The three-dimensional conformation and activity of mitochondria in syncytial male germ line-cysts of medicinal leeches.

We studied the spatial conformation and activity of mitochondria in the developing syncytial male germline cysts during spermatogenesis of the medicinal leeches using light, fluorescent, transmission electron microscopy, and serial block-face scanning electron microscopy. In cysts with spermatogonia and spermatocytes, mitochondria form networks and are in a dynamic hyperfusion state, while in cysts with spermatids, a single huge mitochondrion is observed. As spermiogenesis progresses, this huge mitochondrion is finally located in the future midpiece. The highest activity, in terms of membrane potential, of the mitochondria in H. medicinalis germline cysts was observed in cysts with spermatocytes; the lowest was in cysts with late elongated spermatids.

Minimal resin embedding of SBF-SEM samples reduces charging and facilitates finding a surface-linked region of interest.

BACKGROUND: For decoding the mechanism of how cells and organs function information on their ultrastructure is essential. High-resolution 3D imaging has revolutionized morphology. Serial block face scanning electron microscopy (SBF-SEM) offers non-laborious, automated imaging in 3D of up to ~ 1 mm3 large biological objects at nanometer-scale resolution. For many samples there are obstacles. Quality imaging is often hampered by charging effects, which originate in the nonconductive resin used for embedding. Especially, if the imaged region of interest (ROI) includes the surface of the sample and neighbours the empty resin, which insulates the object. This extra resin also obscures the sample's morphology, thus making navigation to the ROI difficult. RESULTS: Using the example of small arthropods and a fish roe we describe a workflow to prepare samples for SBF-SEM using the minimal resin (MR) embedding method. We show that for imaging of surface structures this simple approach conveniently tackles and solves both of the two major problems-charging and ROI localization-that complicate imaging of SBF-SEM samples embedded in an excess of overlying resin. As the surface ROI is not masked by the resin, samples can be precisely trimmed before they are placed into the imaging chamber. The initial approaching step is fast and easy. No extra trimming inside the microscope is necessary. Importantly, charging is absent or greatly reduced meaning that imaging can be accomplished under good vacuum conditions, typically at the optimal high vacuum. This leads to better resolution, better signal to noise ratio, and faster image acquisition. CONCLUSIONS: In MR embedded samples charging is minimized and ROI easily targeted. MR embedding does not require any special equipment or skills. It saves effort, microscope time and eventually leads to high quality data. Studies on surface-linked ROIs, or any samples normally surrounded by the excess of resin, would benefit from adopting the technique.

Therapy-Induced Senescent/Polyploid Cancer Cells Undergo Atypical Divisions Associated with Altered Expression of Meiosis, Spermatogenesis and EMT Genes.

Upon anticancer treatment, cancer cells can undergo cellular senescence, i.e., the temporal arrest of cell division, accompanied by polyploidization and subsequent amitotic divisions, giving rise to mitotically dividing progeny. In this study, we sought to further characterize the cells undergoing senescence/polyploidization and their propensity for atypical divisions. We used p53-wild type MCF-7 cells treated with irinotecan (IRI), which we have previously shown undergo senescence/polyploidization. The propensity of cells to divide was measured by a BrdU incorporation assay, Ki67 protein level (cell cycle marker) and a time-lapse technique. Advanced electron microscopy-based cell visualization and bioinformatics for gene transcription analysis were also used. We found that after IRI-treatment of MCF-7 cells, the DNA replication and Ki67 level decreased temporally. Eventually, polyploid cells divided by budding. With the use of transmission electron microscopy, we showed the presence of mononuclear small cells inside senescent/polyploid ones. A comparison of the transcriptome of senescent cells at day three with day eight (when cells just start to escape senescence) revealed an altered expression of gene sets related to meiotic cell cycles, spermatogenesis and epithelial-mesenchymal transition. Although chemotherapy (DNA damage)-induced senescence is indispensable for temporary proliferation arrest of cancer cells, this response can be followed by their polyploidization and reprogramming, leading to more fit offspring.

