE, leading to an increase in CCD. The surface 3DSEM showed that the S.P. creates an uneven pattern on the SE pellet and the structure size is several µm. The structure size increases with the increase in the abrasive grain size. This unevenness improves the contact between SE and Li and suppresses the interface resistance. This interface resistance suppression depends on the structure size, and the lowest interface resistance is achieved for the S.P. with the smallest grain size of 50 µm. As a result of the balance between high compressive residual stress by small size grain and the low interface resistance by large size grain, the highest CCD is obtained by middle size grain of 100 µm.This study shows that S.P. can decrease the interfacial resistance of ASSLiMB and improve CCD. The unevenness caused by S.P. is on the order of several micrometers, and it can be said that S.P. can be applied to separators thinner than 100 micrometers. Figure 1

Connectomic analysis of thalamus-driven disinhibition in cortical layer 4.

Sensory signals are transmitted via the thalamus primarily to layer 4 (L4) of the primary sensory cortices. While information about average neuronal connectivity in L4 is available, its detailed higher-order circuit structure is not known. Here, we used three-dimensional electron microscopy for a connectomic analysis of the thalamus-driven inhibitory network in L4. We find that thalamic input drives a subset of interneurons with high specificity, which in turn target excitatory neurons with subtype specificity. These interneurons create a directed disinhibitory network directly driven by the thalamic input. Neuronal activity recordings show that strong synchronous sensory activation yields about 1.5-fold stronger activation of star pyramidal cells than spiny stellates, in line with differential windows of opportunity for activation of excitatory neurons in the thalamus-driven disinhibitory circuit model. With this, we have identified a high degree of specialization of the microcircuitry in L4 of the primary sensory cortex.

Spatiotemporal organisation of protein processing in the kidney

The kidney regulates plasma protein levels by eliminating them from the circulation. Proteins filtered by glomeruli are endocytosed and degraded in the proximal tubule and defects in this process result in tubular proteinuria, an important clinical biomarker. However, the spatiotemporal organization of renal protein metabolism in vivo was previously unclear. Here, using functional probes and intravital microscopy, we track the fate of filtered proteins in real time in living mice, and map specialized processing to tubular structures with singular value decomposition analysis and three-dimensional electron microscopy. We reveal that degradation of proteins requires sequential, coordinated activity of distinct tubular sub-segments, each adapted to specific tasks. Moreover, we leverage this approach to pinpoint the nature of endo-lysosomal disorders in disease models, and show that compensatory uptake in later regions of the proximal tubule limits urinary protein loss. This means that measurement of proteinuria likely underestimates severity of endocytotic defects in patients.

Structural Diversity within the Endoplasmic Reticulum-From the Microscale to the Nanoscale.

The endoplasmic reticulum (ER) is a continuous, highly dynamic membrane compartment that is crucial for numerous basic cellular functions. The ER stretches from the nuclear envelope to the outer periphery of all living eukaryotic cells. This ubiquitous organelle shows remarkable structural complexity, adopting a range of shapes, curvatures, and length scales. Canonically, the ER is thought to be composed of two simple membrane elements: sheets and tubules. However, recent advances in superresolution light microscopy and three-dimensional electron microscopy have revealed an astounding diversity of nanoscale ER structures, greatly expanding our view of ER organization. In this review, we describe these diverse ER structures, focusing on what is known of their regulation and associated functions in mammalian cells.

NCOA4 drives ferritin phase separation to facilitate macroferritinophagy and microferritinophagy.

A ferritin particle consists of 24 ferritin proteins (FTH1 and FTL) and stores iron ions within it. During iron deficiency, ferritin particles are transported to lysosomes to release iron ions. Two transport pathways have been reported: macroautophagy and ESCRT-dependent endosomal microautophagy. Although the membrane dynamics of these pathways differ, both require NCOA4, which is thought to be an autophagy receptor for ferritin. However, it is unclear whether NCOA4 only acts as an autophagy receptor in ferritin degradation. Here, we found that ferritin particles form liquid-like condensates in a NCOA4-dependent manner. Homodimerization of NCOA4 and interaction between FTH1 and NCOA4 (i.e., multivalent interactions between ferritin particles and NCOA4) were required for the formation of ferritin condensates. Disruption of these interactions impaired ferritin degradation. Time-lapse imaging and three-dimensional correlative light and electron microscopy revealed that these ferritin-NCOA4 condensates were directly engulfed by autophagosomes and endosomes. In contrast, TAX1BP1 was not required for the formation of ferritin-NCOA4 condensates but was required for their incorporation into autophagosomes and endosomes. These results suggest that NCOA4 acts not only as a canonical autophagy receptor but also as a driver to form ferritin condensates to facilitate the degradation of these condensates by macroautophagy (i.e., macroferritinophagy) and endosomal microautophagy (i.e., microferritinophagy).

