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Brain microinfarcts are prevalent in humans, however due to the inherent difficulty of identifying and localizing individual microinfarcts, brain-wide quantification is impractical. In mice, microinfarcts have been created by surgically introducing microemboli into the brain, but a major limitation of this model is the absence of automated methods to identify and localize individual occlusions. We present a novel and semi-automated workflow to identify the anatomical location of fluorescent emboli (microspheres) within the mouse brain through histological processing and atlas registration. By incorporating vibratome block-face imaging with the QuickNII brain registration tool, we show that the anatomical location of microspheres can be accurately registered to brain structures within the Allen mouse brain (AMB) atlas (e.g. somatomotor areas, hippocampal region, visual areas, etc.). Compared to registering images of slide mounted sections to the AMB atlas, microsphere location was more accurately determined when block-face images were utilized. As a proof of principle, using this workflow we compared the distribution of microspheres within the brains of mice that were either perfused or immersion fixed. No significant effect of perfusion on total microsphere number or location was detected. In general, microspheres were distributed brain-wide, with the largest density found in the thalamus. In sum, our block-face imaging workflow, enables efficient characterization of the widespread distribution of fluorescent microemboli, facilitating future investigation into the impact of microinfarct load and location on brain health. Significance Statement Cerebral microemboli are small materials, such as a blood clot or atherosclerotic plaque, that pass through the circulation until being lodged within downstream vasculature and occasionally resulting in localized cell death (microinfarction). The largest limitation of microemboli models of microinfarction in rodents is the lack of efficient methods for accurately quantifying the brain-wide distribution of occlusions in individual subjects. Compared to traditional histology, incorporating our block-face imaging workflow significantly reduces the time required to register all emboli from a single brain (from days to hours). Going forward, this workflow will allow researchers to more efficiently characterize the global distribution of microemboli in mice and facilitate the stratification of mice based on microsphere load or location.

Summary The advancement of serial cryoFIB/SEM offers an opportunity to study large volumes of near-native, fully hydrated frozen cells and tissues at voxel sizes of 10 nm and below. We explored this capability for pathologic characterization of vitrified human patient cells by developing and optimizing a serial cryoFIB/SEM volume imaging workflow. We demonstrate profound disruption of subcellular architecture in primary fibroblasts from a Leigh syndrome patient harboring a disease-causing mutation in USMG5 protein responsible for impaired mitochondrial energy production., Graphical Abstract, Highlights • Developed and optimized a serial cryoFIB/SEM volume imaging workflow • Visualized the 3D structure of an entire cell under native conditions • Revealed a disruption of cellular structures in primary LS patient fibroblasts • Demonstrated the potential for clinical phenotyping of pathogenic tissues, Serial cryoFIB/SEM offers new opportunities for structural analysis of cells and tissues under near-native conditions. Zhu et al. developed and optimized a serial cryoFIB/SEM volume imaging workflow for visualization of entire vitreous cells in 3D, and demonstrated its potential for clinical and pathological applications with primary Leigh syndrome patient cells.

Correlative microscopy and block-face imaging (CoMBI), a method that we previously developed, is characterized by the ability to correlate between serial block-face images as 3-dimensional (3D) datasets and sections as 2-dimensional (2D) microscopic images. CoMBI has been performed for the morphological analyses of various biological specimens, and its use is expanding. However, the conventional CoMBI system utilizes a cryostat, which limits its compatibility to only frozen blocks and the resolution of the block-face image. We developed a new CoMBI system that can be applied to not only frozen blocks but also paraffin blocks, and it has an improved magnification for block-face imaging. The new system, called CoMBI-S, comprises sliding-type sectioning devices and imaging devices, and it conducts block slicing and block-face imaging automatically. Sections can also be collected and processed for microscopy as required. We also developed sample preparation methods for improving the qualities of the block-face images and 3D rendered volumes. We successfully obtained correlative 3D datasets and 2D microscopic images of zebrafish, mice, and fruit flies, which were paraffin-embedded or frozen. In addition, the 3D datasets at the highest magnification could depict a single neuron and bile canaliculus.

