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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.

A number of methods for ultrastructural imaging by scanning electron microscopy (SEM) in three dimensions (3D) have been developed over the last decade. These techniques, which we collectively term 3D SEM, offer 2D imaging of quality approaching that attainable by traditional transmission electron microscopy, but with fine resolution in the third dimension as well. In this article, we cover a number of 3D SEM methodologies, including serial block face SEM (SBF-SEM) and focused ion beam SEM (FIB-SEM), as well as other complementary techniques. In addition, we describe the process from preparing a sample to generating a volume, using data from a murine duodenal epithelial cell junction to exemplify the cellular structural detail that may be studied with 3D SEM.

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.

International audience; We present a new concept of a combined scanning probe microscope (SPM)/ultramicrotome apparatus. It enables “slice-and-view” scanning probe nanotomography measurements and 3D reconstruction of the bulk sample nanostructure from series of SPM images after consecutive ultrathin sections. The sample is fixed on a flat XYZ scanning piezostage mounted on the ultramicrotome arm. The SPM measuring head with a cantilever tip and a laser-photodiode tip detection system approaches the sample for SPM measurements of the block-face surface immediately after the ultramicrotome sectioning is performed. The SPM head is moved along guides that are also fixed on the ultramicrotome arm. Thereby, relative dysfunctional displacements of the tip, the sample, and the ultramicrotome knife are minimized. The design of the SPM head enables open frontal optical access to the sample block-face adapted for high-resolution optical lenses for correlative SPM/optical microscopy applications. The new system can be used in a wide range of applications for the study of 3D nanostructures of biological objects, biomaterials, polymer nanocomposites, and nanohybrid materials in various SPM and optical microscopy measuring modes.

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.

A block face was generated by dividing a drawn face into a matrix of rectangular cells and then making the brightness of each cell equal to the average of the brightnesses within the facial area circumscribed by a cell. A wavelet analysis was performed on the numbers representing the brightnesses of the cells in each row of the matrix, and from each row, the wavelet was deleted which corresponded to the fundamental or the lowest frequency sine wave of a complex wave. The appearance of the block face was not substantially altered by the deletion of the wavelet corresponding to the fundamental.

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.

Recently, finite-element computer simulation of airflow in rat nasal airways has been used to predict local and global xenobiotic mass transport. These computer simulations have the capability to facilitate the extrapolation of experimental data from rats to humans for risk assessment. Implementation of similar models of other species requires accurate and detailed geometrical coordinates of airways. This article presents a sectioning method for the acquisition of airway coordinates and morphometric data from fixed tissues. Using this method, airway tissue specimens are embedded in a frozen, contrast-enhancing medium and serially sectioned. Video images are digitized from photographs of the block face, and image analysis is used to obtain geometrical coordinates of the nasal passage perimeters. Calibration images and embedded alignment marks are used to scale and align sequential sections. Data resulting from the application of this method to a rhesus monkey specimen are reported, including a thre...

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.

A methodology for registering histological slides and ex vivo MRI of prostate is proposed. After such registration is performed, spatial correspondence between histology slides and ex vivo MRI is established, thus histological truth can be propagated to the ex vivo MRI space. We employ the well established registration approach based on mutual information (MI) and thin-plate splines (TPS), which is automatic after user's initial placement of control points. Directly registering histology slides onto ex vivo MRI is challenging because 1) it is a difficult 2D (slice) to 3D (volume) registration problem and 2) there is a big difference in information content as histology slides are typically taken at much higher resolution than ex vivo MRI. Here we propose to overcome this challenge by breaking the difficult direct registration task into easier registration sub-tasks. For this purpose, we acquire digital photographs of prostate specimen as it is sectioned, which are referred to as block face photos. First, we register histology slides onto block face photos and then register block face photos onto ex vivo MRI. Results from two registrations tasks are combined to establish registration between histology slides and ex vivo MRI. Before the second registration task, we stack the block face photos into a volume so that registration onto the ex vivo MRI is a more stable 3D (volume) to 3D (volume) registration.

