3D Transmission Electron Microscopy Research Articles

Imbalanced mitochondrial dynamics contributes to the pathogenesis of X-linked adrenoleukodystrophy.

The peroxisomal disease adrenoleukodystrophy (X-ALD) is caused by loss of the transporter of very-long-chain fatty acids (VLCFAs), ABCD1. An excess of VLCFAs disrupts essential homeostatic functions crucial for axonal maintenance, including redox metabolism, glycolysis and mitochondrial respiration. As mitochondrial function and morphology are intertwined, we set out to investigate the role of mitochondrial dynamics in X-ALD models. Using quantitative 3D transmission electron microscopy, we revealed mitochondrial fragmentation in corticospinal axons in Abcd1- mice. In patient fibroblasts, an excess of VLCFAs triggers mitochondrial fragmentation through the redox-dependent phosphorylation of DRP1 (DRP1S616). The blockade of DRP1-driven fission by the peptide P110 effectively preserved mitochondrial morphology. Furthermore, mRNA inhibition of DRP1 not only prevented mitochondrial fragmentation but also protected axonal health in a Caenorhabditis elegans model of X-ALD, underscoring DRP1 as a potential therapeutic target. Elevated levels of circulating cell-free mtDNA in patients' CSF align this leukodystrophy with primary mitochondrial disorders. Our findings underscore the intricate interplay between peroxisomal dysfunction, mitochondrial dynamics and axonal integrity in X-ALD, shedding light on potential avenues for therapeutic intervention.

3D orientation field transform

Vascular structure enhancement is very useful in image processing and computer vision. The enhancement of the presence of the structures like tubular networks in given images can improve image-dependent diagnostics and can also facilitate tasks like segmentation. The two-dimensional (2D) orientation field transform has been proved to be effective at enhancing 2D contours and curves in images by means of top-down processing. It, however, has no counterpart in 3D images due to the extremely complicated orientation in 3D against 2D. Given the rising demand and interest in handling 3D images, we experiment with modularising the concept and generalise the algorithm to 3D curves. In this work, we propose a 3D orientation field transform. It is a vascular structure enhancement algorithm that can cleanly enhance images having very low signal-to-noise ratio, and push the limits of 3D image quality that can be enhanced computationally. This work also utilises the benefits of modularity and offers several combinative options that each yield moderately better enhancement results in different scenarios. In principle, the proposed 3D orientation field transform can naturally tackle any number of dimensions. As a special case, it is also ideal for 2D images, owning a simpler methodology compared to the previous 2D orientation field transform. The concise structure of the proposed 3D orientation field transform also allows it to be mixed with other enhancement algorithms, and as a preliminary filter to other tasks like segmentation and detection. The effectiveness of the proposed method is demonstrated with synthetic 3D images and real-world transmission electron microscopy tomograms ranging from 2D curve enhancement to, the more important and interesting, 3D ones. Extensive experiments and comparisons with existing related methods also demonstrate the excellent performance of the proposed 3D orientation field transform.

The application and development of electron microscopy for three-dimensional reconstruction in life science: a review.

Imaging technologies have played a pivotal role in advancing biological research by enabling visualization of biological structures and processes. While traditional electron microscopy (EM) produces two-dimensional images, emerging techniques now allow high-resolution three-dimensional (3D) characterization of specimens in situ, meeting growing needs in molecular and cellular biology. Combining transmission electron microscopy (TEM) with serial sectioning inaugurated 3D imaging, attracting biologists seeking to explore cell ultrastructure and driving advancement of 3D EM reconstruction. By comprehensively and precisely rendering internal structure and distribution, 3D TEM reconstruction provides unparalleled ultrastructural insights into cells and molecules, holding tremendous value for elucidating structure-function relationships and broadly propelling structural biology. Here, we first introduce the principle of 3D reconstruction of cells and tissues by classical approaches in TEM and then discuss modern technologies utilizing TEM and on new SEM-based as well as cryo-electron microscope (cryo-EM) techniques. 3D reconstruction techniques from serial sections, electron tomography (ET), and the recent single-particle analysis (SPA) are examined; the focused ion beam scanning electron microscopy (FIB-SEM), the serial block-face scanning electron microscopy (SBF-SEM), and automatic tape-collecting lathe ultramicrotome (ATUM-SEM) for 3D reconstruction of large volumes are discussed. Finally, we review the challenges and development prospects of these technologies in life science. It aims to provide an informative reference for biological researchers.