The present protocol describes the process of cryosectioning OCT-embedded fresh frozen pig heart tissue, along with the acquisition of block face images, followed by Nissl staining of the tissue sections and microscopic imaging of the sections for localizing the fluorescently-labeled neurons in the tissue. This tissue section preparation workflow was optimized for use in laser capture microdissection of single neurons from the pig heart tissue, which are used in downstream transcriptomic analysis and mapping of the neuronal location and molecular data to a 3D reconstructed contour stack of block face images.

This study was carefully designed to determine the section compression of paraffin embedded sections. Two sections (one with thickness 5 µm and one 10 µm set by microtome) were cut from each of 2 sets of 12 testicular tissue (adult rats) blocks and stained with hematoxylin. Using scanned images, the area and the vertical (along the sectioning direction) and horizontal diameters of the block face were measured and compared with those of the unstained, stained or coverslipped section. Using the coverslipped section, the vertical and horizontal diameters of round spermatid nuclear profiles and the actual thickness of section were measured with light microscopy. Overall, the area of the coverslipped section was reduced by 5.5%-8.6% (on average) in comparison with that of the block face, with 69.5%-84.4% of the reduction being contributed by section compression in the process of section cutting, mounting and drying. The vertical (linear) compression of section, the primary cause of section area compression, was 5.9%-8.9%. The vertical compression of nuclear profiles was 1.5%-2.3% in 2 sets of sections and 5.2%-5.7% in other sections, indicating a non-uniform compression of structures within some sections depending on procedures of section drying. The measured mean thickness of sections decreased by 3.1%-5.0%.

The railway infrastructure is closely connected with economic development. The degree of its influence on a country?s economy is interrelated to its condition. The article analyses a situation in two post-Communist countries: Serbia and the Czech Republic in comparison with a number of selected European countries. The investments into the railway infrastructure are substantially subject to governmental activities as private investments are close to non-existent in the post-communist countries. The EU recognises the importance of the railway infrastructure and allocates subsidies by the way of structural funds into the construction of TEN-T network as well as into the reconstruction of the regional tracks. In the Balkan states, however, this role is adopted by the Russian Federation and newly by China; both the countries have realised the strategic position of the Balkan states in Europe while strengthening their role in this region. The countries of the former Eastern Block face a massive decline in the use of the railway, especially in passenger transportation. While the situation has been slowly improving in the Czech Republic, Serbia has been fighting a huge drop of interest in the railway since the beginning of the 1990s, which was mainly caused by its poor condition. The article points out the consequences that have arisen due to underestimated investments into the railway infrastructure in recent years deriving from the economic development of these economies. It highlights the necessity to consider the investment into the railway infrastructure from a long-term perspective and analyses the consequences of infrastructure destruction or sudden investment failures that have an immediate negative impact on the economy of the whole country.* This research was supported by Internal Grant Agency, FNH, VSE, IGA 506010.

Because of the significance of electron microscope tomography in the investigation of biological structure at nanometer scales, ongoing improvement efforts have been continuous over recent years. This is particularly true in the case of software developments. Nevertheless, verification of improvements delivered by new algorithms and software remains difficult. Current analysis tools do not provide adaptable and consistent methods for quality assessment. This is particularly true with images of biological samples, due to image complexity, variability, low contrast and noise. We report an electron tomography (ET) simulator with accurate ray optics modeling of image formation that includes curvilinear trajectories through the sample, warping of the sample and noise. As a demonstration of the utility of our approach, we have concentrated on providing verification of the class of reconstruction methods applicable to wide field images of stained plastic-embedded samples. Accordingly, we have also constructed digital phantoms derived from serial block face scanning electron microscope images. These phantoms are also easily modified to include alignment features to test alignment algorithms. The combination of more realistic phantoms with more faithful simulations facilitates objective comparison of acquisition parameters, alignment and reconstruction algorithms and their range of applicability. With proper phantoms, this approach can also be modified to include more complex optical models, including distance-dependent blurring and phase contrast functions, such as may occur in cryotomography.

