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Mixed matrix membranes (MMMs) have emerged as promising materials for various separation processes due to their tunable properties, enhanced separation performance and reproducibility. In this review paper, we provide a comprehensive overview of the methodologies, challenges, and applications associated with the characterization of MMMs using two advanced imaging techniques: Focused Ion Beam Scanning Electron Microscopy (FIB‐SEM) and Transmission Electron Microscopy (TEM). We begin by outlining the principles and capabilities of FIB‐SEM and TEM, emphasizing their suitability for studying the microstructure, morphology, and composition of MMMs at nanoscale resolution. Subsequently, we discuss the specific challenges and limitations encountered in the characterization of MMMs using these techniques, including sample preparation, image acquisition, and data interpretation. Furthermore, we review the diverse applications of FIB‐SEM and TEM in elucidating the structure‐property relationships of MMMs. Through illustrative examples, we highlight the valuable insights gained from these imaging techniques in optimizing MMMs for various separation applications. Finally, we propose future directions and emerging trends in MMM characterization, including the integration of lasers into FIB‐SEM and in situ characterization techniques, to address current challenges and push the boundaries of MMM design and performance. Overall, this review provides a comprehensive overview of the state‐of‐the‐art methodologies for characterizing MMMs using FIB‐SEM and TEM, identifies key challenges, and offers insights into future research directions aimed at harnessing the full potential of MMMs for sustainable separation technologies. [ABSTRACT FROM AUTHOR]

This review considers the use of fractal concepts to improve the development, fabrication, and characterisation of catalytic materials and supports. First, the theory of fractals is discussed, as well as how it can be used to better describe often highly complex catalytic materials and enhance structural characterisation via a variety of different methods, including gas sorption, mercury porosimetry, NMR, and several imaging modalities. The review then surveys various synthesis and fabrication methods that can be used to create catalytic materials that are fractals or possess fractal character. It then goes on to consider how the fractal properties of catalysts affect their performance, especially their overall activity, selectivity for desired products, and resistance to deactivation. Finally, this review describes how the optimum fractal catalyst material for a given reaction system can be designed on a computer. [ABSTRACT FROM AUTHOR]

Collagen-based hydrogels are commonly used in mechanobiology to mimic the extracellular matrix. A quantitative analysis of the influence of collagen concentration and properties on the structure and mechanics of the hydrogels is essential for tailored design adjustments for specific in vitro conditions. We combined focused ion beam scanning electron microscopy and rheology to provide a detailed quantitative atlas of the mechanical and nanoscale three-dimensional structural alterations that occur when manipulating different hydrogel's physicochemistry. Moreover, we study the effects of such alterations on the phenotype of breast cancer cells and their mechanical interactions with the extracellular matrix. Regardless of the microenvironment's pore size, porosity or mechanical properties, cancer cells are able to reach a stable mesenchymal-like morphology. Additionally, employing 3D traction force microscopy, a positive correlation between cellular tractions and ECM mechanics is observed up to a critical threshold, beyond which tractions plateau. This suggests that cancer cells in a stable mesenchymal state calibrate their mechanical interactions with the ECM to keep their migration and invasiveness capacities unaltered. The paper presents a thorough study on the mechanical microenvironment in breast cancer cells during their interaction with collagen based hydrogels of different compositions. The hydrogels' microstructure were obtained using state-of-the-art 3D microscopy, namely focused ion beam-scanning electron microscope (FIB-SEM). FIB-SEM was originally applied in this work to reconstruct complex fibered collagen microstructures within the nanometer range, to obtain key microarchitectural parameters. The mechanical microenvironment of cells was recovered using Traction Force Microscopy (TFM). The obtained results suggest that cells calibrate tractions such that they depend on mechanical, microstructural and physicochemical characteristics of the hydrogels, hence revealing a steric hindrance. We hypothesize that cancer cells studied in this paper tune their mechanical state to keep their migration and invasiveness capacities unaltered. [Display omitted] [ABSTRACT FROM AUTHOR]

The oxygen effective diffusion and proton effective conductivity tensors of a cathode catalyst layer (CCL) are computed using a two-scale approach on CCL microstructure three-dimensional images obtained by focused ion beam-scanning electron microscopy (FIB-SEM) after reconstruction of the ionomer distribution. The results show that the obtained tensors are not isotropic. It is also found that some off-diagonal components are not negligible in the case of the proton effective conductivity tensor. The simulations confirm that Knudsen diffusion has a significant impact on oxygen diffusion. The results are comparable to those obtained in previous works on synthetic images as regards the oxygen effective diffusion through-plane component but differ as regards the proton conductivity through-plane component. Contrary to previous works using synthetic images which tend to underestimate the proton conductivity, they are globally in better agreement with experimental data. The results also show that the effective proton conductivity is heterogeneous at the scale of the computational domains typically considered in several previous works due to the variability in the ionomer distribution at this scale. The impact of liquid water in the primary pores on the proton transport is explored and found to have a significant impact. • The oxygen effective diffusion tensor is computed from 3D digital images of a PEMFC catalyst layer (CL). • The proton effective conductivity tensor is computed from 3D digital images of the CL microstructure. • Liquid water in the primary pores has a noticeable impact on the proton transport. • Results on the proton transport validate the considered ionomer spatial distribution. [ABSTRACT FROM AUTHOR]