Statistical 3D morphology characterization of vaterite microspheres produced by engineered Escherichia coli

Hollow vaterite microspheres are important materials for biomedical applications such as drug delivery and regenerative medicine owing to their biocompatibility, high specific surface area, and ability to encapsulate a large number of bioactive molecules and compounds. We demonstrated that hollow vaterite microspheres are produced by an Escherichia coli strain engineered with a urease gene cluster from the ureolytic bacteria Sporosarcina pasteurii in the presence of bovine serum albumin. We characterized the 3D nanoscale morphology of five biogenic hollow vaterite microspheres using 3D high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) tomography. Using automated high-throughput HAADF-STEM imaging across several sample tilt orientations, we show that the microspheres evolved from a smaller more ellipsoidal shape to a larger more spherical shape while the internal hollow core increased in size and remained relatively spherical, indicating that the microspheres produced by this engineered strain likely do not contain the bacteria. The statistical 3D morphology information demonstrates the potential for using biogenic calcium carbonate mineralization to produce hollow vaterite microspheres with controlled morphologies. Statement of significanceThe nanoscale 3D structures of biomaterials determine their physical, chemical, and biological properties, however significant efforts are required to obtain a statistical understanding of the internal 3D morphology of materials without damaging the structures. In this study, we developed a non-destructive, automated technique that allows us to understand the nanoscale 3D morphology of many unique hollow vaterite microspheres beyond the spectroscopy methods that lack local information and microscopy methods that cannot interrogate the full 3D structure. The method allowed us to quantitatively correlate the external diameters and aspect ratios of vaterite microspheres with their hollow internal structures at the nanoscale. This work demonstrates the opportunity to use automated transmission electron microscopy to characterize nanoscale 3D morphologies of many biomaterials and validate the chemical and biological functionality of these materials.

2D and 3D TEM Characterisation of Benzene and Ethylene Soot

Flame-generated soot particles from two different fuels, benzene (B) and ethylene (E), at different ageing conditions, were analysed to assess their morphological and structural features. Samples were collected at 6, 10 and 14 mm from the nozzle location. Traditional 2D transmission electron microscopy (TEM) and a novel 3D TEM were used to investigate morphology variations. High-resolution TEM (HRTEM) was used to capture structural characteristics. Samples were then placed on lacey carbon microgrids. A field emission gun TEM was used to capture images of the agglomerates. Tilt series of ±60 degrees were acquired at 1-degree steps to perform the 3D reconstruction. IMOD software and backwards projection were used to reconstruct the tomogram from the tilt series. The 2D analysis revealed that soot particles’ agglomerations for both fuels change from a bundle to a chain-like structure as they “age”, i.e., extracted at a higher “flame height”. In B, the primary particle diameter increases as they get “older,” whereas in E, the opposite happens, and overall, E particles are bigger than B ones. The nanostructure presents ordered regions with parallel-stacked layers of carbon lamellae. This is more evident in the aged soot samples, with the difference that in E a thick amorphous layer is present at the edge of the particles, which is not observable for B. A nanostructure analysis suggests a trend of increasing fringes length as the soot ages, going from 1.04 nm of B6 to 1.22 nm of B10 to 1.05 nm of B14 and from 1.139 nm of E6 to 1.20 nm of E14 (±0.02 nm). The tortuosity does not vary greatly across all the samples, ranging between 1.132 and 1.149 (±0.004). Separation is also quite similar everywhere, with 0.404 nm of B6, 0.392 nm of B10, 0.399 nm of B14, 0.397 nm of E6 and 0.396 nm of E14 (±0.002 nm). Ring structures and particle overlaps, two examples of geometrical characteristics concealed in 2D, can be seen in the 3D reconstructions. Furthermore, the comparison between 3D and 2D volume and surface area raises questions about the reliability of those parameters as derived from 2D measurements. This study advances knowledge of how soot structure can be affected by the fuel type and emphasises the significance of how soot is investigated.

