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Cryomicroscopy is commonly hampered by frost accumulation, reducing the visual clarity of the specimen. Pulling a vacuum or purging with nitrogen gas can greatly reduce the sample chamber's humidity, but at cryogenic temperatures, even minute concentrations of water vapor can still result in frost deposition. Here, a hygroscopic ice frame was created around the specimen to suppress frost growth during cryomicroscopy. Specifically, fluorescently tagged rat brain vessels were frozen on a silicon nitride window with an ice frame, and the luminescence of the fluorescent tag was improved by a factor of 6 compared to a similar specimen in only a nitrogen purge environment. These findings suggest that the simple implementation of a hygroscopic ice frame surrounding the specimen can substantially improve the visual clarity for cryomicroscopy, beyond that of a vacuum or nitrogen purge system. Center of BioMolecular Structure (CBMS); National Institutes of Health, the National Institute of General Medical Sciences (NIGMS), through a Center Core P30 Grant [P30GM133893]; DOE Office of Biological and Environmental Research (DOE-BER) [KP1607011]; DOE-BER Published version This work was supported by the Center of BioMolecular Structure (CBMS). CBMS is primarily supported by the National Institutes of Health, the National Institute of General Medical Sciences (NIGMS), through a Center Core P30 Grant (P30GM133893), and by the DOE Office of Biological and Environmental Research (DOE-BER) (KP1607011). The BioImaging Core within the CMBS was supported entirely by the DOE-BER.

Human induced pluripotent stem cell-derived kidney organoids have potential for disease modeling and to be developed into clinically transplantable auxiliary tissue. However, they lack a functional vasculature, and the sparse endogenous endothelial cells (ECs) are lost upon prolonged culture in vitro, limiting maturation and applicability. Here, we use intracoelomic transplantation in chicken embryos followed by single-cell RNA sequencing and advanced imaging platforms to induce and study vasculogenesis in kidney organoids. We show expansion of human organoid-derived ECs that reorganize into perfused capillaries and form a chimeric vascular network with host-derived blood vessels. Ligand-receptor analysis infers extensive potential interactions of human ECs with perivascular cells upon transplantation, enabling vessel wall stabilization. Perfused glomeruli display maturation and morphogenesis to capillary loop stage. Our findings demonstrate the beneficial effect of vascularization on not only epithelial cell types, but also the mesenchymal compartment, inducing the expansion of ´on target´ perivascular stromal cells, which in turn are required for further maturation and stabilization of the neo-vasculature. The here described vasculogenic capacity of kidney organoids will have to be deployed to achieve meaningful glomerular maturation and kidney morphogenesis in vitro. ispartof: NPJ REGENERATIVE MEDICINE vol:7 issue:1 ispartof: location:United States status: published

The discovery of Langerhans and microscopic description of islets in the pancreas were crucial steps in the discovery of insulin. Over the past 150 years, many discoveries in islet biology and type 1 diabetes have been made using powerful microscopic techniques. In the past decade, combination of new probes, animal and tissue models, application of new biosensors and automation of light and electron microscopic methods and other (sub)cellular imaging modalities have proven their potential in understanding the beta cell under (patho)physiological conditions. The imaging evolution, from fluorescent jellyfish to real-time intravital functional imaging, the revolution in automation and data handling and the increased resolving power of analytical imaging techniques are now converging. Here, we review innovative approaches that address islet biology from new angles by studying cells and molecules at high spatiotemporal resolution and in live models. Broad implementation of these cellular imaging techniques will shed new light on cause/consequence of (mal)function in islets of Langerhans in the years to come.

The discovery of Langerhans and microscopic description of islets in the pancreas were crucial steps in the discovery of insulin. Over the past 150 years, many discoveries in islet biology and type 1 diabetes have been made using powerful microscopic techniques. In the past decade, combination of new probes, animal and tissue models, application of new biosensors and automation of light and electron microscopic methods and other (sub)cellular imaging modalities have proven their potential in understanding the beta cell under (patho)physiological conditions. The imaging evolution, from fluorescent jellyfish to real‐time intravital functional imaging, the revolution in automation and data handling and the increased resolving power of analytical imaging techniques are now converging. Here, we review innovative approaches that address islet biology from new angles by studying cells and molecules at high spatiotemporal resolution and in live models. Broad implementation of these cellular imaging techniques will shed new light on cause/consequence of (mal)function in islets of Langerhans in the years to come., Innovative microscopic approaches that allow to address islet biology from new angles by studying cells and molecules at high spatiotemporal resolution and in live models are reviewed in this article. Broad implementation of these cellular imaging techniques will shed new light into the cause/consequence of (mal)function of islet of Langerhans in the years to come.