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

The first “cutting machine” for preparing sections of soft tissues was introduced by Cummings in 1770. The prototype, a metal cylinder that contained the specimen, usually a plant, was screwed by hand into the path of a knife to make a tissue slice. In 1835, Pritchard clamped a similar instrument to a table for greater stability. A section was then prepared with a two-handled knife by cutting across the specimen. Eventually, Chevalier introduced the name “microtome” for these devices in 1839. The development of a sliding mechanism for cutting machines began as early as 1798 as a method to section large tissue specimens. With hand-held knives barely adequate for cutting small specimens, rotary microtomes were introduced by scientific investigators in 1883 and 1886 for greater precision and uniform section thickness. In this case, the specimen passed through into a stable stationary knife. The Spencer Lens Company, between 1901 and 1910, manufactured the first clinical microtome and a larger, precision laboratory microtome.8 However, these early devices were inadequate for sectioning mineralized tissues unless the mineral could be removed by a decalcification process. At the time, mineralized bone had to be cut with a saw into relatively thick sections, and ground to a desired thickness.

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

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

Extended abstract of a paper presented at MC 2007, 33rd DGE Conference in Saarbrücken, Germany, September 2 – September 7, 2007

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2006

In immunohistochemistry, the availability of a steadily increasing number of antibodies allows for a more comprehensive characterization of tissue components. Often, very large numbers of slides are easily generated when panels of antibodies are applied to a series of cases, as in clinical research.

Two different kinds of cryo techniques for immunolabelling will be discussed in this chapter. The first, the Tokuyasu cryo-sectioning method, has now become a widely used method for immunolabelling at both the light- and electron microscopy level. The second category — freeze fracture and replica methods — deals with a group of techniques that are primarily used in a small number of laboratories that have specialized in these approaches.

A simple technique is described that allows thin sections to be taken from four different areas of a large block face. This is achieved simply by successive slight tilting of the block face around the horizontal and vertical axes. Thin sections can be obtained from four contiguous quadrants of the original large block face. The sections will differ only slightly from each other in orientation

The ability to view objects, including subcellular structures, in three-dimensions is crucial to understanding their form and function. For example, the three-dimensional (3-D) structure of the brain and its neurons has been the central focus of neuroanatomists for well over 100 years. Yet, what role a nerve cell’s three dimensional structure plays in controlling the flow of information within the nervous system remains a mystery. Part of the problem has been the difficulty in obtaining an accurate 3-D picture of a nerve cell’s shape as well as documenting its synaptic interconnections. Although many neurobiologists have turned to biochemical studies in recent years, it is becoming clear that precise structure and network diagrams will be necessary to relate the work of cell biologists with that of computational neuroscientists.

A number of techniques are presented which permit precise selection and efficient preparation of individual cultured cells for electron microscopy. Techniques described include marking of the living and embedded cells, drilling and mounting cores of embedded material, and improved technique for marking of the selected area on the block face before trimming.

SUMMARY The principle of using reflected light to align knife to specimen block proposed by Gorycki (1965) is applied to the LKB ultrotome. A small mirror mounted on the knife holder base at an angle of 5° reflects light from the fluorescent lamp on to the front of the knife from where it is reflected on to the block face. The width of this band of reflected light at the bottom edge of the block is reduced as the knife approaches the block. At the point at which it is no longer visible the block is 2–3 μm from the knife. When the knife is parallel with the block in the cutting plane the line of reflection is parallel with the knife edge. Using reflected light facilitates alignment between block and knife and makes closing the gap prior to the initial cut a simple and certain operation.

SUMMARY A simple procedure for estimating the depth to which a ribbon of ultra-thin sections cuts into a block is described. The average section increment is obtained by dividing the depth by the number of sections in the ribbon. Comparison with measurements of individual sections made using interference microscopy suggests: (a) that loss of resin brought about by the cutting process is insignificant, i.e. that the average section increment equals the average section thickness, and (b) that there can be considerable variation in section thickness along a ribbon. Provided that such variation is taken into account, the method based on measurements of the block face is simple, avoids time-consuming alternative procedures, and is appropriate when seeking to determine the depth dimension to be used in making three-dimensional reconstructions from long sequences of sections.