Serial block face SEM visualization of unusual plant nuclear tubular extensions in a carnivorous plant (Utricularia, Lentibulariaceae).

Background and Aims: In Utricularia nelumbifolia , the nuclei of placental nutritive tissue possess unusually shaped projections not known to occur in any other flowering plant. The main aim of the study was to document the morphology and ultrastructure of these unusual nuclei. In addition, the literature was searched to find examples of nuclear tubular projections in other plant groups, and the nuclei of closely related species of Utricularia (i.e. sects Iperua , Orchidioides , Foliosa and Utricularia ) were examined. Methods: To visualize the complexity of the nuclear structures, transmission electron microscopy (TEM) was used, and 3-D ultrastructural reconstructions were made using the serial block face scanning electron microscopy (SBEM) technique. The nuclei of 11 Utricularia species, i.e. U. nelumbifolia , U. reniformis , U. cornigera , U. nephrophylla (sect. Iperua ), U. asplundii , U. alpina , U. quelchii (sect. Orchidioides ), U. longifolia (sect. Foliosa ), U. intermedia , U. minor and U. gibba (sect. Utricularia ) were examined. Key Results: Of the 11 Utricularia species examined, the spindle-like tubular projections (approx. 5 µm long) emanating from resident nuclei located in placental nutritive tissues were observed only in U. nelumbifolia . These tubular nuclear extensions contained chromatin distributed along hexagonally shaped tubules. The apices of the projections extended into the cell plasma membrane, and in many cases also made contact at the two opposing cellular poles, and with plasmodesmata via a short cisterna of the cortical endoplasmic reticulum. Images from the SBEM provide some evidence that the nuclear projections are making contact with those of neighbouring cells. Conclusions: The term chromatubules (chromatin-filled tubules) for the nuclear projections of U. nelumbifolia placental tissue was proposed here. Due to the apparent association with the plasma membrane and plasmodesmata, it was also speculated that chromatubules are involved in nucleus-cell-cell communication. However, further experimental evidence is required before any functional hypothesis can be entertained.

Challenges of microtome-based serial block-face scanning electron microscopy in neuroscience.

Serial block-face scanning electron microscopy (SBEM) is becoming increasingly popular for a wide range of applications in many disciplines from biology to material sciences. This review focuses on applications for circuit reconstruction in neuroscience, which is one of the major driving forces advancing SBEM. Neuronal circuit reconstruction poses exceptional challenges to volume EM in terms of resolution, field of view, acquisition time and sample preparation. Mapping the connections between neurons in the brain is crucial for understanding information flow and information processing in the brain. However, information on the connectivity between hundreds or even thousands of neurons densely packed in neuronal microcircuits is still largely missing. Volume EM techniques such as serial section TEM, automated tape-collecting ultramicrotome, focused ion-beam scanning electron microscopy and SBEM (microtome serial block-face scanning electron microscopy) are the techniques that provide sufficient resolution to resolve ultrastructural details such as synapses and provides sufficient field of view for dense reconstruction of neuronal circuits. While volume EM techniques are advancing, they are generating large data sets on the terabyte scale that require new image processing workflows and analysis tools. In this review, we present the recent advances in SBEM for circuit reconstruction in neuroscience and an overview of existing image processing and analysis pipelines.

Exploration of fs-laser ablation parameter space for 2D/3D imaging of soft and hard materials by tri-beam microscopy.

Tri-beam microscopes comprising a fs-laser beam, a Xe+ plasma focused ion beam (PFIB) and an electron beam all in one chamber open up exciting opportunities for site-specific correlative microscopy. They offer the possibility of rapid ablation and material removal by fs-laser, subsequent polishing by Xe-PFIB milling and electron imaging of the same area. While tri-beam systems are capable of probing large (mm) volumes providing high resolution microscopical characterisation of 2D and 3D images across exceptionally wide range of materials and biomaterials applications, presenting high quality/low damage surfaces to the electron beam can present a significant challenge, especially given the large parameter space for optimisation. Here the optimal conditions and artefacts associated with large scale volume milling, mini test piece manufacture, serial sectioning and surface polishing are investigated, both in terms of surface roughness and surface quality for metallic, ceramic, mixed complex phase, carbonaceous, and biological materials. This provides a good starting place for those wishing to examine large areas or volumes by tri-beam microscopy across a range of materials.