The three-dimensional (3D) morphology, anatomy, and in-situ chemical composition analysis of fossils are crucial for systematic paleontology and determining their phylogenetic positions. Scanning electron microscopy (SEM) coupled with energy-dispersive X-ray spectroscopy (EDS), offers valuable structural and chemical information for the analysis of fossils. However, its primary limitation is the restriction to two-dimensional surface data, which limits the exploration of fossils' 3D complexities. Conversely, 3D X-ray microscopy (3D-XRM), also known as a novel form of micro-computed tomography (micro-CT) facilitates the non-destructive 3D reconstruction of fossil specimens. Nevertheless, it lacks the capability to provide in-situ compositional data. Acknowledging the constraints inherent in these individual techniques, and in response to the evolving requirements of paleontological research, this study introduces an integrated approach that combines 3D-XRM with EDS-coupled focused ion beam scanning electron microscopy (FIB-SEM). This innovative strategy is designed to synergize the advantages of both techniques, thereby addressing challenges that conventional methods cannot. It enables the rapid identification of regions of interest (ROI) within fossil specimens at micrometer resolution. Subsequently, this method collects detailed data on both 3D structures and chemical compositions at the nanometer scale for the identified ROI. This integrated approach represents a significant advancement in paleontological and geological research methodologies, promising to meet the increasing demands of these fields. [ABSTRACT FROM AUTHOR]

In this work, we investigate the optical properties variation of thermally grown W oxides during the exposure to low energy deuterium (D) plasma. In-situ ellipsometry in the 400–1000 nm range was coupled to ex-situ diagnostics, such as X-ray photoelectron spectroscopy (XPS) and focused ion beam coupled to scanning electron microscopy (FIB-SEM), to probe the evolution of chemical and morphological properties of the W oxides. First, a 70 nm thick WO3 layer was exposed to D plasma at a surface temperature of 650 K. An important reduction of the oxide layer was observed by FIB-SEM, and in-situ ellipsometry showed the evolution of the optical constants n and k of the oxide towards the ones of pure W metal. Secondly, a 300 nm thick WO3 layer was exposed to D plasma at a surface temperature below 373 K. In order to follow the oxide evolution step by step, we alternated in-situ ellipsometry and XPS surface characterization. A quite similar evolution of the optical constants was observed, in particular an increase of the extinction coefficient k in the near infrared, which was linked to a progressive reduction of the oxidation level at the surface, as seen by XPS. Interestingly, the reduction of the oxide at 373 K was below the resolution of the FIB-SEM. This observation indicates that ellipsometry in the 400–1000 nm range is able to follow in-situ the reduction of WO3 oxides by D plasma with high surface sensitivity.

One of the engineering challenges in developing more reliable electrodes in solid oxide fuel cells is to reduce residual stresses generated during electrode fabrication. The Young's modulus of porous ceramic materials employed in the electrodes is a fundamental property essential for evaluating the residual stresses of electrodes. In this study, the correlation between Young's modulus and the microstructure of the cathode material La0.6Sr0.4Co0.2Fe0.8O3-δ was evaluated numerically using a voxel-based finite element method in conjunction with the three-dimensional mesoscale techniques, including a kinetic Monte Carlo and a discrete element method. The results of simulations utilizing the FIB-SEM reconstructed microstructures are in good agreement with those of micro-indentation experiments, indicating that our methodology is effective for estimating the Young's modulus of porous ceramics at room temperature. Furthermore, we reveal that the porosity dependence of Young’s modulus can be well described by an empirical power law based on the percolation model. In particular, our findings indicate that the dependencies on porosity are highly related to the homogeneity of the initial powder microstructures before sintering. Finally, our results suggest that the effective Young’s modulus of the porous materials at any porosity has a correlation with the tortuosity factor.

Electron microscopy (EM), in its various flavors, has significantly contributed to our understanding of lipid droplets (LD) as central organelles in cellular metabolism. For example, EM has illuminated that LDs, in contrast to all other cellular organelles, are uniquely enclosed by a single phospholipid monolayer, revealed the architecture of LD contact sites with different organelles, and provided near‐atomic resolution maps of key enzymes that regulate neutral lipid biosynthesis and LD biogenesis. In this review, we first provide a brief history of pivotal findings in LD biology unveiled through the lens of an electron microscope. We describe the main EM techniques used in the context of LD research and discuss their current capabilities and limitations, thereby providing a foundation for utilizing suitable EM methodology to address LD‐related questions with sufficient level of structural preservation, detail, and resolution. Finally, we highlight examples where EM has recently been and is expected to be instrumental in expanding the frontiers of LD biology. [ABSTRACT FROM AUTHOR]

The brain contains thousands of millions of synapses, exhibiting diverse structural, molecular, and functional characteristics. However, synapses can be classified into two primary morphological types: Gray's type I and type II, corresponding to Colonnier's asymmetric (AS) and symmetric (SS) synapses, respectively. AS and SS have a thick and thin postsynaptic density, respectively. In the cerebral cortex, since most AS are excitatory (glutamatergic), and SS are inhibitory (GABAergic), determining the distribution, size, density, and proportion of the two major cortical types of synapses is critical, not only to better understand synaptic organization in terms of connectivity, but also from a functional perspective. However, several technical challenges complicate the study of synapses. Potassium ferrocyanide has been utilized in recent volume electron microscope studies to enhance electron density in cellular membranes. However, identifying synaptic junctions, especially SS, becomes more challenging as the postsynaptic densities become thinner with increasing concentrations of potassium ferrocyanide. Here we describe a protocol employing Focused Ion Beam Milling and Scanning Electron Microscopy for studying brain tissue. The focus is on the unequivocal identification of AS and SS types. To validate SS observed using this protocol as GABAergic, experiments with immunocytochemistry for the vesicular GABA transporter were conducted on fixed mouse brain tissue sections. This material was processed with different concentrations of potassium ferrocyanide, aiming to determine its optimal concentration. We demonstrate that using a low concentration of potassium ferrocyanide (0.1%) improves membrane visualization while allowing unequivocal identification of synapses as AS or SS. [ABSTRACT FROM AUTHOR]

