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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]

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]

The present study was carried out to investigate the synaptic ultrastructure of the mouse cerebellum using focused ion beam scanning electron microscopy (FIB-SEM). The experiment were performed using seven weeks old wild type mice. In the present work, we used a newly established morphological investigation method of axospinous synapses (FIB-SEM). This method enables fully automated acquisition of serial ultrastructural data at a spatial resolution of 10 nm in z-axis without the ultrastructure distortions. FIB-SEM enabled easily identification of axospinous synapses in the mouse cerebellum where individual synaptic ultra-structure can be easily identified and measured. The results indicated that, the cerbellum axospinous synapses have presynaptic and postsynaptic compartment. The presynaptic compartment consists of the presynaptic axonal varicosity, synaptic vesicles and mitochondria. The postsynaptic compartment consists of the dendritic spine, postsynaptic density and smooth endoplasmic reticulum and spine apparatus. The volume of presynaptic and postsynaptic compartments was varaible. The results indicated a significant positive correlation between presynaptic compartments as well as between postsynaptic compartments. Moreover, this study represented novel correlations between presynaptic and postsynaptic parameters. [ABSTRACT FROM AUTHOR]

Introduction: Osteocytes play a role as mechanosensory cells by sensing flow-induced mechanical stimuli applied on their cell processes. High-resolution imaging of osteocyte processes and the canalicular wall are necessary for the analysis of this mechanosensing mechanism. Focused ion beam-scanning electron microscopy (FIB-SEM) enabled the visualization of the structure at the nanometer scale with thousands of serial-section SEM images. We applied machine learning for the automatic semantic segmentation of osteocyte processes and canalicular wall and performed a morphometric analysis using three-dimensionally reconstructed images. Materials and methods: Six-week-old-mice femur were used. Osteocyte processes and canaliculi were observed at a resolution of 2 nm/voxel in a 4 × 4 μm region with 2000 serial-section SEM images. Machine learning was used for automatic semantic segmentation of the osteocyte processes and canaliculi from serial-section SEM images. The results of semantic segmentation were evaluated using the dice similarity coefficient (DSC). The segmented data were reconstructed to create three-dimensional images and a morphological analysis was performed. Results: The DSC was > 83%. Using the segmented data, a three-dimensional image of approximately 3.5 μm in length was reconstructed. The morphometric analysis revealed that the median osteocyte process diameter was 73.8 ± 18.0 nm, and the median pericellular fluid space around the osteocyte process was 40.0 ± 17.5 nm. Conclusion: We used machine learning for the semantic segmentation of osteocyte processes and canalicular wall for the first time, and performed a morphological analysis using three-dimensionally reconstructed images. [ABSTRACT FROM AUTHOR]

Keywords: Monazite; fission tracks; etching; FIB-SEM; etch-anneal-etch EN Monazite fission tracks etching FIB-SEM etch-anneal-etch 1065 1073 9 06/01/22 20220601 NES 220601 Introduction A series of experiments presented by [9] led to a first-order understanding of monazite fission track etching properties. [Extracted from the article]

The structural complexity of nervous tissue makes it very difficult to unravel the connectivity between neural elements at different scales. Numerous methods are available to trace long-range projections at the light microscopic level, and to identify the actual synaptic connections at the electron microscopic level. However, correlating mesoscopic and nanoscopic scales in the same cell, cell population or brain region is a problematic, laborious and technically demanding task. Here we present an effective method for the 3D reconstruction of labeled subcellular structures at the ultrastructural level, after single-neuron labeling in fixed tissue. The brain is fixed by intracardial perfusion of aldehydes and thick vibratome sections (250 μm) are obtained. Single cells in these vibratome sections are intracellularly injected with horseradish peroxidase (HRP), so that the cell body and its processes can be identified. The thick sections are later flat-embedded in epoxy resin and re-sectioned into a series of thinner (7 μm) sections. The sections containing the regions of interest of the labeled cells are then imaged with automated focused ion beam milling and scanning electron microscopy (FIB-SEM), acquiring long series of high-resolution images that can be reconstructed, visualized, and analyzed in 3D. With this methodology, we can accurately select any cellular segment at the light microscopic level (e.g., proximal, intermediate or distal dendrites, collateral branches, axonal segments, etc.) and analyze its synaptic connections at the electron microscopic level, along with other ultrastructural features. Thus, this method not only facilitates the mapping of the synaptic connectivity of single-labeled neurons, but also the analysis of the surrounding neuropil. Since the labeled processes can be located at different layers or subregions, this method can also be used to obtain data on the differences in local synaptic organization that may exist at different portions of the labeled neurons. [ABSTRACT FROM AUTHOR]

[Display omitted] • Microbial cellulose production with a new Kombucha consortium. • High cellulose production amount (18.68 g/L). • A significant cost reduction (29.74 %) was achieved. • Obtained cellulose has excellent physicochemical properties. Bacterial cellulose (BC) is a natural biopolymer, used in biomedical, food, paper, electronics and other industrial processes. The objective of this research was to improve the productivity of cellulose by using a new kombucha consortium (Komagataeibacter saccharivorans LN886705, Brettanomyces bruxellensis MH393498 and Brettanomyces anomalus KY103303) with a low-cost nitrogen source (black tea). Accordingly, BC can be highly produced (18.68 g/L BC) in optimized media (containing 1% black tea and 6% glucose, pH 6) at 30 °C, for 10 days under static cultivation. Results demonstrated that cellulose production enhanced 4.06 times in optimized medium when compared with Hestrin-Schramm medium (4.59 g/L BC) with a 29.74% cost reduction. Characterization of obtained cellulose was performed by using Focussed Ion Beam Scanning Electron Microscopy (FIB-SEM),Fouirer Transform Infrared (FT-IR) Spectrometer, Thermogravimetric Analysis (TGA-DSC) and X-Ray Diffraction (XRD). Cellulose fibrils showed a typical porous, cellulose type I structure and exhibits high thermal stability with a total weight loss of 76%. Results confirmed that, it was possible to obtain high value cellulose with excellent physicochemical properties and cost-reduction by using low-cost nitrogen source. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum exit sites (ERESs) are tubular outgrowths of endoplasmic reticulum that serve as the earliest station for protein sorting and export into the secretory pathway. How these structures respond to different cellular conditions remains unclear. Here, we report that ERESs undergo lysosome-dependent microautophagy when Ca2+ is released by lysosomes in response to nutrient stressors such as mTOR inhibition or amino acid starvation in mammalian cells. Targeting and uptake of ERESs into lysosomes were observed by super-resolution live-cell imaging and focus ion beam scanning electron microscopy (FIB-SEM). The mechanism was ESCRT dependent and required ubiquitinated SEC31, ALG2, and ALIX, with a knockout of ALG2 or function-blocking mutations of ALIX preventing engulfment of ERESs by lysosomes. In vitro, reconstitution of the pathway was possible using lysosomal lipid-mimicking giant unilamellar vesicles and purified recombinant components. Together, these findings demonstrate a pathway of lysosome-dependent ERES microautophagy mediated by COPII, ALG2, and ESCRTS induced by nutrient stress. [Display omitted] • Nutrient stress induces ER exit site delivery into the lysosome • Uptake of ER exit sites into lysosomes occurs by lysosomal engulfment • ER exit site delivery into lysosomes is mediated by COPII, ALG2, and ESCRTs machinery ER exit sites are the earliest station for protein sorting and export from the ER into the secretory pathway. Liao et al. report that under nutrient stresses, ER exit sites are delivered into lysosomes using a mechanism involving COPII, ALG2, and ESCRTs. [ABSTRACT FROM AUTHOR]