Microstructure, mechanical and thermal properties of YSZ thermal barrier coatings deposited by axial suspension plasma spraying

Yttrium-stabilized zirconia (YSZ) thermal barrier coatings (TBCs) are indispensable elements of present-day turbine propulsion systems. The ones deposited with atmospheric plasma spraying (APS) are characterized by required low thermal conductivity, but they are unable to survive frequent thermomechanical loading and therefore their application is limited to parts remaining stationary. Expanding capability of TBCs is sought in various areas, but the one realized through modification of most proliferated apparatus used for plasma spraying (PS) (from radial to axial injection) and substituting micrometric powders with the nano-structured suspension needs least changes in the industry established procedures and offers the highest property improvement. The present experiment covered the deposition of ZrO2-8Y2O3 YSZ TBC using both atmospheric and suspension PS processes. They were performed with commercial micrometric and nano-structured YSZ (8% Y2O3) powders. The coatings morphology and microstructure were characterized with 3D profilometry, scanning and transmission electron microscopy (SEM/TEM) methods. Finally, the coating’s hardness and heat conductivity were measured. This complex approach allowed to state that PS of micrometric t’-ZrO2 powder having an admixture of m-ZrO2 phase is capable of only partial improvement in its homogenization. However, the suspension PS process of nano-structured powder eliminated any traces of the monoclinic phase from the coating. The TEM microstructure observations indicated that the suspension PS coating is built by in-flight solidified droplets as well as by the melted ones flattened on arrival. A surface layer of liquefied material on solid droplets increases their adhesion to surface asperities promoting pseudo-columnar growth of the coating. The preservation of monotonic slow increase of thermal conductivity during heating of the suspension PS coating means, that its pseudo-columnar microstructure is better suited to withstand high strains during such treatment.

Glyoxal-induced disruption of tumor cell progression in breast cancer.

Breast cancer is the most common malignant tumor in women and remains a major global challenge, with ~1.4 million cases per year, worldwide. Numerous studies have shown that changes in cell metabolism are associated with the regulation of tumor progression. In the present study, the anti-cancer properties of glyoxal (GO), which is the smallest dialdehyde formed in the oxidation-reduction reaction and involved in electron transfer and energy metabolism, in breast cancer was investigated. The biological functions and molecular mechanisms of GO were investigated in breast cancer cell lines using MTT and crystal violet assays, flow cytometry, western blot analysis, 3D laser scanning confocal microscopy and transmission electron microscopy. The results showed that GO strongly inhibited cell proliferation, promoted cell apoptosis and cell cycle G2/M arrest, induced the disappearance of cellular microvilli, and enlarged mitochondria. In addition, the protein expression level of AKT, mTOR and p70-S6K decreased in the AKT-mTOR pathway, accompanied by an increase in p-ERK and p-MEK in the MAPK pathway. The results from the present study indicate that GO suppressed breast cancer progression via the MAPK and AKT-mTOR pathways. Taken together, these results provide the basis for a potential therapeutic strategy for breast cancer.

Biomimetic Keratin-Coated Gold Nanoparticles for Photo-Thermal Therapy in a 3D Bioprinted Glioblastoma Tumor Model.

Before entering human clinical studies to evaluate their safety and effectiveness, new drugs and novel medical treatments are subject to extensive animal testing that are expensive and time-consuming. By contrast, advanced technologies enable the development of animal-free models that allow the efficacy of innovative therapies to be studied without sacrificing animals, while providing helpful information and details. We report on the powerful combination of 3D bioprinting (3DB) and photo-thermal therapy (PTT) applications. To this end, we realize a 3DB construct consisting of glioblastoma U87-MG cells in a 3D geometry, incorporating biomimetic keratin-coated gold nanoparticles (Ker-AuNPs) as a photo-thermal agent. The resulting plasmonic 3DB structures exhibit a homogeneous cell distribution throughout the entire volume while promoting the localization of Ker-AuNPs within the cells. A 3D immunofluorescence assay and transmission electron microscopy (TEM) confirm the uniform distribution of fluorescent-labeled Ker-AuNPs in the volume and their capability to enter the cells. Laser-assisted (λ = 532 nm) PTT experiments demonstrate the extraordinary ability of Ker-AuNPs to generate heating, producing the highest temperature rise of about 16 °C in less than 2 min.