Single-particle reconstruction can be used to perform three-dimensional (3D) imaging of homogeneous populations of nano-sized objects, in particular viruses and proteins. Here, it is demonstrated that it can also be used to obtain 3D reconstructions of heterogeneous populations of inorganic nanoparticles. An automated acquisition scheme in a scanning transmission electron microscope is used to collect images of thousands of nanoparticles. Particle images are subsequently semi-automatically clustered in terms of their properties and separate 3D reconstructions are performed from selected particle image clusters. The result is a 3D dataset that is representative of the full population. The study demonstrates a methodology that allows 3D imaging and analysis of inorganic nanoparticles in a fully automated manner that is truly representative of large particle populations.

Biosensors and nanoscale analytical tools have shown huge growth in literature in the past 20 years, with a large number of reports on the topic of ‘ultrasensitive’, ‘cost-effective’, and ‘early detection’ tools with a potential of ‘mass-production’ cited on the web of science. Yet none of these tools are commercially available in the market or practically viable for mass production and use in pandemic diseases such as coronavirus disease 2019 (COVID-19). In this context, we review the technological challenges and opportunities of current bio/chemical sensors and analytical tools by critically analyzing the bottlenecks which have hindered the implementation of advanced sensing technologies in pandemic diseases. We also describe in brief COVID-19 by comparing it with other pandemic strains such as that of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) for the identification of features that enable biosensing. Moreover, we discuss visualization and characterization tools that can potentially be used not only for sensing applications but also to assist in speeding up the drug discovery and vaccine development process. Furthermore, we discuss the emerging monitoring mechanism, namely wastewater-based epidemiology, for early warning of the outbreak, focusing on sensors for rapid and on-site analysis of SARS-CoV2 in sewage. To conclude, we provide holistic insights into challenges associated with the quick translation of sensing technologies, policies, ethical issues, technology adoption, and an overall outlook of the role of the sensing technologies in pandemics.

Phylotranscriptomic studies of the past decade have repeatedly placed Oweniidae together with Magelonidae, as the sister group to remaining annelids. This newly established placement clearly makes them a key-lineage for understanding annelid evolution and morphology. One of the most prominent morphological features of all annelids are their chaetae. The arrangement and formation process (chaetogenesis) of these chitinous bristles have been studied extensively in hooked chaetae that are arranged in rows. However, the information on other types of chaetae is still scarce. In this study, we investigated the scaled capillary notochaetae of Owenia fusiformis, looking both into the formation process that causes the scaly surface ornamentation and into their arrangement within tight bundles. Our results demonstrate the incredible plasticity of chaetogenesis that allows forming a vast array of three-dimensional structures. The capillary chaetae of Owenia fusiformis are unique in lacking an enamel coating and the scales covering the apical surface of each chaeta are formed by a single microvillus of the chaetoblast. Furthermore, the bundle of chaetae has a peripherally located formative site and a central degenerative site and it appears to result from a secondary curling of the chaetal sac.

The strain rate (epsilon ) and temperature ( T ) dependent mechanical response of single crystal magnesium (Mg) micropillars compressed along the [ 2 over line 110 ] direction ( a-axis) is investigated from room temperature to 573 K and from 10-3 to 100 s-1 . The loading direction was chosen to disfavour basal slip activation by a low Schmid factor, allowing the investigation of the rate-sensitivities of extension twinning and prismatic slip. For T 423 K and for epsilon 10 s-1 , however, the accommodation of the plastic deformation by activation of prismatic slip is not enough to match the applied deformation rate, favouring again deformation twinning. The first part of this work provides a complete overview of the mutual effects of T and epsilon on the transition points of deformation modes in Mg at the microscale. In a second stage, the influence of thermal and kinetic contributions on the evolution of the flow stress leading to the slip to twin transition at 573 K has been assessed in more detail. Within the slip-dominated plasticity regime, this work provides a quantitative assessment of the increases in the saturation stress (stage III) with epsilon at high temperature, showing how the strain rate dependency of the dislocation generation rate in the pillar and escape rate at the free surfaces of the structure controls the stress evolution in Mg microcrystals.(c) 2022 The Authors. Published by Elsevier Ltd on behalf of Acta Materialia Inc. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )

Scanning transmission electron microscopy (STEM) is suitable for visualizing the inside of a relatively thick specimen than the conventional transmission electron microscopy, whose resolution is limited by the chromatic aberration of image forming lenses, and thus, the STEM mode has been employed frequently for computed electron tomography based three-dimensional (3D) structural characterization and combined with analytical methods such as annular dark field imaging or spectroscopies. However, the image quality of STEM is severely suffered by noise or artifacts especially when rapid imaging, in the order of millisecond per frame or faster, is pursued. Here we demonstrate a deep-learning-assisted rapid STEM tomography, which visualizes 3D dislocation arrangement only within five-second acquisition of all the tilt-series images even in a 300 nm thick steel specimen. The developed method offers a new platform for various in situ or operando 3D microanalyses in which dealing with relatively thick specimens or covering media like liquid cells are required.

2D materials have proved their potential in nearly every area of material science and chemistry. Unfortunately, large‐scale production of nanosheets is not straightforward. Current methods suffer from low yield, uncontrollable defects, and requires a high‐energy input. There is always a tradeoff between high quality and high yield. In this review, the alternative is highlighted to existing methods of 2D nanosheet production – 1D dissolution, historically known as osmotic swelling. As a thermodynamically driven, and therefore spontaneous, process it provides numerous benefits such as high aspect ratio and defect‐free nanosheets with a quantitative yield. In this review, the theory behind this process is discussed, compare it with the existing methods, and highlight the key features that allow to extend 1D dissolution to different charged layered materials. Moreover, the applications in which nanosheets obtained by 1D dissolution proved to be advantageous due to their unique, processing‐related features are discussed.

Single-molecule localization microscopy (SMLM) describes a family of powerful imaging techniques that dramatically improve spatial resolution over standard, diffraction-limited microscopy techniques and can image biological structures at the molecular scale. In SMLM, individual fluorescent molecules are computationally localized from diffraction-limited image sequences and the localizations are used to generate a super-resolution image or a time course of super-resolution images, or to define molecular trajectories. In this Primer, we introduce the basic principles of SMLM techniques before describing the main experimental considerations when performing SMLM, including fluorescent labelling, sample preparation, hardware requirements and image acquisition in fixed and live cells. We then explain how low-resolution image sequences are computationally processed to reconstruct super-resolution images and/or extract quantitative information, and highlight a selection of biological discoveries enabled by SMLM and closely related methods. We discuss some of the main limitations and potential artefacts of SMLM, as well as ways to alleviate them. Finally, we present an outlook on advanced techniques and promising new developments in the fast-evolving field of SMLM. We hope that this Primer will be a useful reference for both newcomers and practitioners of SMLM. This Primer explains the central concepts of single-molecule localization microscopy (SMLM) before discussing experimental considerations regarding fluorophores, optics and data acquisition, processing and analysis. The Primer further describes recent high-impact discoveries made by SMLM techniques and concludes by discussing emerging methodologies.

International audience; Many cellular organelles, including endosomes, show compartmentalization into distinct functional domains, which, however, cannot be resolved by diffraction-limited light microscopy. Single molecule localization microscopy (SMLM) offers nanoscale resolution but data interpretation is often inconclusive when the ultrastructural context is missing. Correlative light electron microscopy (CLEM) combining SMLM with electron microscopy (EM) enables correlation of functional subdomains of organelles in relation to their underlying ultrastructure at nanometer resolution. However, the specific demands for EM sample preparation and the requirements for fluorescent single-molecule photo-switching are opposed. Here, we developed a novel superCLEM workflow that combines triple-color SMLM (dSTORM & PALM) and electron tomography using semi-thin Tokuyasu thawed cryosections. We applied the superCLEM approach to directly visualize nanoscale compartmentalization of endosomes in HeLa cells. Internalized, fluorescently labeled Transferrin and EGF were resolved into morphologically distinct domains within the same endosome. We found that the small GTPase Rab5 is organized in nanodomains on the globular part of early endosomes. The simultaneous visualization of several proteins in functionally distinct endosomal sub-compartments demonstrates the potential of superCLEM to link the ultrastructure of organelles with their molecular organization at nanoscale resolution.