A metho d is proposed for measuring high-voltage charging potentials of dielectrics under ion irradiation by shifting the boundary of the bremsstrahlung X-ray spectrum. Since there is no bremsstrahlung output during Xe+ ion irradiation, it was proposed to use a probing electron beam to generate bremsstrahlung X-rays. To eliminate the effect of charge compensation on the surface, the value of the current of the probing probe of electrons was selected. The values of the equilibrium charging potentials of Al2O3 ceramics, Al2O3 sapphire, SiO2, and Teflon are obtained at different ion irradiation energies. The data obtained are compared with the results of spectrometric studies. [ABSTRACT FROM AUTHOR]

The porous-cracked microstructure of plasma sprayed ceramics coatings directly influences their macroscopic mechanical response. A 3D micromechanical model based on the discrete element method (DEM) is developed to reproduce their behavior. 3D observations are conducted with FIB-SEM nano-tomography and image analysis is used to extract and discretize the microstructure. A modeling strategy is developed to incorporate both pores and cracks into the model while preserving the computation performance. The lattice nature of DEM is used for the modeling of crack thickness that are smaller than the discrete element size. A method to compute the crack thickness from gray level images is proposed. Virtual quasi-static tests are performed on a sample of yttria-stabilized-zirconia. The results are in accordance with the literature data, such as the anisotropy and the non-linear behavior. The model is helpful to precisely investigate the role of the microscopic components, and micro-cracking on the macroscopic failure. • Crack thickeness is computed by image analysis and modeled in a numerical model. • Non-linear behavior in compression is due to crack closure with increasing pressure. • Cracks are responsible for transverse isotropy. • A coupling effect between pores and cracks is identified. • A DEM tensile failure criterion allows modelling macroscopic failure in compression. [ABSTRACT FROM AUTHOR]

Magnetic signals in igneous rocks arise from assemblages of iron‐oxide bearing minerals that differ in for example, size, shape, and chemistry. Paleomagnetic measurements on bulk samples measure millions of such grains simultaneously, producing a statistical ensemble of the magnetic moments of the individual grains. Scanning magnetometry techniques such as the Quantum Diamond Microscope (QDM) measure magnetic signals on micrometer scales, allowing the identification of magnetic moments of individual grains in a sample using for example, Micromagnetic Tomography (MMT). Here we produce a grain‐size distribution of iron‐oxides in a typical Hawaiian basalt from the superparamagnetic threshold (∼40 nm) to grains with a diameter of 10 µm. This grain‐size distribution is obtained by combining FIB‐SEM and MicroCT data from sister specimens, and normalizing them to the mineral surface area of non‐magnetic minerals. Then we use this grain‐size distribution to determine the contributions of individual magnetic carriers to bulk magnetic measurements and surface magnetometry. We found that measurements on bulk samples are sensitive to relatively small grain sizes in the realm of single domain or vortex states (<200 nm), while signals in surface magnetometry arise mainly from larger grains with diameters >1 µm. This implies that bulk measurements cannot be compared straightforwardly to signals from surface magnetometry from the same sample. Moreover, our observations explain why MMT results are insensitive to the presence of many small grains in a sample that intuitively should hamper their outcome. Plain Language Summary: Magnetic grains in lavas acquire a magnetic signal while cooling in presence of Earth's magnetic field. However, not all grains preserve the signal well, meaning that both good and bad recorders are present. Classical paleomagnetic techniques measure the magnetic signal of all recorders together, that is, the bulk signal. New scanning magnetometry techniques such as Micromagnetic Tomography acquire the signal from individual recorders in the lava, enabling the selection of potentially good recorders and the rejection of signals from bad recorders. Here we found that these two types of magnetic measurements do not measure the same grains that are present in the sample: classical techniques emphasize small grains (<200 nm), while signals in surface magnetometry arise mainly from larger grains with diameters >1 μm. This means that measurements from both techniques performed on the same sample material cannot be compared straightforwardly. Furthermore, our results explain why Micromagnetic Tomography results often are successful, even when many small magnetic grains that intuitively should hamper this technique are present in a sample. Key Points: We determine contributions of individual magnetic carriers to bulk magnetic measurements and surface magnetometryMeasurements on bulk samples are sensitive to small grains (<200 nm); surface magnetometry emphasizes signals from larger grains (>1 μm)Our observations explain why undetected ghost grains in MMT experiments have an unintuitively low impact on the accuracy of MMT results [ABSTRACT FROM AUTHOR]