OS08.2.A Three layers of neuronal-like mechanisms driving brain tumor invasion

Abstract Background Glioblastoma are characterized by their infiltration into the whole brain. Membrane protrusions of glioma cells called tumor microtubes are subcellular structures contributing to glioma cell invasion. They are the anatomical building block to build a functional and therapy-resistant tumor cell network interconnected by tumor microtubes (TMs) that characterizes a glioblastoma cell subpopulation, while other subpopulations appear unconnected to other glioma cells. The biological role of glioblastoma cells lacking connections with each other remains unclear. Furthermore, how neurogliomal synapses influence this subpopulation is unclear. Material and Methods Time-lapse in vivo two-photon microscopy and single-cell RNA-sequencing are combined to characterize functional different subpopulations of glioblastoma cells. Intravital, augmented microscopy, three-dimensional calcium imaging, electrophysiology and volume electron microscopy are used to investigate the role of neuronal invasion mechanisms and the role of neurogliomal synapses for tumor microtube generation and dynamics. Results In-vivo imaging revealed that glioblastoma cells lacking connections to other tumor cells and astrocytes were the main subpopulation driving glioblastoma invasion invasion. These glioblastoma cells were characterised with single-cell RNA-sequencing. This revealed that this subpopulation is enriched for neuronal, neural progenitor-like, and non-mesenchymal-like cell states as previously described. Sparse regions enriched with such tumor-cell and astrocyte-unconnected, invasive glioblastoma cells evolve over time into regions with tumor-cell and astrocyte-connected, glioblastoma cell networks reflected by molecular cell state changes that are reflected in cell state changes in different regions of human glioblastoma. In addition, mechanisms of glioblastoma cell invasion resembled neuronal and neural progenitor patterns during brain development. Lastly, neuronal activity stimulated neurogliomal synapses and subsequently increased glioblastoma cell invasivieness by stimulating generation of new tumor microtubes and enhanced tumor microtube dynamics. Conclusion This study uncovers three novel layers of neuronal features driving glioblastoma cell invasion. We are able to connect molecular, cellular heterogeneity and functional glioblastoma cell states interlinking heterogeneity and dissemination of glioblastoma, two important hallmarks of this disease. Lastly, this study delineates a potential roadmap to clinical translation with the multidimensional characterisation of human glioblastoma.

Three-Dimensional Electron Microscopy of Human Umbilical Cord Tissue Allograft Pre and Post Processing: A Literature Comparison

One in four adults in the US suffer from cartilage degeneration of the Intervertebral Disc (DDD) or load bearing joints (DJD). Since cartilage is avascular, it has a limited regenerative capacity. Conventional non-surgical treatments provide brief symptomatic relief, have sided effects, and do not address the cartilage defect itself. As such, new alternatives are needed. Perinatal birth tissue allografts are a novel frontier for bio-mechanical cartilage engineering research. The tissues of interest include umbilical cord-derived Wharton’s Jelly (WJ). This study assessed WJ tissue samples via ZEISS Supra 55VP FieldEmission Scanning Electron Microscope (SEM) at 100 and 300 nm resolution scales. The captured images of pre and post-processed structural tissue matrices in WJ allografts were analyzed against themselves and peer-reviewed SEM images of articular cartilage, intervertebral disc cartilage, and muscle fascia. SEM images of post-processed WJ structural tissue matrices were found to be comparable to structural tissue matrices in human articular cartilage, intervertebral disc cartilage, and muscle fascia on a qualitative and quantitative level. This is the first study that we are aware of, to demonstrate that structural collagen matrices in post-processed WJ allografts are analogous in structure to the cartilage in articular joints, intervertebral discs, and muscle fascia.