Recently hBN has become an interesting platform for quantum optics due to the peculiar defect-related luminescence properties. Concomitantly, hBN was established as the ideal insulating support for realizing 2D materials device, where, on the contrary, defects can affect the device performance. In this work, we study the light emission properties of hBN flakes obtained by mechanical exfoliation with particular focus on extended defects generated in the process. In particular, we tackle different issues as the light emission in hBN flakes of different thicknesses in the range of hundreds of nm, revealing a higher concentration of deep level emission in thinner area of the flake. We recognize the effect of crystal deformation in some areas of the flake with an important blue-shift (130 meV) of the room temperature near band edge emission of hBN and the concurrent presence of a novel emission at 2.36 eV related to the formation of array of dislocations. We studied the light emission properties by means of cathodoluminescence and sub-bandgap excitation photoluminescence of thickness steps with different crystallographic orientations, revealing the presence of different concentration of radiative centers. CL mapping allows to detect buried thickness steps, invisible to the SEM and AFM morphological analysis., 27 pages, 6 figures, 1 table

The endoplasmic reticulum (ER) is a multipurpose organelle comprising dynamic structural subdomains, such as ER sheets and tubules, serving to maintain protein, calcium, and lipid homeostasis. In neurons, the single ER is compartmentalized with a careful segregation of the structural subdomains in somatic and neurite (axodendritic) regions. The distribution and arrangement of these ER subdomains varies between different neuronal types. Mutations in ER membrane shaping proteins and morphological changes in the ER are associated with various neurodegenerative diseases implying significance of ER morphology in maintaining neuronal integrity. Specific neurons, such as the highly arborized dopaminergic neurons, are prone to stress and neurodegeneration. Differences in morphology and functionality of ER between the neurons may account for their varied sensitivity to stress and neurodegenerative changes. In this review, we explore the neuronal ER and discuss its distinct morphological attributes and specific functions. We hypothesize that morphological heterogeneity of the ER in neurons is an important factor that accounts for their selective susceptibility to neurodegeneration.

Autophagy contributes to the selective degradation of liquid droplets, including the P-Granule, Ape1-complex and p62/SQSTM1-body, although the molecular mechanisms and physiological relevance of selective degradation remain unclear. In this report, we describe the properties of endogenous p62-bodies, the effect of autophagosome biogenesis on these bodies, and the in vivo significance of their turnover. p62-bodies are low-liquidity gels containing ubiquitin and core autophagy-related proteins. Multiple autophagosomes form on the p62-gels, and the interaction of autophagosome-localizing Atg8-proteins with p62 directs autophagosome formation toward the p62-gel. Keap1 also reversibly translocates to the p62-gels in a p62-binding dependent fashion to activate the transcription factor Nrf2. Mice deficient for Atg8-interaction-dependent selective autophagy show that impaired turnover of p62-gels leads to Nrf2 hyperactivation in vivo. These results indicate that p62-gels are not simple substrates for autophagy but serve as platforms for both autophagosome formation and anti-oxidative stress., Liquid-liquid phase separation of p62/SQSTM1 has been previously described, although the significance in vivo remains unclear. Here the authors show p62 droplets contain ubiquitin, autophagy-related proteins and Keap1 to serve as platform of not only autophagosome formation but also Nrf2 activation.