Controlling FIB-SBEM slice thickness by monitoring the transmitted ion beam.

Serial block-face electron microscopy with focused ion beam cutting suffers from cutting artefacts caused by changes in the relative position of beam and sample, which are, for example, inevitable when reconditioning the ion gun. The latter has to be done periodically, which limits the continuous stack-acquisition time to several days. Here, we describe a method for controlling the ion-beam position that is based on detecting that part of the ion beam that passes the sample (transmitted beam). We find that the transmitted-beam current decreases monotonically as the beam approaches the sample and can be used to determine the relative position of beam and sample to an accuracy of around one nanometre. By controlling the beam approach using this current as the feedback parameter, it is possible to ion-mill consecutive 5 nm slices without detectable variations in thickness even in the presence of substantial temperature fluctuations and to restart the acquisition of a stack seamlessly. In addition, the use of a silicon junction detector instead of the in-column detector is explored.

3D electron microscopy and volume-based bouton sorting reveal the selectivity of inputs onto geniculate relay cell and interneuron dendrite segments.

Introduction: The visual signals evoked at the retinal ganglion cells are modified and modulated by various synaptic inputs that impinge on lateral geniculate nucleus cells before they are sent to the cortex. The selectivity of geniculate inputs for clustering or forming microcircuits on discrete dendritic segments of geniculate cell types may provide the structural basis for network properties of the geniculate circuitry and differential signal processing through the parallel pathways of vision. In our study, we aimed to reveal the patterns of input selectivity on morphologically discernable relay cell types and interneurons in the mouse lateral geniculate nucleus. Methods: We used two sets of Scanning Blockface Electron Microscopy (SBEM) image stacks and Reconstruct software to manually reconstruct of terminal boutons and dendrite segments. First, using an unbiased terminal sampling (UTS) approach and statistical modeling, we identified the criteria for volume-based sorting of geniculate boutons into their putative origins. Geniculate terminal boutons that were sorted in retinal and non-retinal categories based on previously described mitochondrial morphology, could further be sorted into multiple subpopulations based on their bouton volume distributions. Terminals deemed non-retinal based on the morphological criteria consisted of five distinct subpopulations, including small-sized putative corticothalamic and cholinergic boutons, two medium-sized putative GABAergic inputs, and a large-sized bouton type that contains dark mitochondria. Retinal terminals also consisted of four distinct subpopulations. The cutoff criteria for these subpopulations were then applied to datasets of terminals that synapse on reconstructed dendrite segments of relay cells or interneurons. Results: Using a network analysis approach, we found an almost complete segregation of retinal and cortical terminals on putative X-type cell dendrite segments characterized by grape-like appendages and triads. On these cells, interneuron appendages intermingle with retinal and other medium size terminals to form triads within glomeruli. In contrast, a second, presumed Y-type cell displayed dendrodendritic puncta adherentia and received all terminal types without a selectivity for synapse location; these were not engaged in triads. Furthermore, the contribution of retinal and cortical synapses received by X-, Y- and interneuron dendrites differed such that over 60% of inputs to interneuron dendrites were from the retina, as opposed to 20% and 7% to X- and Y-type cells, respectively. Conclusion: The results underlie differences in network properties of synaptic inputs from distinct origins on geniculate cell types.

Serial block-face scanning electron microscopy of Schmidtea mediterranea.

The flatworm planarian, Schmidtea mediterranea (Smed) is a master at regenerating and rebuilding whole animals from fragments. A full understanding of Smed's regenerative capabilities requires a high-resolution characterization of organs, tissues, and the adult stem cells necessary for regeneration in their native environment. Here, we describe a serial block face scanning electron microscopy (SBF-SEM) protocol, optimized for Smed specifically, for visualizing the ultrastructure of membranes and condensed chromosomes in this model organism.