[Display omitted] • Melanin granules in black hair samples were investigated by FIB-SEM. • The three-dimensional distributions of melanin granules in the hair were visualized. • The morphometric parameters of melanin granules were calculated. • The morphologies of melanin granules in Asian and Caucasian hair were compared. • The morphologies of melanin granules differed significantly between the two races. Melanin granules (melanosomes) in Asian and Caucasian black hairs were investigated by focused ion beam scanning electron microscopy (FIB-SEM). This technique facilitates a direct evaluation of the three-dimensional distribution and morphology of melanin granules without requiring their isolation from hair. Three-dimensional reconstructed images of melanin granule distribution in hair samples were obtained using serial SEM images observed by FIB-SEM. Melanin granules in black hair tended to be three-dimensionally dense in the outer periphery of the cortex. The morphometric parameters of melanin granules were calculated using the reconstructed three-dimensional images. The results confirmed that melanin granules in Caucasian black hair were much smaller those in Asian black hair. Moreover, it was indicated that the relative frequency distribution of the volume of melanin granules was significantly different between Asians and Caucasians. [ABSTRACT FROM AUTHOR]

Developing a fuel cell model with fundamental structural properties such as distribution of pore size, geometrical network of individual phase, and volume-specific interfacial area are critical in evaluating the accurate cell performance. Therefore, herein, by Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) tomography, three-dimensional (3-D) microstructure of CLs is reconstructed from two real-time samples: (i) High tortuosity humidifying catalyst layer (HTH CL) and (ii) standard catalyst layer. From the reconstructed microstructure, water imbibition behavior at different levels of capillary pressure is simulated and the effective transport properties such as gas permeability, gas diffusivity, surface area and water permeability are derived as well. By coupling the effective structural and transport properties, a 2D model is developed to predict the performances of the two CLs, at relative humidity (RH) levels of 20% and 100%. Since the effective transport properties are derived from real-time samples, this 2D model is expected to have a greater accuracy in predicting the fuel cell performance. Finally, the mechanism of self-humidifying MEA at lower and higher RH conditions (20% RH and 100% RH) is demonstrated as a function of liquid water saturation in the cathode CL and water dry-out in the anode CL. • Realistic model for prediction of water transport characteristics with high accuracy. • Accelerating back diffusion of water to prevent anode dehydration at lower humidity. • Lower water permeation behavior of HTH CL makes it ideal for dry-condition operation. [ABSTRACT FROM AUTHOR]

Investigating the pore structure of shale rocks plays an important role in understanding the mechanism of shale oil accumulation and migration; thus, it has a notable influence on the exploration and development of shale oil resources. In the present study, the microstructure of oil shale samples extracted from the Shahejie Formation in the Dongying Depression was investigated through the joint usage of focused ion beam (FIB) and scanning electron microscopy (SEM). Herein, we selected four representative samples. The SEM images of the sample surfaces milled by FIB indicated that pores were found in both the organic and inorganic matter. Organic pores were less developed in the samples because of the low thermal maturity of the organic matter. Continuous 3D digital rock images of shale samples were generated by FIB grinding and SEM in situ imaging. Based on multithreshold segmentation, the shale rocks were separated into different parts including kerogen, inorganic matter, and pores. The pore connectivity and pore size distributions were analyzed via Avizo software and in‐house developed MATLAB scripts. The pore size and corresponding volume distributions suggested that mesoscale pores (2‐50 nm) were abundant in the samples but they contributed limitedly to the total pore volume. Macropores (>50 nm) accounted for a small proportion but they had a greater contribution to the overall pore space volume. The coordination numbers of the subject four samples were mainly 0 and 1, which means that isolated pores accounted for the vast majority of the pores. These observations are critical to conduct reliable petrophysical simulations in the oil shales and understand their intrinsic properties accurately. [ABSTRACT FROM AUTHOR]

Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) tomography provides a stack of images that represent serial slices of the sample. These images are displaced relatively to each other, and an alignment procedure is required. Traditional methods for alignment of a 3D image are based on a comparison of two adjacent slices. However, such algorithms are easily confused by anisotropy in the sample structure or even experiment geometry in the case of porous media. This may lead to significant distortions in the pore space geometry, if there are no stable fiducial marks in the frame. In this paper, we propose a new method, which meaningfully extends existing alignment procedures. Our technique allows the correction of random misalignments between slices and, at the same time, preserves the overall geometrical structure of the specimen. We consider displacements produced by existing alignment algorithms as a signal and decompose it into low and high-frequency components. Final transformations exclude slow variations and contain only high frequency variations that represent random shifts that need to be corrected. The proposed algorithm can operate with not only translations but also with arbitrary affine transformations. We demonstrate the performance of our approach on a synthetic dataset and two real FIB-SEM images of natural rock [ABSTRACT FROM AUTHOR]

The paper deals with particle analysis and the evaluation of the shape and size distribution of micro- to nano-sized quartz particles as a key parameter for the evaluation of quality in many industrial fields, particularly for high-purity applications. Samples are analyzed by laser diffraction, the most commonly used method for determining the size of the particles; for precise measurement with the possibility of high-resolution imaging of particles, scanning electron microscopy was applied. For morphological and size analysis of electrically non-conductive micro- to nano-particles under environmentally compatible conditions of elevated gas pressure and humidity, environmental scanning electron microscopy was selected as the most suitable. Moreover, the high-end methods of focused ion beam scanning electron microscopy and serial block-face scanning electron microscopy enabled the analysis of particles in the epoxy resin volume including spatial imaging. The application of image post-processing as an important part of the electron microscopy workflow is also discussed. [Display omitted] • Shape analysis and particle size distribution of nano to micro quartz particles. • Comparison of laser diffraction and other advanced electron microscopic methods. • Image post-processing is an important part of analysis when using microscopic methods. • Environmental scanning electron microscopy used as a complex particle analysis method. [ABSTRACT FROM AUTHOR]

• High resolution 3D segmentation of human skeletal muscle mitochondria. • High-throughput, easy-to-use and versatile segmentation pipeline. • Orders of magnitude increase in acquisition and segmentation of large volumes. • Large scale statistical analysis of sub-cellular organelles. Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) is an imaging approach that enables analysis of the 3D architecture of cells and tissues at resolutions that are 1–2 orders of magnitude higher than that possible with light microscopy. The slow speeds of data collection and manual segmentation are two critical problems that limit the more extensive use of FIB-SEM technology. Here, we present an easily accessible robust method that enables rapid, large-scale acquisition of data from tissue specimens, combined with an approach for semi-automated data segmentation using the open-source machine learning Weka segmentation software, which dramatically increases the speed of image analysis. We demonstrate the feasibility of these methods through the 3D analysis of human muscle tissue by showing that our process results in an improvement in speed of up to three orders of magnitude as compared to manual approaches for data segmentation. All programs and scripts we use are open source and are immediately available for use by others. [ABSTRACT FROM AUTHOR]