Imaging of liquids and cryogenic biological materials by electron microscopy has been recently enabled by innovative approaches for specimen preparation and the fast development of optimised instruments for cryo-enabled electron microscopy (cryo-EM). Yet, Cryo-EM typically lacks advanced analytical capabilities, in particular for light elements. With the development of protocols for frozen wet specimen preparation, atom probe tomography (APT) could advantageously complement insights gained by cryo-EM. Here, we report on different approaches that have been recently proposed to enable the analysis of relatively large volumes of frozen liquids from either a flat substrate or the fractured surface of a wire. Both allowed for analysing water ice layers which are several microns thick consisting of pure water, pure heavy-water and aqueous solutions. We discuss the merits of both approaches, and prospects for further developments in this area. Preliminary results raise numerous questions, in part concerning the physics underpinning field evaporation. We discuss these aspects and lay out some of the challenges regarding the APT analysis of frozen liquids., Comment: submitted for publication assocaited to the APT&M 2020 conference

Analytical chemistry 93(34), 11660 - 11668 (2021). doi:10.1021/acs.analchem.1c00579, An optimized micro-X-ray fluorescence confocal imaging (��XRF-CI) analytical method has been developed to determine the 2D distribution of elemental composition in small (1���3 mm) biological objects at a 10���20 ��m spatial resolution. Plants take up chemical elements from soil, and the vascular system transports them toward shoots. In order to obtain biochemical information related to this biological process, 2D distributions of chemical elements in roots and in hypocotyls of cucumber plants were analyzed by synchrotron radiation based on micro-X-ray fluorescence computer tomography and ��XRF-CI techniques. The experiments were carried out at HASYLAB Beamline L of the DORIS-III storage ring in Hamburg, a facility that provided optimal physical conditions for developing and performing these unique analyses: high flux monochromatic synchrotron beam, X-ray optical elements, precision moving stages, and silicon drift detectors. New methodological improvements and experimental studies were carried out for applicability of lyophilized samples and cryo-cooling. Experimental parameters were optimized to maximize the excitation yield of arsenic K�� radiation and improvement of the spatial resolution of the ��XRF-CI analytical method., Published by American Chemical Society, Columbus, Ohio

Microtubules play a major role in intracellular trafficking of vesicles in endocrine cells. Detailed knowledge of microtubule organization and their relation to other cell constituents is crucial for understanding cell function. However, their role in insulin transport and secretion is currently under debate. Here, we use Fib-Semto image islet beta cells in their entirety with unprecedented resolution. We reconstruct mitochondria, Golgi apparati, centrioles, insulin secretory granules and micro-tubules of seven beta cells, and generate a comprehensive spatial map of microtubule-organelle interactions. We find that micro-tubules form non-radial networks that are predominantly not connected to either centrioles or endomembranes. Microtubule number and length, but not microtubule polymer density, vary with glucose stimulation. Furthermore, insulin secretory granules are enriched near the plasma membrane where they associate with microtubules. In summary, we provide the first 3D reconstructions of complete microtubule networks in primary mammalian cells together with evidence regarding their importance for insulin secretory granule positioning and thus supportive role in insulin secretion.

The fluorescent metabolic labeling of microorganisms genome is an advanced imaging technique to observe and study the native shapes, structural changes, functions, and tracking of nucleic acids in single cells or tissues. We have attempted to visualize the newly synthesized DNA within the intact nucleoid of ice-embedded proliferating cells of Escherichia coli K-12 (thymidine-requiring mutant, strain N4316) via correlative light-electron microscopy. For that purpose, erythrosine-11-dUTP was synthesized and used as a modified analog of the exogenous thymidine substrate for metabolic incorporation into the bacterial chromosome. The formed fluorescent genomic DNA during in cellulo polymerase reaction caused a minimal cellular arrest and cytotoxicity of E. coli at certain controlled conditions. The stained cells were visualized in typical red emission color via an epifluorescence microscope. They were further ice-embedded and examined with a Hilbert differential contrast transmission electron microscopy. At high-resolution, the ultrastructure of tagged nucleoid appeared with significantly higher electron dense in comparison to the unlabeled one. The enhanced contrast areas in the chromosome were ascribed to the presence of iodine contents from erythrosine dye. The presented labeling approach might be a powerful strategy to reveal the structural and dynamic changes in natural DNA replication including the relationship between newly synthesized in vivo nucleic acid and the physiological state of the cell.