(Invited, Digital Presentation) Sub-Nanoscale Electrocatalyst Design for Enhanced CO2 Conversion

The majority of today’s research in electrochemical catalysis is centered in designing catalyst materials and processes that can surpass the current benchmarks of the classical catalysis. However, developments of the performance parameters of the important electrochemical reaction such as CO2 reduction reaction has been sluggish due to the intrinsic limitations such as adsorption energy scaling relations of the intermediate species.1 Recently, theoretical and experimental studies have revealed that the electrochemical reactions confined within a very small space with a wall separation under 1 nanometer outperforms the reactions without confinement.2-4 Theoretical models suggested that the enhancement from the nanoscopic confinement arises from the electronic interaction of the adsorbed species with multiple surfaces, which breaks the adsorption energy scaling relations of the important intermediate species.2 As a proof-of-concept, we synthesized novel copper oxide (CuOx) nanoparticles (NP) and tin oxide (SnOx) NP with highly controlled sub-nanoscale interplanar gaps of widths <1 nm via the lithium electrochemical tuning method. Transmission electron microscopy (TEM) and 3D tomo-scanning TEM (STEM) analysis confirm the presence of a distinct segmentation pattern and the newly engineered interparticle confined space in the designed catalysts. Atomic-gap controlled to 5-6 Å CuOx allows a current density exceeding that of unmodified CuOx nanoparticles by about 12 folds and a Faradaic efficiency of ≈80% to C2+.3 Separately, lithiated SnOx exhibits a significant increase in CO2RR vs. hydrogen evolution selectivity by a factor of ~5 with 20% higher formate selectivity relative to pristine SnO2 NPs at −1.2 VRHE.4 Density functional theory calculations indicate that the enhanced performance is attributable to a gap-stabilization of the rate-limiting *COOH and/or *OCHO intermediates. These results highlight the potential of controlled atomic spaces in directing electrochemical reaction selectivity and the design of highly optimized catalytic materials. Keywords : Electrocatalyst, CO2 reduction, Space Confinement, Sub-Nano Spacing Kwon et al., ACS Energy Lett., 2020, 5, 2987-2994.Lu and Cheng et al., J. Phys. Chem. Lett., 2020, 11, 1896-1902.Kwon et al., Adv. Energy Mater., 2020, 19034423.Kwon et al., Adv. Funct. Mater., 2021, 2107349.

Evaporation-Induced Self-Assembly of Metal Oxide Inverse Opals: From Synthesis to Applications.

ConspectusInverse opals (IOs) are highly interconnected three-dimensional macroporous structures with applications in a variety of disciplines from optics to catalysis. For instance, when the pore size is on the scale of the wavelength of visible light, IOs exhibit structural color due to diffraction and interference of light rather than due to absorption by pigments, making these structures valuable as nonfading paints and colorants. When IO pores are in an ordered arrangement, the IO is a 3D photonic crystal, a structure with a plethora of interesting optical properties that can be used in a multitude of applications, from sensors to lasers. IOs also have interesting fluidic properties that arise from the re-entrant geometry of the pores, making them excellent candidates for colorimetric sensors based on fluid surface tension. Metal oxide IOs, in particular, can also be photo- and thermally catalytically active due to the catalytic activity of the background matrix material or of functional nanoparticles embedded within the structure.Evaporation-induced self-assembly of sacrificial particles has been developed as a scalable method for forming IOs. The pore size and shape, surface chemistry, matrix material, and the macroscopic shape of the IO, as well as the inclusion of functional components, can be designed through the choice of deposition conditions such as temperature and humidity, types and concentrations of components in the self-assembly mixture, and the postassembly processing. These parameters allow researchers to tune the optical, mechanical, and thermal transport properties of IOs for optimum functionality.In this Account, we focus on experimental and theoretical studies to understand the self-assembly process and properties of metal oxide IOs without (bare) and with (hybrid) plasmonic or catalytic metal nanoparticles incorporated. Several synthetic approaches are first presented, together with a discussion of the various forces involved in the assembly process. The visualization of the deposition front with time-lapse microscopy is then discussed together with analytical theory and numerical simulations to determine the conditions needed for the deposition of a continuous IO film. Subsequently, we present high-resolution scanning electron microscopy (SEM) of assembled colloids over large areas, which provides a detailed view of the evolution of the assembly process, showing that the organization of the colloids is initially dictated by the meniscus of the evaporating suspension on the substrate, but that gradually all grains rotate to occupy the thermodynamically most favorable orientation. High-resolution 3D transmission electron microscopy (TEM) is then presented together with analysis of the wetting of the templating colloids by the matrix precursor to provide a detailed picture of the embedding of metallic nanoparticles at the pore–matrix interface. Finally, the resulting properties and applications in optics, wetting, and catalysis are discussed, concluding with an outlook on the future of self-assembled metal-oxide-based IOs.