A typical Longmaxi (LMX) shale sample from Sichuan basin, China, is investigated in three dimensional through multiscale tomography methods spanning pixel sizes from 3.25 μm to 10 nm, combining X-ray computed tomography and focused ion beam-scanning electron microscopy imaging techniques. Pores, organic matter (OM) and pyrite were segmented and reconstructed at multiple scales and the representative elementary volume (REV) of each phase at different scales based on the convergence of error bars in the volume fraction are determined. Results show that REVs at each scale varied between different phases. The determination of REV defined the applicable scale of different methods as well as the appropriate scales of specific structures. Porosity of LMX shale determined at different scales shows significant difference at two orders of magnitude. This difference likely was caused by structural heterogeneity together with the limitations of methods. This study confirms that REV is a feasible and powerful approach in modeling shale using multiscale 3D imaging. The application of multiscale 3D imaging techniques to worldwide shale gas reservoirs could allow for significant insights into the assessment of migration mechanisms within low-permeability systems. Image 1 • Longmaxi shale is visualized in 3-D across the mm to nm scale using complementary methods. • Integrated imaging illustrates microstructural complexities of increasing heterogeneity at higher resolutions. • REVs at multiscale are determined, which defines the applicable scale of different methods. • The porosity changes with the observing scale and is likely to peak at about 250 μm within the mm scale. [ABSTRACT FROM AUTHOR]

Circuit-level alternations in patients of autism spectrum disorder (ASD) is under active investigation and detailed characterization of synapse morphology in ASD model mice should be informative. We utilized focused ion beam milling and scanning electron microscopy (FIB-SEM) to obtain three-dimensional images of synapses in the layer 2/3 of the somatosensory cortex from a mouse model for ASD with human 15q11-13 chromosomal duplication (15q dup mice). We found a trend of higher spine density and a higher fraction of astrocytic contact with both spine and shaft synapses in 15q dup mice. Measurement of spine synapse structure indicated that the size of the post-synaptic density (PSD), spine head volume, spine head width and spine neck width were smaller in 15q dup mice. Categorization of spine synapses into five classes suggested a trend of less frequent mushroom spines in 15q dup mice. These results suggest relative increase in excitatory synapses with immature morphology but more astrocytic contacts in 15q dup mice, which may be linked to enhanced synapse turnover seen in ASD mouse models. [ABSTRACT FROM AUTHOR]

Coated paper is an example of a multi-layer porous medium, involving a coating layer along the two surfaces of the paper and a fibrous layer in the interior of the paper. The interface between these two media needs to be characterized in order to develop relevant modeling tools. After careful cutting of the paper, a cross section was imaged using focused ion beam scanning electron microscopy. The resulting image was analyzed to characterize the coating layer and its transition to the fibrous layer. Such image analysis showed that the coating layer thickness is highly variable, with a significant fraction of it being thinner than a minimum thickness required to keep ink from invading into the fibrous layer. The overall structure of the coating and fibrous layers observed in this analysis provide insights into how the system should be modeled, with the resulting conclusion pointing to a specific kind of multi-scale modeling approach. [ABSTRACT FROM AUTHOR]

[Display omitted] • The material of sturgeon scales comprises two main layers containing collagen and carbonated hydroxyapatite as well as lacunae and canaliculi, which are all characteristics of bone. • In both layers collagen fibril bundles orientated at various angles in a single plane is the predominant motif, which is reminiscent of dentin. Collagen fiber bundles also emanate from the plane. • The sturgeon scale may be an example of an intermediate mineralized material which is neither bone nor dentin but contains characteristics of both materials, reflecting the close association between the evolution of bone and dentin. Scales are structures composed of mineralized collagen fibrils embedded in the skin of fish. Here we investigate structures contributing to the bulk of the scale material of the sturgeon (Acipencer guldenstatii) at the millimeter, micrometer and nanometer length scales. Polished and fracture surfaces were prepared in each of the three anatomic planes for imaging with light and electron microscopy, as well as focused ion beam – scanning electron microscopy (FIB-SEM). The scale is composed of three layers, upper and lower layers forming the bulk of the scale, as well as a thin surface layer. FTIR shows that the scale is composed mainly of collagen and carbonated hydroxyapatite. Lacunae are present throughout the structure. Fracture surfaces of all three layers are characterized by large diameter collagen fibril bundles (CFBs) emanating from a plane comprising smaller diameter CFBs orientated in different directions. Fine lineations seen in polished surfaces of both major layers are used to define planes called here the striation planes. FIB-SEM image stacks of the upper and lower layers acquired in planes aligned with the striation planes, show that CFBs are oriented in various directions within the striation plane, with larger CFBs emanating from the striation plane. Fibril bundles oriented in different directions in the same plane is reminiscent of a similar organization in orthodentin. The large collagen fibril bundles emanating out of this plane are analogous to von Korff fibrils found in developing dentin with respect to size and orientation. Scales of the sturgeon are unusual in that their mineralized collagen fibril organization contains structural elements of both dentin and bone. The sturgeon scale may be an example of an early evolved mineralized material which is neither bone nor dentin but contains characteristics of both materials, however, the fossil data required to confirm this is missing. [ABSTRACT FROM AUTHOR]

Scanning electron microscopy has been used to observe and study parasitic protozoa for at least 40 years. However, field emission electron sources, as well as improvements in lenses and detectors, brought the resolution power of scanning electron microscopes (SEM) to a new level. Parallel to the refinement of instruments, protocols for preservation of the ultrastructure, immunolabeling, exposure of cytoskeleton and inner structures of parasites and host cells were developed. This review is focused on protozoan parasites of medical and veterinary relevance, e.g., Toxoplasma gondii , Tritrichomonas foetus , Giardia intestinalis , and Trypanosoma cruzi , compilating the main achievements in describing the fine ultrastructure of their surface, cytoskeleton and interaction with host cells. Two new resources, namely, Helium Ion Microscopy (HIM) and Slice and View, using either Focused Ion Beam (FIB) abrasion or Microtome Serial Sectioning (MSS) within the microscope chamber, combined to backscattered electron imaging of fixed (chemically or by quick freezing followed by freeze substitution and resin embedded samples is bringing an exponential amount of valuable information. In HIM there is no need of conductive coating and the depth of field is much higher than in any field emission SEM. As for FIB- and MSS-SEM, high resolution 3-D models of areas and volumes larger than any other technique allows can be obtained. The main results achieved with all these technological tools and some protocols for sample preparation are included in this review. In addition, we included some results obtained with environmental/low vacuum scanning microscopy and cryo-scanning electron microscopy, both promising, but not yet largely employed SEM modalities. [ABSTRACT FROM AUTHOR]