International audience; Single-particle cryo-electron microscopy (cryo-EM) reconstructs the three-dimensional (3D) structure of bio-molecules from a large set of 2D projection images with random and unknown orientations. A crucial step in the single-particle cryo-EM pipeline is 3D refinement, which resolves a high-resolution 3D structure from an initial approximate volume by refining the estimation of the orientation of each projection. In this work, we propose a new approach that refines the projection angles on the continuum. We formulate the optimization problem over the density map and the orientations jointly. The density map is updated using the efficient alternating-direction method of multipliers, while the orientations are updated through a semi-coordinate-wise gradient descent for which we provide an explicit derivation of the gradient. Our method eliminates the requirement for a fine discretization of the orientation space and does away with the classical but computationally expensive template-matching step. Numerical results demonstrate the feasibility and performance of our approach compared to several baselines.

International audience; To better understand the physiology and acclimation capability of the cell, one of the great challenges of the future is to access the interior of a cell and unveil its chemical landscape (composition and distribution of elements and molecules). Chemical imaging has greatly improved in sensitivity and spatial resolution to visualize and quantify nutrients, metabolites, toxic elements, and drugs in single cells at the subcellular level. This review aims to present the current potential of these emerging imaging technologies and to guide biologists towards a strategy for interrogating biological processes at the nanoscale. We also describe various solutions to combine multiple imaging techniques in a correlative way and provide perspectives and future directions for integrative subcellular imaging across different disciplines

Gold is ubiquitous in the human environment and most people are in contact with it through wearing jewellery, dental devices, implants or therapies for rheumatoid arthritis. Gold is not a nutrient but people are exposed to it as a food colorant and in food chains. The present review discusses the hazards faced in personal and domestic use of gold and the far greater risks presented through occupational exposure to the metal in mining and processing gold ores. In the last situation, regular manual contact or inhalation of toxic or carcinogenic materials like mercury or arsenic respectively, presents far greater hazard and greatly complicates the evaluation of gold toxicity. The uses and risks presented by new technology and use of nanoparticulate gold in anti-cancer therapies and diagnostic medicine forms a major consideration in gold toxicity, where tissue uptake and distribution are determined largely by particle size and surface characteristics. Many human problems arise through the ability of metallic gold to induce allergic contact hypersensitivity. Whilst gold in jewellery can evoke allergic reactions, other metals such as nickel, chromium and copper present in white gold or alloys exhibit more serious clinical problems. It is concluded that toxic risks associated with gold are low in relation to the vast range of potential routes of exposure to the metal in everyday life.

Significance: The physiological relevance of contacts between the sarcoplasmic reticulum (SR), a specialized domain of the endoplasmic reticulum (ER) in skeletal muscle, and mitochondria is still not clear. Recent Advances: An extensive close proximity of these two organelles is a late developmental event, which suggests that it does not have an essential function. Critical Issues: The intimate association of SR/mitochondria develops during murine postnatal differentiation and the recovery of denervated atrophic muscle, which suggests that this is a highly regulated process with a specific function. Analyses of mouse models for muscle diseases suggest that impaired ER/SR-mitochondrial contacts may be due to ER stress and lead to defective bioenergetics and insulin signaling. Future Directions: Future studies are necessary to identify the molecular determinants weakening insulin signaling upon impairment of ER/mitochondrial contacts in skeletal muscles as well as to analyze the distance between SR/ER and mitochondria in muscle diseases associated with ER stress.

Entry of enveloped viruses relies on insertion of hydrophobic residues of the viral fusion protein into the host cell membrane. However, the intermediate conformations during fusion remain unknown. Here, we address the fusion mechanism of Rift Valley fever virus. We determine the crystal structure of the Gn glycoprotein and fit it with the Gc fusion protein into cryo-electron microscopy reconstructions of the virion. Our analysis reveals how the Gn shields the hydrophobic fusion loops of the Gc, preventing premature fusion. Electron cryotomography of virions interacting with membranes under acidic conditions reveals how the fusogenic Gc is activated upon removal of the Gn shield. Repositioning of the Gn allows extension of Gc and insertion of fusion loops in the outer leaflet of the target membrane. These data show early structural transitions that enveloped viruses undergo during host cell entry and indicate that analogous shielding mechanisms are utilized across diverse virus families., Viral fusion proteins undergo extensive conformational changes during entry but intermediate conformations often remain unknown. Here, the authors show how Gn of Rift Valley fever virus fusion protein shields hydrophobic fusion loops of Gc and how these loops embed in the target membrane at acidic conditions.