Generation and Staging of Human Retinal Organoids Based on Self-Formed Ectodermal Autonomous Multi-Zone System.

Methods for stem cell-derived, three-dimensional retinal organoids induction have been established and shown great potential for retinal development modeling and drug screening. Herein, we reported an exogenous-factors-free and robust method to generate retinal organoids based on “self-formed ectodermal autonomous multi-zone” (SEAM) system, a two-dimensional induction scheme that can synchronously generate multiple ocular cell lineages. Characterized by distinct morphological changes, the differentiation of the obtained retinal organoids could be staged into the early and late differentiation phases. During the early differentiation stage, retinal ganglion cells, cone photoreceptor cells (PRs), amacrine cells, and horizontal cells developed; whereas rod PRs, bipolar cells, and Müller glial cells were generated in the late differentiation phase, resembling early-phase and late-phase retinogenesis in vivo. Additionally, we modified the maintenance strategy for the retinal organoids and successfully promoted their long-term survival. Using 3D immunofluorescence image reconstruction and transmission electron microscopy, the substantial mature PRs with outer segment, inner segment and ribbon synapse were demonstrated. Besides, the retinal pigment epithelium (RPE) was induced with distinct boundary and the formation of ciliary margin was observed by co-suspending retina organoids with the zone containing RPE. The obtained RPE could be expanded and displayed similar marker expression, ultrastructural feature and functional phagocytosis to native RPE. Thus, this research described a simple and robust system which enabled generation of retina organoids with substantial mature PRs, RPE and the ciliary margin without the need of exogenous factors, providing a new platform for research of retinogenesis and retinal translational application.

Hierarchical Microphase Behaviors of Chiral Block Copolymers under Kinetic and Thermodynamic Control

Hierarchical Microphase Behaviors of Chiral Block Copolymers under Kinetic and Thermodynamic Control

Doublecortin-like expressing astrocytes of the suprachiasmatic nucleus are implicated in the biosynthesis of vasopressin and influences circadian rhythms.

We have recently identified a novel plasticity protein, doublecortin‐like (DCL), that is specifically expressed in the shell of the mouse suprachiasmatic nucleus (SCN). DCL is implicated in neuroplastic events, such as neurogenesis, that require structural rearrangements of the microtubule cytoskeleton, enabling dynamic movements of cell bodies and dendrites. We have inspected DCL expression in the SCN by confocal microscopy and found that DCL is expressed in GABA transporter‐3 (GAT3)‐positive astrocytes that envelope arginine vasopressin (AVP)‐expressing cells. To investigate the role of these DCL‐positive astrocytes in circadian rhythmicity, we have used transgenic mice expressing doxycycline‐induced short‐hairpin (sh) RNA's targeting DCL mRNA (DCL knockdown mice). Compared with littermate wild type (WT) controls, DCL‐knockdown mice exhibit significant shorter circadian rest‐activity periods in constant darkness and adjusted significantly faster to a jet‐lag protocol. As DCL‐positive astrocytes are closely associated with AVP‐positive cells, we analyzed AVP expression in DCL‐knockdown mice and in their WT littermates by 3D reconstructions and transmission electron microscopy (TEM). We found significantly higher numbers of AVP‐positive cells with increased volume and more intensity in DCL‐knockdown mice. We found alterations in the numbers of dense core vesicle‐containing neurons at ZT8 and ZT20 suggesting that the peak and trough of neuropeptide biosynthesis is dampened in DCL‐knockdown mice compared to WT littermates. Together, our data suggest an important role for the astrocytic plasticity in the regulation of circadian rhythms and point to the existence of a specific DCL+ astrocyte‐AVP+ neuronal network located in the dorsal SCN implicated in AVP biosynthesis.