Application of the mirror technique for block-face scanning electron microscopy.

The mirror technique adapted for electron microscopy allows correlating neuronal structures across the cutting plane of adjoining light microscopic sections which, however, have a limited thickness, typically less than 100 µm (Talapka et al. in Front Neuroanat, 2021, https://doi.org/10.3389/fnana.2021.652422 ). Here, we extend the mirror technique for tissue blocks in the millimeter range and demonstrate compatibility with serial block-face electron microscopy (SBEM). An essential step of the methodological improvement regards the recognition that unbound resin must be removed from the tissue surface to gain visibility of surface structures. To this, the tissue block was placed on absorbent paper during the curing process. In this way, neuronal cell bodies could be unequivocally identified using epi-illumination and confocal microscopy. Thus, the layout of cell bodies which were cut by the sectioning plane can be correlated with the layout of their complementary part in the adjoining section processed for immunohistochemistry. The modified mirror technique obviates the spatial limit in investigating synaptology of neurochemically identified structures such as neuronal processes, dendrites and axons.

The complex synaptic pathways onto a looming-detector neuron revealed using serial block-face scanning electron microscopy.

The ability of locusts to detect looming stimuli and avoid collisions or predators depends on a neuronal circuit in the locust's optic lobe. Although comprehensively studied for over three decades, there are still major questions about the computational steps of this circuit. We used fourth instar larvae of Locusta migratoria to describe the connection between the lobula giant movement detector 1 (LGMD1) neuron in the lobula complex and the upstream neuropil, the medulla. Serial block-face scanning electron microscopy (SBEM) was used to characterize the morphology of the connecting neurons termed trans-medullary afferent (TmA) neurons and their synaptic connectivity. This enabled us to trace neurons over several hundred micrometers between the medulla and the lobula complex while identifying their synapses. We traced two different TmA neurons, each from a different individual, from their synapses with the LGMD in the lobula complex up into the medulla and describe their synaptic relationships. There is not a simple downstream transmission of the signal from a lamina neuron onto these TmA neurons; there is also a feedback loop in place with TmA neurons making outputs as well as receiving inputs. More than one type of neuron shapes the signal of the TmA neurons in the medulla. We found both columnar and trans-columnar neurons connected with the traced TmA neurons in the medulla. These findings indicate that there are computational steps in the medulla that have not been included in models of the neuronal pathway for looming detection.

Integrating single cell transcriptomics and volume electron microscopy confirms the presence of pancreatic acinar-like cells in sea urchins.

The identity and function of a given cell type relies on the differential expression of gene batteries that promote diverse phenotypes and functional specificities. Therefore, the identification of the molecular and morphological fingerprints of cell types across taxa is essential for untangling their evolution. Here we use a multidisciplinary approach to identify the molecular and morphological features of an exocrine, pancreas-like cell type harbored within the sea urchin larval gut. Using single cell transcriptomics, we identify various cell populations with a pancreatic-like molecular fingerprint that are enriched within the S. purpuratus larva digestive tract. Among these, in the region where they reside, the midgut/stomach domain, we find that populations of exocrine pancreas-like cells have a unique regulatory wiring distinct from the rest the of the cell types of the same region. Furthermore, Serial Block-face scanning Electron Microscopy (SBEM) of the exocrine cells shows that this reported molecular diversity is associated to distinct morphological features that reflect the physiological and functional properties of this cell type. Therefore, we propose that these sea urchin exocrine cells are homologous to the well-known mammalian pancreatic acinar cells and thus we trace the origin of this particular cell type to the time of deuterostome diversification. Overall, our approach allows a thorough characterization of a complex cell type and shows how both the transcriptomic and morphological information contribute to disentangling the evolution of cell types and organs such as the pancreatic cells and pancreas.

Defining the ultrastructure of the hematopoietic stem cell niche by correlative light and electron microscopy.