Summary: Since the recent boost in the usage of electron microscopy in life‐science research, there is a great need for new methods. Recently minimal resin embedding methods have been successfully introduced in the sample preparation for focused‐ion beam scanning electron microscopy (FIB‐SEM). In these methods several possibilities are given to remove as much resin as possible from the surface of cultured cells or multicellular organisms. Here we introduce an alternative way in the minimal resin embedding method to remove excess of resin from two widely different cell types by the use of Mascotte filter paper. Our goal in correlative light and electron microscopic studies of immunogold‐labelled breast cancer SKBR3 cells was to visualise gold‐labelled HER2 plasma membrane proteins as well as the intracellular structures of flat and round cells. We found a significant difference (p < 0.001) in the number of gold particles of selected cells per 0.6 μm2 cell surface: on average a flat cell contained 2.46 ± 1.98 gold particles, and a round cell 5.66 ± 2.92 gold particles. Moreover, there was a clear difference in the subcellular organisation of these two cells. The round SKBR3 cell contained many organelles, such as mitochondria, Golgi and endoplasmic reticulum, when compared with flat SKBR3 cells. Our next goal was to visualise crosswall associated organelles, septal pore caps, of Rhizoctonia solani fungal cells by the combined use of a heavy metal staining and our extremely thin layer plastification (ETLP) method. At low magnifications this resulted into easily finding septa which appeared as bright crosswalls in the back‐scattered electron mode in the scanning electron microscope. Then, a septum was selected for FIB‐SEM. Cross‐sectioned views clearly revealed the perforate septal pore cap of R. solani next to other structures, such as mitochondria, endoplasmic reticulum, lipid bodies, dolipore septum, and the pore channel. As the ETLP method was applied on two widely different cell types, the use of the ETLP method will be beneficial to correlative studies of other cell model systems and multicellular organisms. [ABSTRACT FROM AUTHOR]

The aim of this study is to demonstrate the application of focused ion beam-scanning electron microscopy, FIB-SEM for revealing the three-dimensional features of osteocytic cytoplasmic processes in metaphyseal (immature) and diaphyseal (mature) trabeculae. Tibiae of eight-week-old male mice were fixed with aldehyde solution, and treated with block staining prior to FIB-SEM observation. While two-dimensional backscattered SEM images showed osteocytes’ cytoplasmic processes in a fragmented fashion, three-dimensional reconstructions of FIB-SEM images demonstrated that osteocytes in primary metaphyseal trabeculae extended their cytoplasmic processes randomly, thus maintaining contact with neighboring osteocytes and osteoblasts. In contrast, diaphyseal osteocytes extended thin cytoplasmic processes from their cell bodies, which ran perpendicular to the bone surface. In addition, these osteocytes featured thick processes that branched into thinner, transverse cytoplasmic processes; at some point, however, these transverse processes bend at a right angle to run perpendicular to the bone surface. Osteoblasts also possessed thicker cytoplasmic processes that branched off as thinner processes, which then connected with cytoplasmic processes of neighboring osteocytes. Thus, FIB-SEM is a useful technology for visualizing the three-dimensional structures of osteocytes and their cytoplasmic processes. [ABSTRACT FROM AUTHOR]

Here we report on ultrastructural features of brain synapses in the fly Drosophila melanogaster and outline a perspective for the study of their functional significance. Images taken with the aid of focused ion beam-scanning electron microscopy (EM) at 20 nm intervals across olfactory glomerulus DA2 revealed that some synaptic boutons are penetrated by protrusions emanating from other neurons. Similar structures in the brain of mammals are known as synaptic spinules. A survey with transmission EM (TEM) disclosed that these structures are frequent throughout the antennal lobe. Detailed neuronal tracings revealed that spinules are formed by all three major types of neurons innervating glomerulus DA2 but the olfactory sensory neurons (OSNs) receive significantly more spinules than other olfactory neurons. Double-membrane vesicles (DMVs) that appear to represent material that has pinched-off from spinules are also most abundant in presynaptic boutons of OSNs. Inside the host neuron, a close association was observed between spinules, the endoplasmic reticulum (ER) and mitochondria. We propose that by releasing material into the host neuron, through a process triggered by synaptic activity and analogous to axonal pruning, synaptic spinules could function as a mechanism for synapse tagging, synaptic remodeling and neural plasticity. Future directions of experimental work to investigate this theory are proposed. [ABSTRACT FROM AUTHOR]

Semithin sections are commonly used to examine large areas of tissue with an optical microscope, in order to locate and trim the regions that will later be studied with the electron microscope. Ideally, the observation of semithin sections would be from mesoscopic to nanoscopic scales directly, instead of using light microscopy and then electron microscopy (EM). Here we propose a method that makes it possible to obtain high-resolution scanning EM images of large areas of the brain in the millimeter to nanometer range. Since our method is compatible with light microscopy, it is also feasible to generate hybrid light and electron microscopic maps. Additionally, the same tissue blocks that have been used to obtain semithin sections can later be used, if necessary, for transmission EM, or for focused ion beam milling and scanning electron microscopy (FIB-SEM). [ABSTRACT FROM AUTHOR]

[Display omitted] • FIB SEM of the hypermineralized petrous bone reveals two different 3D structures. • The inner pig petrous bone has a woven structure, whereas the outer bone has a lamellar structure. • Directionality evaluation of the structures of this hypermineralized bone was achieved using an anisotropy analysis. • The woven structure is consistent with the inner petrous bone being of fetal origin. • The near absence of nano-pores, may also account for better DNA preservation in fossils. We report on the 3D ultrastructure of the mineralized petrous bone of mature pig using focused ion beam – scanning electron microscopy (FIB-SEM). We divide the petrous bone into two zones based on the degree of mineralization; one zone close to the otic chamber has higher mineral density than the second zone further away from the otic chamber. The hypermineralization of the petrous bone results in the collagen D-banding being poorly revealed in the lower mineral density zone (LMD), and absent in the high mineral density zone (HMD). We therefore could not use D-banding to decipher the 3D structure of the collagen assembly. Instead we exploited the anisotropy option in the Dragonfly image processing software to visualize the less mineralized collagen fibrils and/or nanopores that surround the more mineralized zones known as tesselles. This approach therefore indirectly tracks the orientations of the collagen fibrils in the matrix itself. We show that the HMD bone has a structure similar to that of woven bone, and the LMD is composed of lamellar bone with a plywood-like structural motif. This agrees with the fact that the bone close to the otic chamber is fetal bone and is not remodeled. The lamellar structure of the bone further away from the otic chamber is consistent with modeling/remodeling. The absence of the less mineralized collagen fibrils and nanopores resulting from the confluence of the mineral tesselles may contribute to shielding DNA during diagenesis. We show that anisotropy evaluation of the less mineralized collagen fibrils could be a useful tool to analyze bone ultrastructures and in particular the directionality of collagen fibril bundles that make up the bone matrix. [ABSTRACT FROM AUTHOR]