Visualizing whole cells at many scales Cells need to compartmentalize thousands of distinct proteins, but the nanoscale spatial relationship of many proteins to overall intracellular ultrastructure remains poorly understood. Correlated light and electron microscopy approaches can help. Hoffman et al. combined cryogenic super-resolution fluorescence microscopy and focused ion beam–milling scanning electron microscopy to visualize protein-ultrastructure relationships in three dimensions across whole cells. The fusion of the two imaging modalities enabled identification and three-dimensional segmentation of morphologically complex structures within the crowded intracellular environment. The researchers observed unexpected relationships within a variety of cell types, including a web-like protein adhesion network between juxtaposed cerebellar granule neurons. Science , this issue p. eaaz5357

Kinship verification is a challenging problem that recently attracted much interest in computer vision, this system has a number of applications such as organizing large collections of images and recognizing resemblances among humans and search for lost people. In this work, we propose a new method based on different descriptors mixed such as (LBP, LPQ, BSIF), and the Multi-Block (MB) representation. and we investigate the effect of different features representation for kinship verification, Moreover, the use of TTest to reduce the number of features and the support vector machine (SVM) for the kinship classification. Our approach consists of five stages: (1) features extraction, (2) face representation (3) features representation, (4) features selection and (5) classification. Our approach is tested on five datasets (Cornell, UB Kin Face, Familly 101, KinFac W-I and W-II). Our results are good comparable with other approaches.

Hardened tricalcium silicate (C3S) pastes cured for different times from 12 h to 28 days were studied by serial block-face scanning electron microscopy (SBFSEM) with the region of interest (ROI) of (2.0 × 10 (Nicoleau and Nonat, 2016 [4])) μm3 and their three-dimensional (3D) spatial structures with a voxel size down to 16.6 nm × 16.6 nm × 20 nm were quantitatively analyzed. From these 3D images, the morphological characteristics of different components of the hardened C3S pastes in three-dimensions are observed directly, such as the connected pores and closed pores. Additionally, the degree of hydration and porosity of the measured samples are measured as well. Results show that the evolution of the degree of hydration reveals the high reactivity of C3S with water. The formation of the pore network is a complex process which includes not only the partition of the open pores but also the continued hydration of C3S in the closed pores, especially the newly formed closed pores during hydration. The quantitative analysis from SBFSEM measurements were compared with those from traditional TG-DSC and MIP, and it proves that SBFSEM has good applicability in the field of cement-based materials.

Serial block face imaging with the scanning electron microscope has been developed as an alternative to serial sectioning and transmission electron microscopy for the ultrastructural analysis of the three-dimensional organization of cells and tissues. An ultramicrotome within the microscope specimen chamber permits sectioning and imaging to a depth of many microns within resin-embedded specimens. The technology has only recently been adopted by plant microscopists and here we describe some specimen preparation procedures suitable for plant tissue, suggested microscope imaging parameters and discuss the software required for image reconstruction and analysis.

We have developed an imaging method designated as correlative light microscopy and block-face imaging (CoMBI), which contributes to improve the reliability of morphological analyses. This method can collect both the frozen sections and serial block-face images in a single specimen. The frozen section can be used for conventional light microscopic analysis to obtain 2-dimensional (2D) anatomical and molecular information, while serial block-face images can be used as 3-dimensional (3D) volume data for anatomical analysis. Thus, the sections maintain positional information in the specimen, and allows the correlation of 2D microscopic data and 3D volume data in a single specimen. The subjects can vary in size and type, and can cover most specimens encountered in biology. In addition, the required system for our method is characterized by cost-effectiveness. Here, we demonstrated the utility of CoMBI using specimens ranging in size from several millimeters to several centimeters, i.e., mouse embryos, human brainstem samples, and stag beetle larvae, and present successful correlation between the 2D light microscopic images and 3D volume data in a single specimen.