The blood system is supported by hematopoietic stem and progenitor cells (HSPCs) found in a specialized microenvironment called the niche. Many different niche cell types support HSPCs, however how they interact and their ultrastructure has been difficult to define. Here, we show that single endogenous HSPCs can be tracked by light microscopy, then identified by serial block-face scanning electron microscopy (SBEM) at multiscale levels. Using the zebrafish larval kidney marrow (KM) niche as a model, we followed single fluorescently labeled HSPCs by light sheet microscopy, then confirmed their exact location in a 3D SBEM dataset. We found a variety of different configurations of HSPCs and surrounding niche cells, suggesting there could be functional heterogeneity in sites of HSPC lodgement. Our approach also allowed us to identify dopamine beta-hydroxylase (dbh) positive ganglion cells as a previously uncharacterized functional cell type in the HSPC niche. By integrating multiple imaging modalities, we could resolve the ultrastructure of single rare cells deep in live tissue and define all contacts between an HSPC and its surrounding niche cell types.

Early invasion of the bladder wall by solitary bacteria protects UPEC from antibiotics and neutrophil swarms in an organoid model.

Recurrence of uropathogenic Escherichia coli (UPEC) infections has been attributed to reactivation of quiescent intracellular reservoirs (QIRs) in deep layers of the bladder wall. QIRs are thought to arise late during infection following dispersal of bacteria from intracellular bacterial communities (IBCs) in superficial umbrella cells. Here, we track the formation of QIR-like bacteria in a bladder organoid model that recapitulates the stratified uroepithelium within a volume suitable for high-resolution live-cell imaging. Bacteria injected into the organoid lumen enter umbrella-like cells and proliferate to form IBC-like bodies. In parallel, single bacteria penetrate deeper layers of the organoid wall, where they localize within or between uroepithelial cells. These "solitary" bacteria evade killing by antibiotics and neutrophils and are morphologically distinct from bacteria in IBCs. We conclude that bacteria with QIR-like properties may arise at early stages of infection, independent of IBC formation and rupture.

Expression of TRPC1 and SBEM protein in breast cancer tissue and its relationship with clinicopathological features and prognosis of patients.

This study investigated the relationship of the expression of transient receptor potential channel 1 (TRPC1), small breast epithelial mucin (SBEM) in breast cancer tissues with clinical pathological features and prognosis of patients. Altogether 50 patients with breast cancer who were treated in Weifang People's hospital from April 2017 to November 2018 were selected, and the mRNA and protein differences of TRPC1 and SBEM in breast cancer patients and normal breast cancer tissues were detected by qRT-PCR and Western blot. Spearman test was used for correlation analysis. Logistic univariate and multivariate analysis were performed on the risk factors related to breast cancer metastasis in breast cancer patients. The expression of TRPC1 and SBEM in breast cancer tissues was significantly higher than that in normal breast tissues (P<0.001). The mRNA expression of TRPC1, SBEM and protein was not related to age, tumor size and tissue grade of breast cancer patients, but related to TNM stage, clinical stage and lymph node metastasis (P<0.001). The relative expression of TRPC1 was positively correlated with clinical stage of breast cancer (r=0.992, P<0.001). The relative expression of SBEM was positively correlated with the clinical stage of breast cancer (r=0.853, P<0.001). The relative expression of TRPC1 was positively correlated with TNM staging of breast cancer (r=0.860, P<0.001). The relative expression of SBEM was positively correlated with TNM staging of breast cancer (r=0.880, P<0.001). Multivariate conditional Logistic regression analysis showed that TNM staging, TRPC1, SBEM were independent risk factors for malignant breast cancer metastasis. On the contrary, expression of TRPC1 and SBEM in breast cancer tissues was up-regulated. TRPC1 and SBEM may be involved in the process of breast cancer occurrence, development and metastasis, and can be used as potential tissue biomarkers in diagnosis of breast cancer metastasis and disease assessment.

Coupled Broad Ion Beam-Scanning Electron Microscopy (BIB-SEM) for polishing and three dimensional (3D) serial section tomography (SST).