China has the largest quantity of technically recoverable shale gas resources in the world. However, the complex, three-dimensional (3D), multi-scale structure of the shale reservoir makes it extremely challenging to fully understand the mechanisms that govern shale gas migration and recovery efficiency. In this study, focused ion beam-scanning electron microscopy (FIB-SEM) was applied to identify the nano- and micron-scale 3D structures, including inter-particle pores, organic matter pores, intra-particle pores and fractures, of shale sampled from the Longmaxi Formation, Sichuan Basin, China. The 3D structural and physical characteristics, such as morphology, porosity, connectivity, and permeability of multi-scale pores and fractures, were analysed using 3D reconstruction techniques and the lattice Boltzmann method (LBM). The results show that the majority of pores in the Longmaxi shale samples varied between 10 and 40 nm in size, although some significantly larger intra-particle pores were also detected. The micro-fractures exhibited higher permeability and gas migration capacity than adjacent nano-pores, and organic matter nano-pores displayed high porosity and good connectivity. This study provides a way to quantitatively characterise the multi-scale structure and its effect on shale gas migration and fracturing potential in the Longmaxi formation, as well as in similar shale reservoirs elsewhere around the world. [ABSTRACT FROM AUTHOR]

The size, shape and distribution of different phases in thermoplastic polyolefin (TPO) blends and composites are critical to the properties of the materials, but can be difficult to characterise. Here we report the combination of heavy metal staining and focused ion beam - scanning electron microscopy (FIB-SEM) to reveal the three-dimensional (3D) structure of an elastomer-modified poly(propylene) and a talc filled elastomer-modified poly(propylene). High-quality, high-resolution serial images were collected and the 3D structures were characterised quantitatively. [ABSTRACT FROM AUTHOR]

The development of focused ion beam-scanning electron microscopy (FIB-SEM) techniques has allowed high-resolution 3D imaging of nanometre-scale porous materials. These systems are of important interest to the oil and gas sector, as well as for the safe long-term storage of carbon and nuclear waste. This work focuses on validating the accurate representation of sample pore space in FIB-SEM-reconstructed volumes and the predicted permeability of these systems from subsequent single-phase flow simulations using a highly homogeneous nanometre-scale, mesoporous (2-50 nm) to macroporous (>50 nm), porous ceramic in initial developments for digital rock physics. The limited volume of investigation available from FIB-SEM has precluded direct quantitative validation of petrophysical parameters estimated from such studies on rock samples due to sample heterogeneity, large variations in recorded sample pore sizes and lack of pore connectivity. By using homogeneous synthetic ceramic samples we have shown that lattice-Boltzmann flow simulations using processed FIB-SEM images are capable of predicting the permeability of a homogeneous material dominated by 10-100 nanometre-scale pores (similar, albeit simpler, to those in natural samples) at the much larger scale where permeability measurements become practical. This result shows the LB flow simulations can be used with confidence in pores at this scale allowing future work to focus on sample preparation techniques for samples sensitive to drying and multiple FIB-SEM site selection for the population of larger-scale models for heterogeneous systems. [ABSTRACT FROM AUTHOR]

Background: Molecular and structural information on avian Trypanosoma spp. throughout Australia is limited despite their intrinsic value in understanding trypanosomatid evolution, diversity, and structural biology. In Western Australia tissue samples (n = 429) extracted from 93 birds in 25 bird species were screened using generic PCR primers to investigate the diversity of Trypanosoma spp. To investigate avian trypanosome structural biology the first 3-dimensional ultrastructural models of a Trypanosoma spp. (Trypanosoma sp. AAT) isolated from a bird (currawong, Strepera spp.) were generated using focussed ion beam milling combined with scanning electron microscopy (FIB-SEM). Results: Here, we confirm four intercontinental species of avian trypanosomes in native Australian birds, and identify a new avian Trypanosoma. Trypanosome infection was identified in 18 birds from 13 different bird species (19%). A single new genotype was isolated and found to be closely related to T. culicavium (Trypanosoma sp. CC2016 B002). Other Trypanosoma spp. identified include T. avium, T. culicavium, T. thomasbancrofti, Trypanosoma sp. TL.AQ.22, Trypanosoma sp. AAT, and an uncharacterised Trypanosoma sp. (group C-III sensu Zidková et al. (Infect Genet Evol 12:102-112, 2012)), all previously identified in Australia or other continents. Serially-sectioning Trypanosoma sp. AAT epimastigotes using FIB-SEM revealed the disc-shaped kinetoplast pocket attached perpendicular to the branching mitochondrion. Additionally, the universal minicircle sequence within the kinetoplast DNA and the associated binding protein were determined in Trypanosoma sp. AAT. Conclusions: These results indicate that bird trypanosomes are relatively conserved across continents, while being locally diverse, which supports the hypothesis that bird trypanosomes exist as fewer species than described in the literature. Evidence exists that avian Trypanosoma spp. are infecting mammals and could be transmitted by haemadipsid leeches. Trypanosoma sp. AAT is most likely a separate species currently found only in Australia and the first 3-dimentional ultrastructural analysis of an avian trypanosome provides interesting information on their morphology and organelle arrangement. [ABSTRACT FROM AUTHOR]

An accurate method for three-dimensional reconstruction of microstructures is developed to analyse mass transport phenomena in microporous layers (MPLs). The proposed method involves the use of focused ion beam–scanning electron microscope tomography. Numerical calculations of the structural and mass transport properties of MPLs are conducted using the reconstruction results and these results are found to be in good agreement with experimental results, demonstrating the accuracy of the new method. [ABSTRACT FROM AUTHOR]

To more finely describe the physical basis of coalbed methane (CBM) accumulation and flow mechanisms, two different rank coals ( R o,m of 0.4% and 0.85%) from the southern Junggar Basin of northwest China were systematically investigated to characterize pore-fracture spaces and their nanoscale connectivity in three dimensions (3D) using focused ion beam scanning electron microscopy (FIB-SEM) tomography. We reconstructed the 3D pore morphology of two different rank coals, quantify the pore size/volumes distribution and developed a novel pore network extraction (PNE) model. Samples of subbituminous coal (SC) and high-volatile bituminous coal (HBC) presented significant pore spaces for CBM adsorption, with total voxel numbers of 28,944 and 83,866, respectively; both samples predominantly consisted of closed-pore diameters of 10–50 nm. The numbers, volumes, and areas of pores (< 100 nm) were less in SC than in HBC, revealing that the SC adsorption capability was weaker than HBC. The porosities acquired by FIB-SEM were 26.06 and 42.85% for the samples SC and HBC, respectively, which could indicate the storage capability of the SC reservoir was lower than that of the HBC reservoir. The SC and HBC samples revealed a significant connectivity for CBM seepage, with a connected pores proportion of ~ 100% in pores > 300 nm. The SC sample allowed pore space connections throughout, whereas the HBC sample partially displayed pore space connections in a 3D pore network. It was found that a throat size of 400–500 nm could possibly dominate coal permeability in the SC sample; the numbers of throats (fractures) decreased with rising throat size. These results show that the gas accumulation capacity of the SC reservoir are lower than that of the HBC, whereas the fluid flow capability of the SC reservoir is higher than that of the HBC reservoir. These findings may be scientifically significant for lucubrating nanoscale pores to affect CBM storage and seepage. This paper comments on the reduction of the gas sorption capability and mass transport rate in CBM development, and we provide recommendations for relevant future work. [ABSTRACT FROM AUTHOR]