We have measured the dependence of the Fe–Mg interdiffusion coefficient, DFe-Mg, on the ferrosilite component in orthopyroxene, which so far has not been experimentally calibrated. Diffusion couples, consisting of approximately 1 µm thin-films were deposited by pulsed laser ablation on orthopyroxene crystals of En91Fs9 composition. Diffusion experiments were carried out at atmospheric pressure in vertical gas mixing furnaces (CO-CO2) at temperatures between 950 and 1100 °C at constant fO2 = 10–7 Pa. Using a focused ion beam-scanning electron microscope (FIB-SEM), FIB-foils were cut from diffusion couples before and after annealing. Diffusion profiles were extracted by using combined backscattered electron (BSE) imaging and energy dispersive X-ray (EDXS) mapping on FIB-foils which allowed to resolve concentration gradients within 20 nm. The microstructure of the diffusion experiments was investigated using transmission electron microscopy (TEM). Using this method, we obtained the first experimentally determined data on the dependence of DFe-Mg on the ferrosilite content in orthopyroxene at different temperatures, appearing to increase with increasing a temperature. For the temperature range (950 – 1100 °C), log fO2 = -7 Pa, along [001] in the composition Fs9, the log DFe-Mg yields the following Arrhenius equation: D F e - M g [ m 2 / s ] = 3.8 · 10 - 9 exp [ - 261.07 ± 24 [ k J / m o l ] / (R / T [ K ]) ] The effect of XFe on DFe-Mg, given by D (X Fe) = D (X Fe = 0.09) · 10 m (X Fe - 0.09) , can be calculated by the following parameterization where m follows a linear regression of m on temperature: m = - 2.711 · 10 4 / T (K) + 23.5408 By considering the DFe-Mg dependence on XFe, the timescales of natural processes obtained from modelling the compositional zoning of natural crystal may considerably differ from previously estimated timescales. [ABSTRACT FROM AUTHOR]

Abstract Magnetic signals in igneous rocks arise from assemblages of iron‐oxide bearing minerals that differ in for example, size, shape, and chemistry. Paleomagnetic measurements on bulk samples measure millions of such grains simultaneously, producing a statistical ensemble of the magnetic moments of the individual grains. Scanning magnetometry techniques such as the Quantum Diamond Microscope (QDM) measure magnetic signals on micrometer scales, allowing the identification of magnetic moments of individual grains in a sample using for example, Micromagnetic Tomography (MMT). Here we produce a grain‐size distribution of iron‐oxides in a typical Hawaiian basalt from the superparamagnetic threshold (∼40 nm) to grains with a diameter of 10 µm. This grain‐size distribution is obtained by combining FIB‐SEM and MicroCT data from sister specimens, and normalizing them to the mineral surface area of non‐magnetic minerals. Then we use this grain‐size distribution to determine the contributions of individual magnetic carriers to bulk magnetic measurements and surface magnetometry. We found that measurements on bulk samples are sensitive to relatively small grain sizes in the realm of single domain or vortex states (1 µm. This implies that bulk measurements cannot be compared straightforwardly to signals from surface magnetometry from the same sample. Moreover, our observations explain why MMT results are insensitive to the presence of many small grains in a sample that intuitively should hamper their outcome.

The brain contains thousands of millions of synapses, exhibiting diverse structural, molecular, and functional characteristics. However, synapses can be classified into two primary morphological types: Gray’s type I and type II, corresponding to Colonnier’s asymmetric (AS) and symmetric (SS) synapses, respectively. AS and SS have a thick and thin postsynaptic density, respectively. In the cerebral cortex, since most AS are excitatory (glutamatergic), and SS are inhibitory (GABAergic), determining the distribution, size, density, and proportion of the two major cortical types of synapses is critical, not only to better understand synaptic organization in terms of connectivity, but also from a functional perspective. However, several technical challenges complicate the study of synapses. Potassium ferrocyanide has been utilized in recent volume electron microscope studies to enhance electron density in cellular membranes. However, identifying synaptic junctions, especially SS, becomes more challenging as the postsynaptic densities become thinner with increasing concentrations of potassium ferrocyanide. Here we describe a protocol employing Focused Ion Beam Milling and Scanning Electron Microscopy for studying brain tissue. The focus is on the unequivocal identification of AS and SS types. To validate SS observed using this protocol as GABAergic, experiments with immunocytochemistry for the vesicular GABA transporter were conducted on fixed mouse brain tissue sections. This material was processed with different concentrations of potassium ferrocyanide, aiming to determine its optimal concentration. We demonstrate that using a low concentration of potassium ferrocyanide (0.1%) improves membrane visualization while allowing unequivocal identification of synapses as AS or SS.

ATG9A is an important membrane protein in mammalian macroautophagy. The formation of autophagosomes and phagophores is blocked in atg9a KO cells. However, it remains possible that residual membrane formation activity exists in these cells. These precursor structures that precede phagophores are, if they exist, rare and may be difficult to find. Here, we introduce the modified volume correlative light and electron microscopy (CLEM) method to analyze these structures three-dimensionally. In addition to target proteins, mitochondria were labeled as a landmark for precise correlation of slice images by a confocal fluorescence microscope and a focused ion beam scanning electron microscope. We found phagophores and small membrane vesicles near SQSTM1/p62 aggregates in atg9a KO cells, indicating that phagophores could be formed in atg9a-deficient cells, although they were immature and inefficient. Furthermore, we found that RB1CC1/FIP200-positive structures formed clusters around SQSTM1/p62 with ferritin and TAX1BP1. Taken together, our method contributes to the understanding of undiscovered fine structures. Abbreviations: CLEM: correlative light and electron microscopy; EM: electron microscopy; ER: endoplasmic reticulum; FIB-SEM: focused ion beam scanning electron microscopy; FM: fluorescence microscopy; GFP: green fluorescent protein; KO: knock out; MEF: mouse embryonic fibroblast; PBS: phosphate-buffered saline; ROI: region of interest; SEM: scanning electron microscopy.