Serial block-face scanning electron microscopy (SBEM) is quickly becoming an important imaging tool to explore three-dimensional biological structure across spatial scales. At probe-beam-electron energies of 2.0 keV or lower, the axial resolution should improve, because there is less primary electron penetration into the block face. More specifically, at these lower energies, the interaction volume is much smaller, and therefore, surface detail is more highly resolved. However, the backscattered electron yield for metal contrast agents and the backscattered electron detector sensitivity are both sub-optimal at these lower energies, thus negating the gain in axial resolution. We found that the application of a negative voltage (reversal potential) applied to a modified SBEM stage creates a tunable electric field at the sample. This field can be used to decrease the probe-beam-landing energy and, at the same time, alter the trajectory of the signal to increase the signal collected by the detector. With decelerated low landing-energy electrons, we observed that the probe-beam-electron-penetration depth was reduced to less than 30 nm in epoxy-embedded biological specimens. Concurrently, a large increase in recorded signal occurred due to the re-acceleration of BSEs in the bias field towards the objective pole piece where the detector is located. By tuning the bias field, we were able to manipulate the trajectories of the primary and secondary electrons, enabling the spatial discrimination of these signals using an advanced ring-type BSE detector configuration or a standard monolithic BSE detector coupled with a blocking aperture.

Serial block face scanning electron microscopy (SBF-SEM) is a powerful method to analyze cells in 3D. Here, working at the resolution limit of the method, we describe a correlative light-SBF-SEM workflow to resolve microtubules of the mitotic spindle in human cells. We present three examples of uses for this workflow which are not practical by light microscopy and/or TEM. First, distinguishing closely associated microtubules within K-fibers; second, resolving bridging fibers in the mitotic spindle; third, visualizing membranes in mitotic cells, relative to the spindle apparatus. Our workflow also includes new computational tools for exploring the spatial arrangement of MTs within the mitotic spindle. We use these tools to show that microtubule order in mitotic spindles is sensitive to the level of TACC3 on the spindle.

Podocytes present a unique 3D architecture specialized for glomerular filtration. However, several 3D morphological aspects on podocyte development remain partially understood because they are difficult to reveal using conventional scanning electron microscopy (SEM). Here, we adopted serial block-face SEM imaging, a powerful tool for analyzing the 3D cellular ultrastructure, to precisely reveal the morphological process of podocyte development, such as the formation of foot processes. Development of foot processes gives rise to three morphological states: the primitive, immature and mature foot processes. Immature podocytes were columnar in shape and connected to each other by the junctional complex, which migrated toward the basal side of the cell. When the junctional complex was close to the basement membrane, immature podocytes started to interdigitate with primitive foot processes under the level of junctional complex. As primitive foot processes lengthened, the junctional complex moved between primitive foot processes to form immature foot processes. Finally, the junctional complex was gradually replaced by the slit diaphragm, resulting in the maturation of immature foot processes into mature foot processes. In conclusion, the developmental process of podocytes is now clearly visualized by block-face SEM imaging.

Block-face imaging is a scanning electron microscopic technique which enables easier acquisition of serial ultrastructural images directly from the surface of resin-embedded biological samples with a similar quality to transmission electron micrographs. In the present study, we analyzed the three-dimensional architecture of podocytes using serial block-face imaging. It was previously believed that podocytes are divided into three kinds of subcellular compartment: cell body, primary process and foot process, which are simply aligned in this order. When the reconstructed podocytes were viewed from their basal side, the foot processes were branched from a ridge-like prominence, which was formed on the basal surface of the primary process and was similar to the usual foot processes in structure. Moreover, from the cell body, the foot processes were also emerged via the ridge-like prominence, as found in the primary process. The ridge-like prominence anchored the cell body and primary process to the glomerular basement membrane and connected the foot processes to the cell body and primary process. In conclusion, serial block-face imaging is a powerful tool for clear understanding the three-dimensional architecture of podocytes through its ability to reveal novel structures which were difficult to determine by conventional transmission and scanning electron microscopes alone.

Neighborhood physical disorder, or the deterioration of urban environments, is associated with negative mental and physical health outcomes. Eleven trained raters used CANVAS, a web-based system for conducting reliable virtual street audits, to collect data on nine indicators of physical disorder using Google Street View imagery of 532 block faces in New York City, New York, USA. We combined the block face indicator data into a disorder scale using item response theory; indicators ranged in severity from presence of litter, a weak indicator of disorder, to abandoned cars, a strong indicator. Using this scale, we estimated disorder at the center point of each sampled block. We then used ordinary kriging to interpolate estimates of disorder levels throughout the city. The resulting map condenses a complex estimation process into an interpretable visualization of the spatial distribution of physical disorder in New York City.