Tendons transmit muscle-generated force through an extracellular matrix of aligned collagen fibrils. The force applied by the muscle at one end of a microscopic fibril has to be transmitted through the macroscopic length of the tendon by mechanisms that are poorly understood. A key element in this structure-function relationship is the collagen fibril length. During embryogenesis short fibrils are produced but they grow rapidly with maturation. There is some controversy regarding fibril length in adult tendon, with mechanical data generally supporting discontinuity while structural investigations favor continuity. This study initially set out to trace the full length of individual fibrils in adult human tendons, using serial block face-scanning electron microscopy. But even with this advanced technique the required length could not be covered. Instead a statistical approach was used on a large volume of fibrils in shorter image stacks. Only a single end was observed after tracking 67.5 mm of combined fibril lengths, in support of fibril continuity. To shed more light on this observation, the full length of a short tendon (mouse stapedius, 125 μm) was investigated and continuity of individual fibrils was confirmed. In light of these results, possible mechanisms that could reconcile the opposing findings on fibril continuity are discussed. Statement of Significance Connective tissues hold all parts of the body together and are mostly constructed from thin threads of the protein collagen (called fibrils). Connective tissues provide mechanical strength and one of the most demanding tissues in this regard are tendons, which transmit the forces generated by muscles. The length of the collagen fibrils is essential to the mechanical strength and to the type of damage the tissue may experience (slippage of short fibrils or breakage of longer ones). This in turn is important for understanding the repair processes after such damage occurs. Currently the issue of fibril length is contentious, but this study provides evidence that the fibrils are extremely long and likely continuous. [ABSTRACT FROM AUTHOR]

Tomographic analysis using focused ion beam-scanning electron microscopy (FIB-SEM) provides three-dimensional information about solid materials with a resolution of a few nanometres and thus bridges the gap between X-ray and transmission electron microscopic tomography techniques. This contribution serves as an introduction and overview of FIB-SEM tomography applied to porous materials. Using two different porous Earth materials, a diatomite specimen, and an experimentally produced amorphous silica layer on olivine, we discuss the experimental setup of FIB-SEM tomography. We then focus on image processing procedures, including image alignment, correction, and segmentation to finally result in a three-dimensional, quantified pore network representation of the two example materials. To each image processing step we consider potential issues, such as imaging the back of pore walls, and the generation of image artefacts through the application of processing algorithms. We conclude that there is no single image processing recipe; processing steps need to be decided on a case-by-case study. [ABSTRACT FROM AUTHOR]

Self-made conventional nickel-yttria-stabilized zirconia composite anodes after reduction and 500 h operation were analyzed by three-dimensional microstructure reconstruction based on focused ion beam-scanning electron microscopy technique. Interfacial area, three-phases-boundary density and phase volume fraction were measured based on the three-dimensional microstructure reconstruction to quantitatively study the statistical characterization of solid oxide fuel cell nickel-yttria-stabilized-zirconia anode microstructure before and after operation. It is found that for anode operated with long time, it is necessary to increase the corresponding three-dimensional reconstruction size to suppress the influence of microstructure variation caused by Ni agglomeration in order to obtain more accurate microstructural quantification information. [ABSTRACT FROM AUTHOR]

The brain is a network of neurons, one that generates behaviour, and knowing the former is crucial to understanding the latter. Identifying the exact network of synaptic connections, or connectome, of the fly’s central nervous system is now a major objective inDrosophilaneurobiology, one that has been initiated in several laboratories, especially the Janelia Research Campus of the Howard Hughes Medical Institute. Progress is most advanced in the optic neuropiles of the visual system. The effort to derive a connectome from these and other neuropile regions is proceeding by various methods of electron microscopy, especially focused-ion beam milling scanning electron microscopy, and relies upon – but is to be carefully distinguished from – published light microscopic methods that reveal the projections of genetically labelled cell types. The latter reveal those neurons that come into close proximity and are therefore candidate synaptic partners. Synaptic partnerships are not in fact reliably revealed by such candidate pairs, anatomical connections often revealing unexpected pathways. Synaptic partnerships identified from ultrastructural features provide a strong heuristic basis to interpret not only functional interactions between identified neurons, but also a powerful means to predict such interactions, and suggest functional pathways not readily predicted from existing experimental evidence. The analysis of circuit function may proceed cell by cell, by examining the behavioural outcome of either interrupting or restoring function to any one element in an anatomically defined circuit, but can be foiled by degeneracy in pathway elements. Circuit information can also be used to identify and analyse circuit motifs, and their role in higher-order network properties. These attempts inDrosophilaanticipate parallel attempts in other systems, notably the inner plexiform layer of the vertebrate retina, and augment the one complete connectome already available to us, that available for 30 years in the nematodeCaenorhabditis elegans. [ABSTRACT FROM PUBLISHER]

In this paper, we propose an empirical formula for i 0,TPB , the exchange current density per unit triple-phase boundary (TPB) length, for porous lanthanum strontium manganite (LSM) cathodes of solid oxide fuel cells (SOFCs); the evaluation of i 0,TPB is of crucial importance in numerical simulations of electrodes based on reconstructed microstructures obtained by a dual beam focused ion beam scanning electron microscopy (FIB-SEM) and tomography techniques. To derive a widely applicable empirical formula for i 0,TPB , electrochemical measurements of porous LSM cathodes are conducted under various oxygen partial pressures (0.05–0.25 atm) and temperatures (800–950 °C). By comparing the derived formula with that derived from a thin and dense patterned LSM electrode used in previous studies, it is found that at an air temperature of 800 °C, i 0,TPB derived from a porous LSM cathode is approximately 40% smaller than that for the patterned electrode. This can be attributed to the fact that the electrochemical reaction in thin and dense electrodes can occur not only at the TPBs but also at the LSM surface owing to the non-negligible ionic conductivity of LSM. The derived formula is also applied to a three-dimensional numerical simulation to confirm its validity. [ABSTRACT FROM AUTHOR]

As part of development of a new assembly technology to achieve bonding for an innovative silicon carbide (SiC) power device used in harsh environments, the aim of this study is to compare two silver sintering profiles and then to define the best candidate for die attach material for this new component. To achieve this goal, the solder joints have been characterized in terms of porosity by determination of the morphological characteristics of the material heterogeneities and estimating their thermal and electrical transport properties. The three dimensional (3D) microstructure of sintered silver samples has been reconstructed using a focused ion beam scanning electron microscope (FIB-SEM) tomography technique. The sample preparation and the experimental milling and imaging parameters have been optimized in order to obtain a high quality of 3D reconstruction. Volume fractions and volumetric connectivity of the individual phases (silver and voids) have been determined. Effective thermal and electrical conductivities of the samples and the tortuosity of the silver phase have been also evaluated by solving the diffusive transport equation. [ABSTRACT FROM AUTHOR]