Osteodentin is a dominant mineralized collagenous tissue in the teeth of many fishes, with structural and histological characteristics resembling those of bone. Osteodentin, like bone, comprises osteons as basic structural building blocks, however, it lacks the osteocytes and the lacuno-canalicular network (LCN), which are known to play critical roles in controlling the mineralization of the collagenous matrix in bone. Although numerous vascular canals exist in osteodentin, their role in tooth maturation and the matrix mineralization process remain poorly understood. Here, high resolution micro-computed tomography (micro-CT) and focused ion beam-scanning electron microscopy (FIB-SEM) were used to obtain 3D structural information of osteodentin in shark teeth at multiple scales. We observed a complex 3D network of primary canals with a diameter ranging from ∼10 µm to ∼120 µm, where the canals are surrounded by osteon-like concentric layers of lamellae, with 'interosteonal' tissue intervening between neighboring osteons. In addition, numerous hierarchically branched secondary canals extended radially from the primary canals into the interosteonal tissue, decreasing in diameter from ∼10 µm to hundreds of nanometers. Interestingly, the mineralization degree increases from the periphery of primary canals into the interosteonal tissue, suggesting that mineralization begins in the interosteonal tissue. Correspondingly, the hardness and elastic modulus of the interosteonal tissue are higher than those of the osteonal tissue. These results demonstrate that the 3D hierarchical canal network is positioned to play a critical role in controlling the gradient mineralization of osteodentin, also providing valuable insight into the formation of mineralized collagenous tissue without osteocytes and LCN. Bone is a composite material with versatile mechanical properties. Osteocytes and their lacuno-canalicular network (LCN) are known to play critical roles during formation of human bone. However, the bone and osteodentin of many fishes, although lacking osteocytes and LCN, exhibit similar osteon-like structure and mechanical functions. Here, using various high resolution 3D characterization techniques, we reveal that the 3D network of primary canals and numerous hierarchically branched secondary canals correlate with the mineralization gradient and micromechanical properties of osteonal and interosteonal tissues of shark tooth osteodentin. This work significantly improves our understanding of the construction of bone-like mineralized tissue without osteocytes and LCN, and provides inspirations for the fabrication of functional materials with hierarchical structure. [Display omitted] [ABSTRACT FROM AUTHOR]

The spatial-temporal relationship between cells, extracellular matrices, and mineral deposits is fundamental for an improved understanding of mineralization mechanisms in vertebrate tissues. By utilizing focused ion beam-scanning electron microscopy with serial surface imaging, normally mineralizing avian tendons have been studied with nanometer resolution in three dimensions with volumes exceeding tens of micrometers in range. These parameters are necessary to yield sufficiently fine ultrastructural details while providing a comprehensive overview of the interrelationships between the tissue structural constituents. Investigation reveals a complex lacuno-canalicular network in highly mineralized tendon regions, where ∼100 nm diameter canaliculi emanating from cell (tenocyte) lacunae surround extracellular collagen fibril bundles. Canaliculi are linked to smaller channels of ∼40 nm diameter, occupying spaces between fibrils. Close to the tendon mineralization front, calcium-rich deposits appear between the fibrils and, with time, mineral propagates along and within them. These close associations between tenocytes, tenocyte lacunae, canaliculi, small channels, collagen, and mineral suggest a concept for the mineralization process, where ions and/or mineral precursors may be transported through spaces between fibrils before they crystallize along the surface of and within the fibrils.

Caudofoveates are benthic organisms that reside in the deep waters of continental slopes in the world. They are considered to be a group that is of phylogenetic and ecological importance for the phylum Mollusca. However, they remain poorly studied. In this work, we revealed the structure of the spicules of Caudofoveatan mollusks Falcidens sp. The spicules presented a hierarchical organization pattern across different length scales. Various imaging and analytical methods related to light and electron microscopy were employed to characterize the samples. The primary imaging methods utilized included: low voltage field emission scanning electron microscopy (FEG-SEM), focused ion beam-scanning electron microscopy (FIB-SEM), high-resolution transmission electron microscopy (HRTEM), atomic force microscopy (AFM), and electron diffraction. In addition, we performed a physicochemical analysis by electron energy loss spectroscopy (EELS) and energy dispersive X-ray spectroscopy (EDS). A crucial factor for successfully obtaining the results was the preparation of lamellae from the spicules without damaging the original structures, achieved using FIB-SEM. This allowed us to obtain diffraction patterns of significant areas of well-preserved sections (lamellae) of the spicules in specific directions and demonstrate for the first time that the bulk of these structures is organized as a single crystal of calcium carbonate aragonite. On the other hand, AFM imaging of the spicules' dorsal surface revealed a wavy appearance, composed of myriads of small, pointed crystallites oriented along the spicules' longer axis (i.e., the c-axis of the aragonite). The organization pattern of these small crystallites, the possible presence of twins, the relationship between confinement conditions and accessory ions in the preference for mineral polymorphs, and the single crystalline appearance of the entire spicule, along with the observation of growth lines, provide support for further studies employing Caudofoveata spicules as a model for biomineralization studies. [ABSTRACT FROM AUTHOR]