Until recently, three-dimensional reconstruction on an ultrastructural level was only possible using serial section transmission electron microscopy (ssTEM). However, ssTEM is highly challenging and prone to artifacts as, e.g., section loss and image distortions. New methods, such as serial block-face scanning electron microscopy (SBFSEM) overcome these limitations and promise a high lateral resolution. However, little is known about the usability of SBFSEM in diminutive, but highly complex cellular systems. We used spider sperm (~3 µm in diameter), which fulfills these conditions, to analyze the potential of SBFSEM compared with ssTEM. Our data suggest that the resolution obtained by SBFSEM allows depicting structures on a cellular level and is sufficient to discriminate subcellular components, but is highly dependent on previous staining procedures and electron density of the target structures.

Summary Over 75,000 permeability measurements were collected from a meter-scale block of Massillon sandstone, characterized by conspicuous crossbedding that forms two distinct nested scales of heterogeneity. With the aid of a gas minipermeameter, spatially exhaustive fields of permeability data were acquired at each of five different sample supports (i.e., sample volumes) from each block face. These data provide a unique opportunity to physically investigate the relationship between the multiscale cross-stratified attributes of the sandstone and the corresponding statistical characteristics of the permeability. These data also provide quantitative physical information concerning the permeability upscaling of a complex heterogeneous medium. Here, a portion of the data taken from a single block face cut normal to stratification is analyzed. The results indicate a strong relationship between the calculated summary statistics and the cross-stratified structural features visibly evident in the sandstone sample. Specifically, the permeability fields and semivariograms are characterized by two nested scales of heterogeneity, including a large-scale structure defined by the cross-stratified sets (delineated by distinct bounding surfaces) and a small-scale structure defined by the low-angle cross-stratification within each set. The permeability data also provide clear evidence of upscaling. That is, each calculated summary statistic exhibits distinct and consistent trends with increasing sample support. Among these trends are an increasing mean, decreasing variance, and an increasing semivariogram range. The results also clearly indicate that the different scales of heterogeneity upscale differently, with the small-scale structure being preferentially filtered from the data while the large-scale structure is preserved. Finally, the statistical and upscaling characteristics of individual cross-stratified sets were found to be very similar because of their shared depositional environment; however, some differences were noted that are likely the result of minor variations in the sediment load and/or flow conditions between depositional events.

Rationale: The organization of the transverse-tubular (t-t) system and relationship to the sarcoplasmic reticulum (SR) underpins cardiac excitation–contraction coupling. The architecture of the SR, and relationship with the t-ts, is not well characterized at the whole-cell level. Furthermore, little is known regarding changes to SR ultrastructure in heart failure. Objective: The aim of this study was to unravel interspecies differences and commonalities between the relationship of SR and t-t networks within cardiac myocytes, as well as the modifications that occur in heart failure, using a novel high-resolution 3-dimensional (3D) imaging technique. Methods and Results: Using serial block face imaging coupled with scanning electron microscopy and image analysis, we have generated 3D reconstructions of whole cardiomyocytes from sheep and rat left ventricle, revealing that the SR forms a continuous network linking t-ts throughout the cell in both species. In sheep, but not rat, the SR has an intimate relationship with the sarcolemma forming junctional domains. 3D reconstructions also reveal details of the sheep t-t system. Using a model of tachypacing-induced heart failure, we show that there are populations of swollen and collapsed t-ts, patches of SR tangling, and disorder with rearrangement of the mitochondria. Conclusions: We provide the first high-resolution 3D structure of the SR network showing that it forms a cell-wide communication pipeline facilitating Ca 2+ diffusion, buffering, and synchronicity. The distribution of the SR within the cell is related to interspecies differences in excitation–contraction coupling, and we report the first detailed analysis of SR remodeling as a result of heart failure.