Pathological structural conversion of α-synuclein at the mitochondria induces neuronal toxicity

Aggregation of alpha-synuclein (α-Syn) drives Parkinson’s disease (PD), although the initial stages of self-assembly and structural conversion have not been directly observed inside neurons. In this study, we tracked the intracellular conformational states of α-Syn using a single-molecule Förster resonance energy transfer (smFRET) biosensor, and we show here that α-Syn converts from a monomeric state into two distinct oligomeric states in neurons in a concentration-dependent and sequence-specific manner. Three-dimensional FRET-correlative light and electron microscopy (FRET-CLEM) revealed that intracellular seeding events occur preferentially on membrane surfaces, especially at mitochondrial membranes. The mitochondrial lipid cardiolipin triggers rapid oligomerization of A53T α-Syn, and cardiolipin is sequestered within aggregating lipid–protein complexes. Mitochondrial aggregates impair complex I activity and increase mitochondrial reactive oxygen species (ROS) generation, which accelerates the oligomerization of A53T α-Syn and causes permeabilization of mitochondrial membranes and cell death. These processes were also observed in induced pluripotent stem cell (iPSC)–derived neurons harboring A53T mutations from patients with PD. Our study highlights a mechanism of de novo α-Syn oligomerization at mitochondrial membranes and subsequent neuronal toxicity.

Real picture of magnetic domain dynamics along the magnetic hysteresis curve inside an advanced permanent magnet

In the long history of permanent magnet research for more than 100 years, three-dimensional magnetic microscopy has been eagerly awaited to elucidate the origin of the magnetic hysteresis of permanent magnets. In this study, we succeeded in observing the three-dimensional magnetic domain structure of an advanced high-coercivity Nd-Fe-B-based permanent magnet throughout the magnetic hysteresis curve using a recently developed hard X-ray magnetic tomography technique. Focused-ion-beam-based three-dimensional scanning electron microscopy was employed to study the relationship between the observed magnetic domains and the microstructure of the magnet for the same observing volume. Thermally demagnetized and coercivity states exhibit considerably different magnetic domain structures but show the same periodicity of 2.3 μm, indicating that the characteristic length of the magnetic domain is independent of the magnetization states. Further careful examination revealed some unexpected magnetic domain behaviors, such as running perpendicular to the magnetic easy axis and reversing back against the magnetic field. These findings demonstrate a wide variety of real magnetic domain behaviors along the magnetic hysteresis inside a permanent magnet.

Three-dimensional microstructure and critical current properties of ultrafine grain Ba(Fe,Co)2As2 bulk superconductors

In iron-based superconductors, randomly oriented grain boundaries have a strong influence on the transport properties via intrinsic weak-link and flux pinning mechanisms. Herein we report the critical current density (Jc) and the three-dimensional microstructure of polycrystalline bulk Co-doped Ba122 (BaFe1.84Co0.16As2) superconductors, with highly dense grain boundaries (grain size smaller than 50 nm), produced by high-energy milling. Three-dimensional electron microscopy revealed that the anomalous growth of secondary particles (aggregation) and the inter-aggregation structures were significantly different in the samples with finer grains, which may have extrinsically limited Jc. These important microstructural features were quantified as two parameters—local thickness and total pore length—by reconstructing the three-dimensional structure of the superconducting phase using the adaptive thresholding method. The results obtained in this study suggest that understanding and controlling the microstructural formation process by sintering are instrumental for improving the Jc properties of 122 polycrystalline materials consisting of ultrafine grains.

Connectomic comparison of mouse and human cortex.

The human cerebral cortex houses 1000 times more neurons than that of the cerebral cortex of a mouse, but the possible differences in synaptic circuits between these species are still poorly understood. We used three-dimensional electron microscopy of mouse, macaque, and human cortical samples to study their cell type composition and synaptic circuit architecture. The 2.5-fold increase in interneurons in humans compared with mice was compensated by a change in axonal connection probabilities and therefore did not yield a commensurate increase in inhibitory-versus-excitatory synaptic input balance on human pyramidal cells. Rather, increased inhibition created an expanded interneuron-to-interneuron network, driven by an expansion of interneuron-targeting interneuron types and an increase in their synaptic selectivity for interneuron innervation. These constitute key neuronal network alterations in the human cortex.