Reliable prediction of the shale fracturing process is a challenging problem in exploiting deep shale oil and gas resources. Complex fracture networks need to be artificially created to employ deep shale oil and gas reserves. Randomly distributed minerals and heterogeneities in shales significantly affect mechanical properties and fracturing behaviors in oil and gas exploitation. Describing the actual microstructure and associated heterogeneities in shales constitutes a significant challenge. The RFPA3D (rock failure process analysis parallel computing program)-based modeling approach is a promising numerical technique due to its unique capability to simulate the fracturing behavior of rocks. To improve traditional numerical technology and study crack propagation in shale on the microscopic scale, a combination of highprecision internal structure detection technology with the RFPA3D numerical simulation method was developed to construct a real mineral structure-based modeling method. First, an improved digital image processing technique was developed to incorporate actual shale microstructures (focused ion beam scanning electron microscopy was used to capture shale microstructure images that reflect the distributions of different minerals) into the numerical model. Second, the effect of mineral inhomogeneity was considered by integrating the mineral statistical model obtained from the mineral nanoindentation experiments into the numerical model. By simulating a shale numerical model in which pyrite particles are wrapped by organic matter, the effects of shale microstructure and applied stress state on microcrack behavior and mechanical properties were investigated and analyzed. In this study, the effect of pyrite particles on fracture propagation was systematically analyzed and summarized for the first time. The results indicate that the distribution of minerals and initial defects dominated the fracture evolution and the failure mode. Cracks are generally initiated and propagated along the boundaries of hard mineral particles such as pyrite or in soft minerals such as organic matter. Locations with collections of hard minerals are more likely to produce complex fractures. This study provides a valuable method for understanding the microfracture behavior of shales. [ABSTRACT FROM AUTHOR]

Natural graphite ores are usually upgraded by froth flotation. However, complex processes with multistage grinding and flotation are required to achieve decent liberation and separation of graphite and gangue minerals. This study reports a short and improved flotation process for fine flake graphite ore by employing a thickening stage. The results indicated that increasing the regrinding concentration via thickening can improve the grinding efficiency and, thus, shorten the separation process. With thickening, a high-grade intermediate concentrate of 96.01% was obtained after three steps of cleaner flotation, which is close to the final concentrate after five steps. Particle size distribution analysis and FIB-SEM-EDS studies suggested that the main contribution of thickening–regrinding was to achieve better abrasion rather than attrition of the graphite flakes, thus liberating the interlayer impurities without reducing the size of the graphite flakes. This study offers a more cost-effective pathway for the simplified flotation of natural graphite ores. [ABSTRACT FROM AUTHOR]

To clarify dynamic changes of organelle microstructures in Chlorella pyrenoidosa cells during photosynthetic growth with CO2 fixation, three-dimensional (3D) organelle microstructures in three growth periods of meristem, elongation, and maturity were quantitatively determined and comprehensively reconstructed with focused ion beam scanning electron microscopy (FIB-SEM). The single round-pancake mitochondria in each cell split into a dumbbell and then into a circular ring, while the barycenter distance of mitochondria to chloroplast and nucleus was reduced to 45.5% and 88.3% to strengthen energy transfer, respectively. The single pyrenoid consisting of a large part and another small part in each chloroplast gradually developed to a mature state in which the two parts were nearly equal in size. The nucleolus progressively became larger with euchromatin replication. The number of starch grains gradually increased, but the mean grain volume remained nearly unchanged. [ABSTRACT FROM AUTHOR]

Porous carbon‐based electrodes are frequently applied in electrochemical energy technologies, for instance in fuel cells and redox flow batteries. In previous work, we observed that the final structure of a fuel cell electrode is dominated by both the morphology of the support material and its processing into a 3D porous structure. Herein, the impact of catalyst support morphology on the performance of polymer electrolyte membrane fuel cells was studied comparing carbon‐supported platinum catalysts only differing in the shape of the carbon support material with otherwise similar features. Carbon‐supported Pt catalysts were obtained by carbonization of polyaniline (PANI) in long fibrous, short fibrous, and granular shape. The chemical identity of the PANI precursors was demonstrated by FTIR spectroscopy and elemental analysis (EA). The final carbon‐supported platinum catalysts were characterized by EA, Raman spectroscopy, XRD, and TEM exhibiting similar degree of carbonization, nanoparticle size, and nanoparticle dispersion. The effect of support morphology and the resulting differences in the 3D structure of the porous electrode were investigated by focused ion beam‐scanning electron microscopy slice and view technique and correlated to their fuel cell performance. [ABSTRACT FROM AUTHOR]

Unicellular euglyphid testate amoeba Paulinella micropora with filose pseudopodia secrete approximately 50 siliceous scales into the extracellular template-free space to construct a shell isomorphic to that of its mother cell. This shell-constructing behavior is analogous to building a house with bricks, and a complex mechanism is expected to be involved for a single-celled amoeba to achieve such a phenomenon; however, the three-dimensional (3D) structure of the shell and its assembly in P. micropora are still unknown. In this study, we aimed to clarify the positional relationship between the cytoplasmic and extracellular scales and the structure of the egg-shaped shell in P. micropora during shell construction using focused ion beam scanning electron microscopy (FIB-SEM). 3D reconstruction revealed an extensive invasion of the electron-dense cytoplasm between the long sides of the positioned and stacked scales, which was predicted to be mediated by actin filament extension. To investigate the architecture of the shell of P. micropora, each scale was individually segmented, and the position of its centroid was plotted. The scales were arranged in a left-handed, single-circular ellipse in a twisted arrangement. In addition, we 3D printed individual scales and assembled them, revealing new features of the shell assembly mechanism of P. micropora. Our results indicate that the shell of P. micropora forms an egg shape by the regular stacking of precisely designed scales, and that the cytoskeleton is involved in the construction process.