Superiority of focused ion beam-scanning electron microscope tomography of cardiomyocytes over standard 2D analyses highlighted by unmasking mitochondrial heterogeneity.

BackgroundCardioprotection by preventing or repairing mitochondrial damage is an unmet therapeutic need. To understand the role of cardiomyocyte mitochondria in physiopathology, the reliable characterization of the mitochondrial morphology and compartment is pivotal. Previous studies mostly relied on two‐dimensional (2D) routine transmission electron microscopy (TEM), thereby neglecting the real three‐dimensional (3D) mitochondrial organization. This study aimed to determine whether classical 2D TEM analysis of the cardiomyocyte ultrastructure is sufficient to comprehensively describe the mitochondrial compartment and to reflect mitochondrial number, size, dispersion, distribution, and morphology.MethodsSpatial distribution of the complex mitochondrial network and morphology, number, and size heterogeneity of cardiac mitochondria in isolated adult mouse cardiomyocytes and adult wild‐type left ventricular tissues (C57BL/6) were assessed using a comparative 3D imaging system based on focused ion beam‐scanning electron microscopy (FIB‐SEM) nanotomography. For comparison of 2D vs. 3D data sets, analytical strategies and mathematical comparative approaches were performed. To confirm the value of 3D data for mitochondrial changes, we compared the obtained values for number, coverage area, size heterogeneity, and complexity of wild‐type cardiomyocyte mitochondria with data sets from mice lacking the cytosolic and mitochondrial protein BNIP3 (BCL‐2/adenovirus E1B 19‐kDa interacting protein 3; Bnip3 −/− ) using FIB‐SEM. Mitochondrial respiration was assessed on isolated mitochondria using the Seahorse XF analyser. A cardiac biopsy was obtained from a male patient (48 years) suffering from myocarditis.ResultsThe FIB‐SEM nanotomographic analysis revealed that no linear relationship exists for mitochondrial number (r = 0.02; P = 0.9511), dispersion (r = −0.03; P = 0.9188), and shape (roundness: r = 0.15, P = 0.6397; elongation: r = −0.09, P = 0.7804) between 3D and 2D results. Cumulative frequency distribution analysis showed a diverse abundance of mitochondria with different sizes in 3D and 2D. Qualitatively, 2D data could not reflect mitochondrial distribution and dynamics existing in 3D tissue. 3D analyses enabled the discovery that BNIP3 deletion resulted in more smaller, less complex cardiomyocyte mitochondria (number: P < 0.01; heterogeneity: C.V. wild‐type 89% vs. Bnip3 −/− 68%; complexity: P < 0.001) forming large myofibril‐distorting clusters, as seen in human myocarditis with disturbed mitochondrial dynamics. Bnip3 −/− mice also show a higher respiration rate (P < 0.01).ConclusionsHere, we demonstrate the need of 3D analyses for the characterization of mitochondrial features in cardiac tissue samples. Hence, we observed that BNIP3 deletion physiologically acts as a molecular brake on mitochondrial number, suggesting a role in mitochondrial fusion/fission processes and thereby regulating the homeostasis of cardiac bioenergetics.

