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Summary A conventional stereo light microscope was used to image polished wood surfaces at cellular resolution over size scales of the growth ring or larger. Bandpass filtering and local area contrast enhancement were used to aid automatic image thresholding and binarisation. An estimate for the location and proportion of cell collapse was introduced based on the distance between uncollapsed cell lumens. Additionally, spatial associations between vessels were determined using a Euclidean distance transform. The analysis of pith to bark cores provided sufficient detail to show significant intra and inter-annual trends in Pinus radiata tracheid dimensions (wall thickness, wall area, and radial widths). These trends were consistent with expectations and in agreement with the literature. Measured cell dimensions may be influenced by cell collapse and deformation as a result of drying. The analysis of air, kiln and oven-dried Eucalyptus nitens showed that cell collapse was highly variable but generally more prominent in the outer third of growth rings. There were significant changes in vessel shape across the growth rings and vessel area was significantly reduced by drying. The technique provides an intermediate step between detailed microscopy and macroscopic imaging that allows spatial analysis at the wood cell level.

We present a deep ultraviolet (DUV) light microscope capable of serial block-face imaging of the whole rodent brain. The capability of the 3D-DUV microscope for quantitative volumetric analysis of the small substructures of habenula is shown. It is expected that large scale wide-field high resolution analysis of the smaller regions would allow for elucidating the functional changes linked with the morphological changes in brain under pathological conditions.

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%.

We describe 3-dimensional histology of fresh or fixed unimbedded tissue using a vibrating microtome with MUSE (microscopy with UV surface excitation) to acquire block-face images that can be reconstructed into 3-dimensional data sets.

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.

Serial-sectioning block-facing (SSBF) imaging is an attractive method to overcome the depth limitations of optical imaging and slice alignment challenges of traditional serial sectioning histology. Despite these advantages, SSBF modalities suffer from reduced axial resolution caused by out of focus sub-surface signals at the block face. In order to restore axial resolution, the sub-surface signal must be removed. In this work, we describe a methodology for restoring the axial resolution through a combination of sample preparation and deconvolution in post-processing. An opacifying agent used during sample preparation, decreases the subsurface signal by absorbing excitation light. From these image stacks we estimate parameters to generate a highly asymmetric point-spread function (PSF), which is then used in a Richardson-Lucy deconvolution algorithm. Whilst our methodology can be widely applied to any SSBF technique, we show its application in multi-fluorescent high resolution episcopic microscopy (MF-HREM), which is a simple and cost-effective alternative to optical sectioning techniques such as two-photon microscopy.

When on-street parking is scarce, the cost of parking includes the extra time and fuel spent searching for a parking space (or cruising). Cruising also unnecessarily contributes to local congestion, vehicle emissions, air pollution, and climate change. The theoretical literature shows that these social costs can be reduced, or even eliminated, if high-quality information on the demand for and supply of parking is used to set parking prices at optimal levels. Not surprisingly, cities plagued by parking shortages, congested streets, and limited financial resources are interested in parking policies that reduce cruising and improve the efficient use of their existing parking and roadway infrastructure. The current study sheds light on the effect of the San Francisco parking pricing program (known as SFpark) on curbside parking search time and distance in urban neighborhoods on non-commuter parking. The study differs from previous empirical evaluations of similar parking pricing programs in its use of direct field measurements of parking search time and distance, rather than simulated data or proxy variables, such as parking availability. We use generalized mixed effect difference-in-difference models with data collected before and after the implementation of SFpark in both treatment and control areas to estimates effects of the San Francisco smart parking project, most importantly the demand responsive parking pricing scheme. The models control for time effects by using data from a separate control area, as opposed to using variables such as block face parking price and employment. The results suggest a significant reduction in average parking search time and distance due to SFpark. Average parking search time and distance declines by approximately 15% and 12%, respectively, from the control to the treatment areas.

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.

Volume electron microscopy allows for the automated acquisition of serial-section imaging data that can be reconstructed in three-dimensions (3D) to provide a detailed, geometrically accurate view of cellular ultrastructure. Two, volume electron microscopy (EM) techniques, serial block face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM), use a similar slice-and-view approach but differ in their fields of view and 3D resolution. This chapter highlights a workflow where the ability of SBF-SEM to image a large field of view is combined with the precise sectioning capability of FIB-SEM to first locate a rare cellular event in a large tissue volume and then inspect the event with higher resolution. Using these two EM platforms in synergy is a powerful technique and can be useful for both simple structural studies as well as correlative studies using both light and electron microscopy.

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.