Flexible graphite (FG) with ρ = 1 g/cm3 density is a type of highly porous and anisotropic graphite, mainly used for gaskets and sealing applications, but also suitable for energy absorption, such as in the beam dumping devices of the Large Hadron Collider (see Heredia 2021 [1]). Knowledge of its microstructure and mechanical properties needs to be developed for the selection of an adequate material model able accurately predict stresses and failure in FG components. Here, the FG microstructure properties available in literature are reviewed, followed by Focused Ion Beam - Scanning Electron Microscopy investigation and compression tests. Specifically, a single 100 μm × 150 μm cross section was obtained, and the 2D pore sizes and shapes were quantified using image segmentation. Monotonic and cyclic out-of-plane compression tests were performed in single and stacked configuration. Stress-strain curves showed three domains: the initial toe, the transition and the densification domain. The cyclic tangent modulus was also calculated from the cyclic tests. Many observations suggested that FG behaves similarly to crushable foams, crumpled materials and compacted powders, and that both crystalline microstructure and crumpled mesostructure play a predominant role in the deformation mechanism.

FlowDenoising is a software tool that implements an adaptive Gaussian denoising filter that preserves visually appreciable structures in volumes of 3D electron microscopy (3DEM). It proceeds by nonrigidly aligning the 2D slices in each dimension, using an optical flow estimator, prior to applying a standard separable (1D) Gaussian filter. FlowDenoising has been developed in Python leveraging well-known public domain libraries, such as OpenCV and NumPy. Furthermore, the software tool exploits data-level parallelism to significantly reduce processing times. Its abilities to denoise huge volumes in just minutes on standard multicore computers makes it a useful tool in 3DEM to explore the interior of cells and tissues at the nanoscale.

Microcrystal electron diffraction (MicroED) leverages the strong interaction between matter and electrons to determine protein structures from vanishingly small crystals. This strong interaction limits the thickness of crystals that can be investigated by MicroED, mainly due to absorption. Recent studies have demonstrated that focused ion-beam (FIB) milling can thin crystals into ideal-sized lamellae; however, it is not clear how to best apply FIB milling for MicroED. Here, the effects of polishing the lamellae, whereby the last few nanometers are milled away using a low-current gallium beam, are explored in both the platinum-precoated and uncoated samples. Our results suggest that precoating samples with a thin layer of platinum followed by polishing the crystal surfaces prior to data collection consistently led to superior results as indicated by higher signal-to-noise ratio, higher resolution, and better refinement statistics. This study lays the foundation for routine and reproducible methodology for sample preparation in MicroED.

Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation typically involves sonication or pipetting a slurry of crystals from a crystallization drop. The resultant crystal fragments are fragile and the quality of the data that can be obtained from them is sensitive to subsequent sample preparation for cryoelectron microscopy as interactions in the water-air interface can damage crystals during blotting. Here, we demonstrate the use of a focused ion beam to generate lamellae of macromolecular protein crystals for continuous rotation MicroED that are of ideal thickness, easy to locate, and require no blotting optimization. In this manner, crystals of nearly any size may be scooped and milled to desired dimensions prior to data collection, thus streamlining the methodology for sample preparation for MicroED.

Abstract Porous carbon‐based electrodes are frequently applied in electrochemical energy technologies, for instance in fuel cells and redox flow batteries. In previous work, we observed that the final structure of a fuel cell electrode is dominated by both the morphology of the support material and its processing into a 3D porous structure. Herein, the impact of catalyst support morphology on the performance of polymer electrolyte membrane fuel cells was studied comparing carbon‐supported platinum catalysts only differing in the shape of the carbon support material with otherwise similar features. Carbon‐supported Pt catalysts were obtained by carbonization of polyaniline (PANI) in long fibrous, short fibrous, and granular shape. The chemical identity of the PANI precursors was demonstrated by FTIR spectroscopy and elemental analysis (EA). The final carbon‐supported platinum catalysts were characterized by EA, Raman spectroscopy, XRD, and TEM exhibiting similar degree of carbonization, nanoparticle size, and nanoparticle dispersion. The effect of support morphology and the resulting differences in the 3D structure of the porous electrode were investigated by focused ion beam‐scanning electron microscopy slice and view technique and correlated to their fuel cell performance.