Over the past two decades, metalloproteinases (MMPs), including MMP‑2, MMP‑3, and MMP‑9, have been implicated as important players in mechanisms underlying various forms of neuroplasticity. In particular, MMP‑3 was found to be involved in both cognitive functions and in plasticity phenomena, but the underlying molecular mechanisms remain largely elusive. In general, it is believed that functional plasticity of neurons is associated with morphological alterations. Interestingly, MMP‑9, in addition to playing a key role in synaptic plasticity, was found to affect plasticity‑related spine morphology changes. Whereas the involvement of MMP‑3 in shaping synapse morphology upon induction of synaptic plasticity awaits determination, it has been demostrated that MMP‑3 knockout results in clearly altered apical dendrite morphology in pyramidal neurons in mouse visual cortex. Considering that the involvement of MMP‑3 in synaptic plasticity has been most extensively documented for the CA1 hippocampal region, we decided to investigate whether genetic deletion of MMP‑3 affects neuronal morphology in this area. To this end, we used Golgi staining to compare dendritic morphology of pyramidal neurons in the CA1 region in MMP‑3‑deficient and wild‑type mice. Surprisingly, in contrast to the results obtained in cortex, extensive analysis of dendritic morphology in the CA1 region revealed no significant differences between MMP‑3 knockout and wild‑type groups. These results suggest that the impact of MMP‑3 on neuronal morphology may be region‑specific. ispartof: Acta Neurobiologiae Experimentalis vol:78 issue:3 pages:281-286 ispartof: location:Poland status: published

A three-dimensional finite element method (FEM) model simulating the electrochemical behaviour of Pt coplanar thin-film electrodes used for liquid cell transmission electron microscopy measurements was developed. The model included reaction kinetics and it was applied for the oxygen evolution reaction (OER). Kinetic parameters of OER in the liquid cell were experimentally acquired and applied to the FEM model. Comparison between the experimental and simulated polarization curves demonstrated the reliability of the FEM predictions. The simulations were used to produce maps of the potential and current density distributions of the working and counter electrodes as well as for calculating the distribution of the current density in the liquid electrolyte. Two distinctive electrode geometries were evaluated with the FEM model. It was predicted that non-symmetrical electrode designs can cause unexpected electrochemical behaviour with respect to the electrolyte current density between working and counter electrodes accompanied by the presence of hot spots. The findings suggest that FEM simulations could be key to designing well-performing electrochemical microcells for liquid phase electron microscopy experiments.

Transport of neo-synthesized influenza A virus (IAV) viral ribonucleoproteins (vRNPs) from the nucleus to the plasma membrane involves Rab 11 but the precise mechanism remains poorly understood. We used metal-tagging and immunolabeling to visualize viral proteins and cellular endomembrane markers by electron microscopy of IAV-infected cells. Unexpectedly, we provide evidence that the vRNP components and the Rab11 protein are present at the membrane of a modified, tubulated endoplasmic reticulum (ER) that extends all throughout the cell, and on irregularly coated vesicles (ICVs). Some ICVs are found very close to the ER and to the plasma membrane. ICV formation is observed only in infected cells and requires an active Rab11 GTPase. Against the currently accepted model in which vRNPs are carried onto Rab11-positive recycling endosomes across the cytoplasm, our findings reveal that the endomembrane organelle that is primarily involved in the transport of vRNPs is the ER., Transport of neo-synthesized influenza A virus viral ribonucleoproteins (vRNPs) from the nucleus to the plasma membrane involves Rab 11 but the mechanism is unclear. Here the authors show that vRNPs are transported through a modified Rab11-positive endoplasmic reticulum and Rab11-dependent vesicles.