Light from the fluorescent lamp on an ultramicrotome can be reflected from a mirror located beneath the knife face so that the knife and edge can be imaged on the block face. It is well known that this image can be used to accurately align the block face to the knife edge and cutting direction. A method is described of pre-aligning the lamp, stereomicroscope, knife, and the mirror, which is fixed with respect to the knife face, so that a bright reflection of the knife face on the block face is obtained only when the block face is brought close to alignment. This initial alignment is an extremely rapid procedure, and is followed by slower, more accurate manipulation of the block and knife for precise alignment. The mirror, easily mounted to a Porter-Blum MT-2 ultramicrotome knife holder, is very simple in design and readily adaptable to any ultramicrotome. Methods to permit small movements of the block for the MT-1 and MT-2 ultramicrotomes are also described.

A combined section-mounting stage and miniature hot plate has been made to be used with the Huxley ultramicrotome. It facilitates precision in the handling of 0.2 to 2 µ sections during mounting and staining for light microscopy. Both processes can be carried out under continuous binocular magnification. The advance mechanism of the microtome has been fitted with a simple bell circuit to give an audible signal at the beginning and end of the full travel of the mechanical advance. This signal may be calibrated by readings on the vernier advance of the knife holder, for re-setting the microtome on a given block face. During prolonged bouts of sectioning the audible signal is a useful warning of full extension of the advance mechanism.

Detailed data on the geographic location of the school age population is of vital importance to school districts for school planning purposes such as: projecting enrollment, planning for the construction of new school buildings and the expansion and modernization of existing facilities, establishing school feeder area boundaries, planning bussing programs, determining teacher assignments. The order of geographical detail needed is the city block or block face. In the most recent application 97.4% of the addresses were recognized as to block face and coded to census tract and block with no editing of original address input.

The construction and operation of a simple, inexpensive illuminator that produces high quality illumination of the ultramicrotome knife edge and the edge to block face gap resembling dark field is described. Use of the illuminator greatly speeds knife adjustment and reduces the likelihood of specimen or knife edge damage. The illuminator uses a grain-of-wheat light bulb and an adjustable bulb holder fashioned from bent paper clips. The holder permits both lateral and axial adjustment of the bulb position, which is necessary to achieve satisfactory illumination with different specimens and knives. The illuminator, with slight modification, can be adapted for use on any ultramicrotome.

This method permits the preparation of Epon sections of rat incisors at 0.5 or 1 μm thickness and up to 1 cm in length. The upper and lower incisors were divided into three or four cross-sectional segments and these segments were split in half, the cut surface becoming the block face. The blocks were trimmed in a specific fashion and cut with routine glass knives on either LKB or Porter-Blum MT1 ultramicrotomes. The entire length of the incisor could then be studied by combining sections from consecutive segments.

The scanning electron microscope (SEM) allows observation of up to 2.5 cm of surface at a magnification that can vary from 20 × to 100,000 ×, unlike the transmission electron microscope (TEM), in which the block face is ordinarily about 1 mm and the actual surface area that can be examined is only 1/40μ. If it is assumed that events occurring at the luminal surface of endothelial cells may be of importance in cerebral blood flow and possibly in atherogenesis, then a model with reproducible surface lesions would be of value. Since the TEM allows high resolution of the interior of cells and intracellular relationships, there are obvious advantages to correlative investigations.

Details are given concerning the construction and use of a simple tool to help align a specimen block face in the vertical axis for subsequent ultrathin sectioning. Further instructions are given for the preparation of cell monolayers for ultrathin sectioning. The advantage is simple, repetitive and quick alignment of the specimen block face for ultrathin sectioning.