The main aim of the present study was to determine if synapses from the exceptionally small brain of the Etruscan shrew show any peculiarities compared to the much larger human brain. We analyzed the cortical synaptic density and a variety of structural characteristics of 7,239 3D reconstructed synapses, using using Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM). We found that some of the general synaptic characteristics are remarkably similar to those found in the human cerebral cortex. However, the cortical volume of the human brain is about 50,000 times larger than the cortical volume of the Etruscan shrew, while the total number of cortical synapses in human is only 20,000 times the number of synapses in the shrew, and synaptic junctions are 35% smaller in the Etruscan shrew. Thus, the differences in the number and size of synapses cannot be attributed to a brain size scaling effect but rather to adaptations of synaptic circuits to particular functions. [ABSTRACT FROM AUTHOR]

For decades, bacteria were thought of as "bags" of enzymes, lacking organelles and significant subcellular structures. This stood in sharp contrast with eukaryotes, where intracellular compartmentalization and the role of large-scale order had been known for a long time. However, the emerging field of Bacterial Cell Biology has established that bacteria are in fact highly organized, with most macromolecular components having specific subcellular locations that can change depending on the cell's physiological state (Barry & Gitai, 2011; Lenz & Søgaard-Andersen, 2011; Thanbichler & Shapiro, 2008). For example, we now know that many processes in bacteria are orchestrated by cytoskeletal proteins, which polymerize into surprisingly diverse superstructures, such as rings, sheets, and tread-milling rods (Pilhofer & Jensen, 2013). These superstructures connect individual proteins, macromolecular assemblies, and even two neighboring cells, to affect essential higher-order processes including cell division, DNA segregation, and motility. Understanding these processes requires resolving the in vivo dynamics and ultrastructure at different functional stages of the cell, at macromolecular resolution and in 3-dimensions (3D). Fluorescence light microscopy (fLM) of tagged proteins is highly valuable for investigating protein localization and dynamics, and the resolution power of transmission electron microscopy (TEM) is required to elucidate the structure of macromolecular complexes in vivo and in vitro. This chapter summarizes the most recent advances in LM and TEM approaches that have revolutionized our knowledge and understanding of the microbial world. [ABSTRACT FROM AUTHOR]

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Focused ion beam scanning electron microscope (FIB-SEM) is one of the most advanced imaging techniques for analyzing and understanding complex pore networks in shale and other fine-grained formations. However, FIB-SEM imaging tends to be time-consuming and labor-intensive and can result in biased interpretations associated with pore analysis. Recently, U-Net or its variants for image segmentation have been applied to capture microscopic pores at higher resolutions. The 'traditional' encoder-decoder-based approaches tend to detect very fine-scale microscopic pores poorly. This study presents an improved convolutional architecture for automatically analyzing pore structures in shale reservoirs using FIB-SEM. It does so by applying an overcomplete convolutional architecture, KiU-Net, to capture very fine-scale microscopic pores by accurately defining their edges in the input FIB-SEM images. The KiU-Net learns low and high-level features by making the model more sensitive to fine-scale microscopic pores in the input images. The purpose of this study is to demonstrate KiU-Net's capabilities by analyzing different shale formations with varying characteristics. The results indicate that KiU-Net is more accurate and efficient than other methods in predicting nanopores in the Longmaxi, Niutitang, Qingshankou, Qianjiang, and Yanchang Formations (China), Bakken shale (Canada), and coal reservoirs (China). Furthermore, KiU-Net demonstrated the advantage of requiring fewer parameters and achieving super convergence compared to the Attention U-Net technique. KiU-Net addresses the challenges of the Edge-Threshold Automatic Processing (ETAP) methods by capturing very fine-scale microscopic pores with accurate edges. This study further enhances the accuracy and efficiency of pore analysis in shales, thereby offering an improved method for understanding shale reservoir quality with the potential to improve petroleum recovery from such formations. • FIB-SEM is an advanced imaging technique used to analyze pore networks in shale and other fine-grained rocks. • An overcomplete convolutional architecture is proposed for analyzing pore structures in shale using FIB-SEM. • KiU-Net detects low- and high-level features by enhancing the sensitivity to fine-scale microscopic pores. • KiU-Net captures very fine-scale microscopic pores, overcoming the limitations of the ETAP method and Attention U-Net. [ABSTRACT FROM AUTHOR]

Titanium hydride is widely considered as an important hydrogen storage material due to its high capacity, stability and low cost. The microstructure of titanium hydride, which is usually induced by the hydrogenation of titanium, strongly influences its storage performance. However, the relationship between the hydrogenation conditions and the derived microstructure of titanium hydride is still unclear, limiting the development of its structure design strategy. In this work, the microstructure of titanium hydride is correlated with its hydrogenation conditions through the thermodynamics and kinetics. By evaluating the effects of the hydrogenation temperature, pressure and cooling rate, three classes of the hydrogenation processes were clarified: kinetic-limited, continuous and stepwise. Besides, according to the SEM and FIB-STEM results, these different processes are confirmed to greatly vary the bulk microstructure. It is concluded that a fast and continuous transition induces a broken bulk morphology, while a stepwise process leads to a larger bulk grain size. In summary, this study presents a framework for designing titanium hydride structures by modifying phase transition processes through careful adjustment of hydrogenation parameters, namely, a condition-process-structure relationship. This relationship offers crucial guidance in managing the grain refinement or coarsening of hydrides within hydrogen storage components and metallurgical applications. • Effect of hydrogenation conditions on the microstructure of TiD x was studied by electron microscopy. • Marked SEM shows the surface morphology of TiD x exhibited negligible change after hydrogenation. • FIB-STEM shows that the cross-section morphology of TiD x varies significantly according to the hydrogenation conditions. • Three classes of the hydrogenation processes were clarified as kinetic-limited, continuous and stepwise. • Fast and continuous phase transition fractures bulk, while a stepwise one induces relative larger grains. [ABSTRACT FROM AUTHOR]