Focused ion beam-scanning electron microscopy (FIB-SEM) is a widely used and easily operational equipment for three-dimensional reconstruction with flexible analysis volume. It has been using successfully and increasingly in the field of solid oxide fuel cell. However, the phase contrast of the SEM images is indistinct in many cases, which will bring difficulties to the image processing. Herein, the phase contrast of a conventional Ni/yttria stabilized zirconia anode is tuned in an FIB-SEM with In-Lens secondary electron (SE) and backscattered electron detectors. Two accessories, tungsten probe and carbon nozzle, are inserted during the observation. The former has no influence on the contrast. When the carbon nozzle is inserted, best and distinct contrast can be obtained by In-Lens SE detector. This method is novel for contrast enhancement. Phase segmentation of the image can be automatically performed. The related mechanism for different images is discussed. [ABSTRACT FROM AUTHOR]

Improved knowledge of the topology of lamellar bodies is a prerequisite for a molecular-level understanding of skin barrier formation, which in turn may provide clues as to the underlying causes of barrier-deficient skin disease. The aim of this study was to examine the key question of continuity vs. discreteness of the lamellar body system using 3 highly specialized and complementary 3-dimensional (3D) electron microscopy methodologies; tomography of vitreous sections (TOVIS), freeze-substitution serial section electron tomography (FS-SET), and focused ion beam scanning electron microscopy (FIB-SEM) tomography. We present here direct evidence that lamellar bodies are not discrete vesicles, but are part of a tubuloreticular membrane network filling out the cytoplasm and being continuous with the plasma membrane of stratum granulosum cells. This implies that skin barrier formation could be regarded as a membrane folding/unfolding process, but not as a lamellar body fusion process. [ABSTRACT FROM AUTHOR]

[Display omitted] This study reports the characterisation of human dental enamel caries using synchrotron nanoscale correlative ptychography and spectroscopic mapping in combination with scanning electron microscopy. A lamella ̴2.4 µm thick was extracted from a carious enamel region of a tooth using focused ion beam-scanning electron microscopy and transferred to two synchrotron beamlines to perform hard X-ray nano-fluorescence spectroscopy simultaneously with differential phase contrast mapping at a beam size of 55 × 45 nm. Soft X-ray ptychography data was then reconstructed with a pixel size of 8 nm. The two dimensional variation in chemistry and structure of carious enamel was revealed at the nanoscale, namely, the organisation of hydroxyapatite nano-crystals within enamel rods was imaged together with the inter-rod region. Correlative use of electron and X-ray scanning microscopies for the same sample allowed visualisation of the connection between structure and composition as presented in a compound image where colour indicates the relative calcium concentration in the sample, as indicated by the calcium K α fluorescence intensity and grey scale shows the nanostructure. This highlights the importance of advanced correlative imaging to investigate the complex structure-composition relationships in nanomaterials of natural or artificial origin. [ABSTRACT FROM AUTHOR]

Effect of frozen non-equilibrium structure for anisotropically deformed natural rubber with the nanomatrix structure on mechanical properties was investigated with respect to morphology analysis through 3D observation. Natural rubber with the nanomatrix structure was prepared by graft copolymerization of styrene onto natural rubber particles in latex stage followed by coagulation of the resulting latex. The morphology was observed by electron tomography and focused ion beam-scanning electron microscopy (FIB-SEM). The nanomatrix structure in isotropic state was precisely analyzed by electron tomography and synchrotron scattering technique. The nanomatrix structure was found to consist of natural rubber particles and polystyrene nanoparticles. The frequency-independent loss tangent at plateau region was attributed to the discontinuous nanomatrix structure. After annealing at 403 K and 7 MPa, the nanomatrix became continuous, which resulted in the increases in stress at strain of one and storage modulus as well as increase in loss tangent. [ABSTRACT FROM AUTHOR]

Nanoindentation tests, spanning various length scales ranging from 200 nm to 5 μm deep, were performed on a sample of organic-rich Woodford shale in both the bedding plane normal and bedding plane parallel directions. Focused ion beam milling, scanning electron microscopy, and energy dispersive X-ray spectroscopy were utilized to characterize the shale at the scale of the nanoindentation testing as being comprised predominantly of clay and other silicate minerals suspended in a mixed organic/clay matrix. The nanoindentation tests reveal the mechanical properties of the relatively homogeneous constituent materials as well as those of the highly heterogeneous composite material. Loads on the order of a few millinewtons produced shallower indents and demonstrated the elastic-plastic deformation response of the constituent materials, whereas higher loads of as much as a few hundred millinewtons produced deeper indents revealing the response of the composite matrix. In both cases, significant creep was observed. We use nonlinear finite element modeling utilizing an isotropic critical state theory with creep to capture the indentation response by calibrating plastic material parameters to the laboratory measurements. The simulations provide a means of extracting plastic material parameters from the nanoindentation measurements and reveal the capabilities as well as limitations of an isotropic model in capturing the response of an inherently anisotropic material. [ABSTRACT FROM AUTHOR]

Trabecular bone is morphologically and functionally different from compact bone at the tissue level, but both are composed of lamellae at the micrometer-scale level. We present a three-dimensional study of the collagenous network of human trabecular lamellar bone from the proximal femur using the FIB-SEM serial surface view method. The results are compared to human compact lamellar bone of the femoral shaft, studied by the same method. Both demineralized trabecular and compact lamellar bone display the same overall structural organization, namely the presence of ordered and disordered materials and the confinement of the canalicular network to the disordered material. However, in trabecular bone lamellae a significant proportion of the ordered collagen fibril arrays is aligned with the long axis of the trabecula and, unlike in compact bone, is not related to the anatomical axis of the whole femur. The remaining ordered collagen fibrils are offset from the axis of a trabecula either by about 30° or 70°. Interestingly, at the tissue scale of millimeters, the most abundant angles between any two connected trabeculae — the inter-trabecular angles - center around 30° and 70°. This implies that within a framework of interconnected trabeculae the same lamellar structure will always have a significant component of the fibrils aligned with the long axes of connected trabeculae. This structural complementarity at different hierarchical levels presumably reflects an adaptation of trabecular bone to function. [ABSTRACT FROM AUTHOR]

Recent developments in electron microscopy largely automate the continuous acquisition of serial electron micrographs (EMGs), previously achieved by laborious manual serial ultrathin sectioning using an ultramicrotome and ultrastructural image capture process with transmission electron microscopy. The new systems cut thin sections and capture serial EMGs automatically, allowing for acquisition of large data sets in a reasonably short time. The new methods are focused ion beam/scanning electron microscopy, ultramicrotome/serial block-face scanning electron microscopy, automated tape-collection ultramicrotome/scanning electron microscopy and transmission electron microscope camera array. In this review, their positive and negative aspects are discussed. [ABSTRACT FROM PUBLISHER]