Notoginsenoside R1 activates the NAMPT-NAD+-SIRT1 cascade to promote postischemic angiogenesis by modulating Notch signaling

Nicotinamide phosphoribosyltransferase (NAMPT) maintains mitochondrial function and protects against cerebral ischemic injury by improving energy metabolism. Notoginsenoside R1 (R1), a unique constituent of Panax notoginseng, has been shown to promote the proliferation and tube formation of human umbilical vein endothelial cells. Whether R1 has proangiogenesis on the activation of NAMPT in ischemic stroke remains unclear. The purpose of this study was to investigate the pharmacodynamic effect and mechanism of R1 on angiogenesis after ischemic stroke. We used male Sprague-Dawley (SD) rats subjected to middle cerebral artery occlusion/reperfusion (MCAO/R). R1 was administered via intraperitoneal (i.p.) injection immediately after ischemia induction. The promotion of R1 on angiogenesis were detected by immunofluorescence staining, 3D stereoscopic imaging and transmission electron microscopy detection. HBMEC cells were pretreated with different concentrations of R1 for 12 h before oxygen-glucose deprivation/reoxygenation (OGD/R) exposure. Afterward, scratch assay, EdU staining and tube formation were determined. Western blot analyses of proteins, including those involved in angiogenesis, NAMPT-SIRT1 cascade, VEGFR-2, and Notch signaling, were conducted. We showed that R1 significantly restored cerebral blood flow, improved mitochondrial energy metabolism and promoted angiogenesis. More importantly, incubation with 12.5–50 μM R1 significantly increased the migration, proliferation and tube formation of HBMECs in vitro. The promotion of R1 on angiogenesis were associated with the NAMPT-NAD+-SIRT1 cascade and Notch/VEGFR-2 signaling pathway, which was partially eliminated by inhibitors of NAMPT and SIRT1. We demonstrated that R1 promotes post-stroke angiogenesis via activating NAMPT-NAD+-SIRT1 cascade. The modulation of Notch signaling and VEGFR-2 contribute to the post-stroke angiogenesis. These findings offer insight for exploring new therapeutic strategies for neurorestoration via R1 treatment after ischemic stroke.

Defects in olivine

Abstract. Olivine, a ferromagnesian orthosilicate, is the most abundant mineral in Earth's upper mantle and is stable down to the olivine–wadsleyite phase transition, which defines the 410 km depth mantle transition zone. Olivine also occurs in crustal environments in metamorphic and hydrothermal rocks and is expected to be the major mineral constituent of the Martian and Venusian mantles. The olivine atomic structure is also used in materials science to manufacture lithium batteries. Like any other crystalline solid, including minerals, olivine never occurs with a perfect crystalline structure: defects in various dimensions are ubiquitous, from point, line, and planar defects to three-dimensional (3-D) inclusions. In this contribution, I review the current state of the art of defects in olivine and several implications for key processes occurring in Earth's mantle. Intrinsic and extrinsic point defects are detailed, exemplifying the astonishing diversity of atomic impurities in mantle-derived olivine. Linear defects, one of the key defect types responsible for ductile deformation in crystalline solids, are examined in light of recent progress in 3-D transmission electron microscopy, which has revealed an important diversity of dislocation slip systems. I summarize the principal characteristics of interface defects in olivine: the free surface, grain and interface boundaries, and internal planar defects. As the least-studied defects to date, interface defects represent an important challenge for future studies and are the main application of numerical simulation methods in materials science. I provide an overview of melt, fluid, and mineral inclusions, which are widely studied in volcanology and igneous petrology. Special attention is given to new crystalline defects that act as deformation agents: disclinations (rotational defects) and the potential occurrence of disconnections in olivine, both of which are expected to occur along or near grain boundaries. Finally, I detail outstanding questions and research directions that will further our understanding of the crystalline specificities and paradoxes of olivine and olivine-rich rocks and ultimately their implications for the dynamics of Earth's upper mantle.

Comparison of microstructure characterization methods by two-point correlation functions and reconstruction of 3D microstructures using 2D TEM images with high degree of phase clustering

Microstructural characterization methods and their performance in virtual microstructure reconstruction were investigated using two-phase Pebax/PBE membranes. In this study, the microstructural characteristics of two-phase Pebax/PBE membranes with random phase distribution were obtained from two-dimensional (2D) transmission electron microscopy (TEM) images to reconstruct three-dimensional (3D) virtual microstructures. The microstructural characteristics of the 2D TEM images with different volume fractions were expressed using a two-point correlation function (P2) and cooccurrence correlation functions (CCFs), and the results were compared. Virtual 3D microstructures were reconstructed through a stochastic optimization process using CCFs obtained from 2D TEM images of the membranes. This study further investigated and evaluated the effects of phase clustering on probabilistic functions, namely, P2 and CCFs. It is confirmed that CCFs accurately represent the characteristics of microstructures with a high degree of phase clustering and that virtual 3D samples can be effectively reconstructed using CCFs obtained from 2D images.