The hippocampal mossy fiber (MF) terminal is among the largest and most complex synaptic structures in the brain. Our understanding of the development of this morphologically elaborate structure has been limited because of the inability of standard electron microscopy techniques to quickly and accurately reconstruct large volumes of neuropil. Here we use serial block-face electron microscopy (SBEM) to surmount these limitations and investigate the establishment of MF connectivity during mouse postnatal development. Based on volume reconstructions, we find that MF axons initially form bouton-like specializations directly onto dendritic shafts, that dendritic protrusions primarily arise independently of bouton contact sites, and that a dramatic increase in presynaptic and postsynaptic complexity follows the association of MF boutons with CA3 dendritic protrusions. We also identify a transient period of MF bouton filopodial exploration, followed by refinement of sites of synaptic connectivity. These observations enhance our understanding of the development of this highly specialized synapse and illustrate the power of SBEM to resolve details of developing microcircuits at a level not easily attainable with conventional approaches.

It has already been shown that the stability of the geotextile reiforced earth walls was improved with the use of rigid facings that brought about the reduction of overall deformation and the amount of tensile force generated in geotextile. To evaluate the effects of facing rigidity, stress-deformation behavior must be analyzed based on the interaction of facings, geotextiles, and soils.A numerical analysis using a two-dimensional elasto-plastic FEM taken account of the construction process was conducted to clarify various aspects of the behavior of a reinforced earth walls measuring 10.0m high. Three types of facings were analyzed: the wrapped-around wall face method, the concrete panel face method, the concrete block face method. In addition, the slope of wall was varied from 1:0.0 to 1:0.8 (pitch of 0.2). The results showed that facings performed using the block and panel face method, which provided higher rigidity than the wrapped-around wall face method, could be increased the stability of the reinforced earth walls under the characteristic bound coditions be satisfied

SUMMARY Electron microscopy of vitrified ultrathin sections allows cell ultrastructure to be studied in the hydrated state. Sectioning of the frozen material is, however, a limiting step, since the cutting forces cause severe mechanical deformation. In order to address this problem, we have investigated the surface of cryosections. It is shown that cryosections have two fundamentally different surfaces. One surface is rough, deformed by cutting-induced deformation lines which are orientated perpendicular to the cutting direction. The other surface, in comparison, is not affected by those deformation lines. Except for knife marks it is smooth. In order to explain the observations, the following model is proposed. The rough relief corresponds to the former block face. Its roughness originates from material that is squeezed out of the section plane when the section is compressed in the cutting direction and bent away from the specimen block. The smooth section surface is the surface in contact with the knife during the sectioning. This contact keeps the surface smooth while imprinting the knife marks.

The mammalian brain comprises interconnected neurons that communicate via synapses to form a bewilderingly complex network. In every microliter of cortical grey matter there is approximately one billion synapses arranged along several kilometers of axons. Visualizing these circuits and all their connections is paramount to being able to understand how the brain works. Synapses are specialized junctions whose sizes range from over a micron in diameter to less than 100nm. Their size means that they can only be identified using the electron microscope, which also allows them to be classified according to their function, ie. inhibitory or excitatory. However, to understand the connectivity between neurons in the cortex, large volumes of neural tissue needs to be imaged at ‘synaptic resolution’ and in three dimensions (3d). Traditionally serial section transmission electron microscopy has been the only method available in which images are acquired from sections that are cut and mounted on grids. These image representing a 3d volume can be used to reconstruct and analyze the structure of the elements within. This method is very labor intensive, requiring a greater deal of manual dexterity, and when small mistakes occur, a continuous dataset is lost. Recent studies have explored an alternative method for serial image acquisition in which the block face of resin-embedded neural tissue was imaged within a FEG SEM [1]. Sections were removed from the imaged face in the microscope using an ultra microtome and then immediately imaged by the SEM. This provides a series of aligned images of the tissue in the block face and has the clear advantage that image acquisition can be fully automated. However the quality of the sectioning is critically dependent on homogenous resin hardness which is difficult to maintain when the electron beam is being used at higher resolution and part of the block suffers from the differential heating effects.

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

Extended abstract of a paper presented at Microscopy and Microanalysis 2009 in Richmond, Virginia, USA, July 26 – July 30, 2009