Naturally occurring nanomatrix structure formed in natural rubber was observed with a field emission scanning electron microscope equipped with a focused ion beam (FIB-SEM), and it was related to viscoelastic properties of the rubber. Film specimen used for the FIB-SEM observation was prepared from serum fraction of natural rubber latex, in which serum rubber contained about 15 w/ w% proteins. Morphology of the film specimen was observed by FIB-SEM, after pinning the nanomatrix structure with glutaraldehyde followed by staining the serum rubber with OsO. Three-dimensional image of the nanomatrix structure was successfully reconstructed in mesoscale without defects and voids, in which rubber particles of 200 nm in diameter were dispersed in the matrix of non-rubber components such as the proteins and phospholipids of several 10 nm in thickness. Storage modulus at plateau region of the serum rubber was about 500 times as high as that of deproteinized natural rubber, suggesting that the nanomatrix of the non-rubber components was completely continuous. [ABSTRACT FROM AUTHOR]

In the present study, correlation between solid oxide fuel cell anode microstructure and electrochemical performance during redox cycles was investigated. Electrolyte-support cell with nickel/yttria stabilized zirconia composite anode was prepared and tested under discharge process with redox cycles. Redox treatment was basically conducted every 20 h during discharge process. Polarization resistance decreased just after redox treatment and increased during discharge process. Enhancement of cell performance after every redox cycles and faster degradation in the following discharge process were observed. Polarization resistance gradually increased as redox cycles were repeated. Focused ion beam-scanning electron microscopy (FIB-SEM) observation was conducted for reconstructing the three dimensional microstructures of the tested samples. From the three dimensional microstructure reconstruction, it is found that the shape of nickel particle got thinner and complicated after redox cycles. Triple phase boundary (TPB) length increased after redox treatment and decreased after discharge process. This TPB change was highly associated with Ni connectivity and Ni specific surface area. These microstructure changes are consistent with the change of cell performance enhancement after redox treatment and degradation after discharge process. However, TPB length density kept on increasing as redox cycles are repeated, which is inconsistent with the gradual degradation of anode performance. [ABSTRACT FROM AUTHOR]

The present study describes the development of a theoretical model for estimating the thickness of homogeneous surface layers on spherical and irregular shaped powder particles using the XPS depth profiling. As opposed to flat surfaces, such an approach for substrates of specific geometry is not straightforward. One needs to consider those geometrical factors associated with the experimental setup and sample's roughness, which impose an angle dependence on the photoelectrons peak intensity, X-ray flux and ion etch rate over the surface in question in order to evaluate it. The novelty of the current model lies in the introduction of geometrical freedom in connection to the experimental arrangement, which can be tailored to match the needs of contemporary instruments. The model was evaluated experimentally by analyzing the surface oxide layers on metal powder grades of different morphology. Complementary analytical techniques such as high resolution (HR) SEM and focused ion beam (FIB) were used in combination to further characterize the surface layers prior to the XPS investigations. The results reveal that the estimation of the oxide/metal interface from the measured relative metal intensity signal as function of etch depth using the model is in extremely good agreement with the corresponding values from direct measurement of HR SEM on FIB cross-sectioned powder samples. The model deviates slightly only for the irregular shaped powder, which can be regarded as means for quantification of surface roughness of the material. The model is used as a basis for a computer software that estimates the thickness of surface layers for powdered materials. [ABSTRACT FROM AUTHOR]

Objectives Implant surface modifications are intended to enhance bone integration. The objective of this study was to assess the effect of different surface treatments on the resistance to hydrothermal degradation, hardness and elastic modulus of a 3Y-TZP ceramic used for dental implants. Methods Samples grouped according to their surface morphologies (AS, as-sintered; C, coated; P, dry-polished; R, roughened; PA, polished and annealed; RA, roughened and annealed) were subjected to accelerated hydrothermal degradation (LTD) by exposure to water steam (134 °C, 2 bars) for 100 h. The t – m phase transformation was quantified by grazing incidence X-ray diffraction (GIXDR) and by combined focused ion beam and scanning electron microscopy (FIB-SEM). Elastic modulus and hardness before- and after prolonged aging (100 h) were assessed by nanoindentation. Results AS and C specimens presented a better resistance to hydrothermal degradation than P and R samples. After prolonged aging, the depth of the monoclinic transformed layer ranged from 11 μm to 14 μm. Hydrothermal degradation led to a significant decrease of elastic modulus and hardness. Significance Surface treatments affected the resistance to hydrothermal degradation of the 3Y-TZP ceramic. Dry mechanical surface modifications should be avoided since a high t – m transformation rate associated to the initial monoclinic content was observed. Annealing was useful to reverse the initial t – m transformation, but did not improve the resistance to hydrothermal degradation. [ABSTRACT FROM AUTHOR]

Abstract: Efficient correlative imaging of small targets within large fields is a central problem in cell biology. Here, we demonstrate a series of technical advances in focused ion beam scanning electron microscopy (FIB–SEM) to address this issue. We report increases in the speed, robustness and automation of the process, and achieve consistent z slice thickness of ∼3nm. We introduce “keyframe imaging” as a new approach to simultaneously image large fields of view and obtain high-resolution 3D images of targeted sub-volumes. We demonstrate application of these advances to image post-fusion cytoplasmic intermediates of the HIV core. Using fluorescently labeled cell membranes, proteins and HIV cores, we first produce a “target map” of an HIV infected cell by fluorescence microscopy. We then generate a correlated 3D EM volume of the entire cell as well as high-resolution 3D images of individual HIV cores, achieving correlative imaging across a volume scale of 109 in a single automated experimental run. [Copyright &y& Elsevier]

Abstract: The vertical growth of C60 microtubes (C60MTs) on anodic aluminum oxide (AAO) membranes was investigated. The C60MT size dependence on isopropyl alcohol (IPA) injection rate, into C60-saturated toluene solutions through AAO membranes, was measured. A longitudinal section of the interface between a vertically grown C60MT (V-C60MT) and a membrane was prepared by focused ion beam processing, and observed with scanning electron microscopy. No cracking was observed along the interface, suggesting good bonding. V-C60MTs exhibited spiral growth. V-C60MT planar density, wall thickness and aspect ratio all decreased with increasing IPA injection rate. The relationships among length, inner and outer diameters of V-C60MTs were also investigated by varying IPA injection rate. [Copyright &y& Elsevier]

Abstract: The degradation of an ethylene–propylene-diene (EPDM) rubber seal used in a water supply system was investigated using focused ion beam-scanning electron microscopy (FIB-SEM). The EPDM rubber seal was used for 3 years within the temperature range 20–40 °C in a city water system. The accretions present on the surface of the EPDM seal after use were observed by SEM and were found to consist of iron and oxygen atoms based on energy dispersive X-ray spectroscopy (EDS) analysis. A cross-sectional depth image of the EPDM rubber was obtained by FIB-SEM, after slicing the EPDM rubber with a focused Gallium ion beam. Iron and oxygen atoms in the cross-section of the EPDM rubber were detected through EDS. The distribution of iron was comparable to that of oxygen derived from the carbonyl groups generated by the degradation of EPDM, suggesting that iron ions may promote the degradation of natural rubber through catalytic effects. [Copyright &y& Elsevier]