The complexity of unconventional rock systems is expressed both in the compositional variance of the microstructure and the extensive heterogeneity of the pore space. Visualizing and quantifying the microstructure of oil shale before and after pyrolysis permits a more accurate determination of petrophysical properties which are important in modeling hydrocarbon production potential. We characterize the microstructural heterogeneity of oil shale using X-ray micro-tomography (µCT), automated ultra-high resolution scanning electron microscopy (SEM), MAPS Mineralogy (Modular Automated Processing System) and Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). The organic-rich Eocene Green River (Mahogany zone) oil shale is characterized using a multi-scale multi-dimensional workflow both before and after pyrolysis. Observations in 2-D and 3-D and across nm-µm-mm length scales demonstrate both heterogeneity and anisotropy at every scale. Image acquisition and analysis using µCT and SEM reveal a microstructure of alternating kerogen-rich laminations interbedded with layers of fine-grained inorganic minerals. MAPS Mineralogy combined with ultrafast measurements reveal mineralogic textures dominated by dolomite, calcite, K-feldspar, quartz, pyrite and illitic clays along with their spatial distribution, augmenting conventional mineral analysis. From high resolution Backscattered electron (BSE) images, intra-organic, inter-organic-mineral, intra- and inter-mineral pores are observed with varying sizes and geometries. By using FIB milling and SEM imaging sequentially and repetitively, 3-D data sets were reconstructed. By setting 3-D gradient and marker-based watershed transforms, the organic matter, minerals and pore phases (including pore-back artifacts) were segmented and visualized and the pore-size distribution was computed. Following pyrolysis, fractures from the mm-to-µm scales were observed with preferential propagation along the kerogen-rich laminations and coalescence leading to an interconnected fracture network. The application of these techniques to worldwide oil shale deposits will allow significant insights into estimating mechanical and chemical proprieties of oil shale formations for modeling and designing oil shale pyrolysis processes.

An efficient and fast pipeline is presented for obtaining near-atomic resolution structures from large single-particle cryo-EM data sets. The approach is virtually reference-free and is therefore less prone to the perils of reference bias., Single-particle cryogenic electron microscopy (cryo-EM) can now yield near-atomic resolution structures of biological complexes. However, the reference-based alignment algorithms commonly used in cryo-EM suffer from reference bias, limiting their applicability (also known as the ‘Einstein from random noise’ problem). Low-dose cryo-EM therefore requires robust and objective approaches to reveal the structural information contained in the extremely noisy data, especially when dealing with small structures. A reference-free pipeline is presented for obtaining near-atomic resolution three-dimensional reconstructions from heterogeneous (‘four-dimensional’) cryo-EM data sets. The methodologies integrated in this pipeline include a posteriori camera correction, movie-based full-data-set contrast transfer function determination, movie-alignment algorithms, (Fourier-space) multivariate statistical data compression and unsupervised classification, ‘random-startup’ three-dimensional reconstructions, four-dimensional structural refinements and Fourier shell correlation criteria for evaluating anisotropic resolution. The procedures exclusively use information emerging from the data set itself, without external ‘starting models’. Euler-angle assignments are performed by angular reconstitution rather than by the inherently slower projection-matching approaches. The comprehensive ‘ABC-4D’ pipeline is based on the two-dimensional reference-free ‘alignment by classification’ (ABC) approach, where similar images in similar orientations are grouped by unsupervised classification. Some fundamental differences between X-ray crystallography versus single-particle cryo-EM data collection and data processing are discussed. The structure of the giant haemoglobin from Lumbricus terrestris at a global resolution of ∼3.8 Å is presented as an example of the use of the ABC-4D procedure.

Drying and wetting induce a number of microstructural changes that could impact transport properties and durability of concrete structures, but their significance is not well-established. This research examines pastes, mortars and concretes with different w/b ratios, binders, aggregate sizes, curing and conditioning regimes. 50 mm thick samples were dried to equilibrium at either 105 °C, 50 °C/7% RH, 21 °C/33% RH or gentle stepwise at 21 °C/93% RH → 3% RH, and then rewetted stepwise by humidification at 21 °C/33% RH → 86% RH and full saturation to produce varying degrees of damage and moisture content. Oxygen diffusivity and permeability, electrical conductivity, microcracking, accessible and total porosity were measured at different conditioning stages over 3-year period to better understand the effects of shrinkage, hysteresis and drying-induced damage on transport properties. The effect of supplementary cementitious materials (GGBS, SF) and implications of drying-wetting on concrete durability are discussed.