Two-dimensional characterization of three-dimensional magnetic bubbles in Fe3Sn2 nanostructures.

We report differential phase contrast scanning transmission electron microscopy (TEM) of nanoscale magnetic objects in Kagome ferromagnet Fe3Sn2 nanostructures. This technique can directly detect the deflection angle of a focused electron beam, thus allowing clear identification of the real magnetic structures of two magnetic objects including three-ring and complex arch-shaped vortices in Fe3Sn2 by Lorentz-TEM imaging. Numerical calculations based on real material-specific parameters well reproduced the experimental results, showing that the magnetic objects can be attributed to integral magnetizations of two types of complex three-dimensional (3D) magnetic bubbles with depth-modulated spin twisting. Magnetic configurations obtained using the high-resolution TEM are generally considered as two-dimensional (2D) magnetic objects previously. Our results imply the importance of the integral magnetizations of underestimated 3D magnetic structures in 2D TEM magnetic characterizations.

Electron tomography: An imaging method for materials deformation dynamics

The combination of in-situ and three-dimensional (3D) in transmission electron microscopy (TEM) is one of the emerging topics of recent advanced electron microscopy research. However, to date, there have been only handful examples of in-situ 3D TEM for material deformation dynamics. In this article, firstly, the authors briefly review technical developments in fast tilt-series dataset acquisition, which is a crucial technique for in-situ electron tomography (ET). Secondly, the authors showcase a recent successful example of in-situ specimen-straining and ET system development and its applications to the deformation dynamics of crystalline materials. The system is designed and developed to explore, in real-time and at sub-microscopic levels, the internal behavior of polycrystalline materials subjected to external stresses, and not specifically targeted for atomic resolution (although it may be possible). Technical challenges toward the in-situ ET observation of 3D dislocation dynamics are discussed for commercial structural crystalline materials, including some of the early studies on in-situ ET imaging and 3D modeling of dislocation dynamics. A short summary of standing technical issues and a proposed guideline for further development in the 3D imaging method for dislocation dynamics are then discussed.

Influence of 3D morphology on the performance of all-polymer solar cells processed using environmentally benign nonhalogenated solvents

Advancing the photovoltaic performance of all-polymer solar cells (APSCs) requires a comprehensive understanding of the dependence of the properties of photoactive blend films on the solvents used to process them. We have studied the correlation between the morphology of PTB7-Th:PNDI2OD-T2 blend films prepared using six different processing solvents. Three-dimensional (3D) transmission electron microscopy tomography (TEMT) revealed the influence of the 3D morphology of the PTB7-Th:PNDI2OD-T2 blend films on their photovoltaic performance. The high-performing toluene (TOL)- and o -xylene (XY)-processed blend films displayed power conversion efficiencies (PCEs) of ~4%, which contained nanoscopically networked fibrous PNDI2OD-T2 polymer domains. The films processed using the other solvents exhibited partly disconnected domains or unfavorable vertical phase separation. In addition, the use of N -dimethylformamide (DMF), N , N -dimethylacetamide (DMAc), or N -methyl-2-pyrrolidone (NMP) as a solvent additive in XY solution resulted in a substantial improvement in the PCEs to 6.1%. This large enhancement was attributed to the formation of the better-interconnected narrower crystalline fibrillary structure of PNDI2OD-T2 chains with the aid of solvent additives, as clearly elucidated by 3D-TEMT imaging. This work highlights that APSCs processed from benign nonhalogenated solvents with appropriate additives can outperform APSCs processed from toxic halogenated solvents by affording a favorable blend morphology and that 3D TEMT imaging is a powerful and essential tool for characterizing the true morphology of photoactive layers. • Three-dimensional transmission electron microscopy revealed nanoscopic morphology. • 3D morphology is a key in determining the performance of all-polymer solar cells. • Environmentally benign nonhalogenated solvents performed better than halogen solvents. • The use of DMAc solvent additive greatly improved the photovoltaic.