Here we describe the first automated fully integrated in-microscope broad ion beam (BIB) system. Ar+-BIB has several advantages over Ga+ focused ion beam (FIB) and Xe+ plasma-FIB (PFIB) methods inducing less beam damage, especially for ion beam sensitive materials. It can mill areas several orders of magnitude larger (up to millimetre scale), and is not confined to the edge of the sample with associated curtaining issues. BIB is shown to have sputter rates up to five times higher than comparable FIB techniques. This new coupled BIB-SEM system (commercial name 'iPrep™II') enables in-microscope surface polishing to remove contaminants or damage for two dimensional (2D) imaging, as well as automated serial section tomography (SST) by milling and imaging hundreds of slices, cost and time efficiently. The milled slice thickness can be controlled from a few nanometers up to a micrometre. A novel sample transfer, handling and interlock system allows automated and sequential BIB polishing, scanning electron microscopy (SEM) and analysis by secondary electron (SE) imaging, electron back scatter diffraction (EBSD) and energy dispersive spectroscopy (EDS) for 3D microstructure analysis. Furthermore, insulating surfaces can be sputter coated after milling each slice to reduce charging during SEM analysis. The performance of the instrument is demonstrated through a series of case studies across the materials, earth and life sciences exploiting the imaging, crystallographic and chemical mapping capabilities. These include the study of butterfly defects in bearing steels, meta-stable intermetallic phases in bronze bearings, shale gas rock, aluminium plasma electrolytic oxide (PEO) coatings as well as liver and mouse brain tissues.

Serial block face scanning electron microscopy in cell biology: Applications and technology.

Three-dimensional electron microscopy (3DEM) is an imaging field containing several powerful modalities such as serial section transmission electron microscopy and electron tomography. However, large-scale 3D studies of biological ultrastructure on a cellular scale have historically been hampered by the difficulty of available techniques. Serial block face scanning electron microscopy (SBFSEM) is a 3DEM technique, developed in 2004, which has greatly increased the reliability, availability and throughput of 3DEM. SBFSEM allows for 3D imaging at resolutions high enough to resolve membranes and small vesicles whilst having the capability to collect data with a large field of view. Since its introduction it has become a major tool for ultrastructural investigation and has been applied in the study of many biological fields, such as connectomics, cellular and matrix biology. In this review, we will discuss biological SBFSEM from a technical standpoint, with a focus on cellular applications and also subsequent image analysis techniques.

Large Volume Electron Microscopy and Neural Microcircuit Analysis.

One recent technical innovation in neuroscience is microcircuit analysis using three-dimensional reconstructions of neural elements with a large volume Electron microscopy (EM) data set. Large-scale data sets are acquired with newly-developed electron microscope systems such as automated tape-collecting ultramicrotomy (ATUM) with scanning EM (SEM), serial block-face EM (SBEM) and focused ion beam-SEM (FIB-SEM). Currently, projects are also underway to develop computer applications for the registration and segmentation of the serially-captured electron micrographs that are suitable for analyzing large volume EM data sets thoroughly and efficiently. The analysis of large volume data sets can bring innovative research results. These recently available techniques promote our understanding of the functional architecture of the brain.

Fast Homogeneous En Bloc Staining of Large Tissue Samples for Volume Electron Microscopy.

Fixation and staining of large tissue samples are critical for the acquisition of volumetric electron microscopic image datasets and the subsequent reconstruction of neuronal circuits. Efficient protocols exist for the staining of small samples, but uniform contrast is often difficult to achieve when the sample diameter exceeds a few hundred micrometers. Recently, a protocol (BROPA, brain-wide reduced-osmium staining with pyrogallol-mediated amplification) was developed that achieves homogeneous staining of the entire mouse brain but requires very long sample preparation times. By exploring modifications of this protocol we developed a substantially faster procedure, fBROPA, that allows for reliable high-quality staining of tissue blocks on the millimeter scale. Modifications of the original BROPA protocol include drastically reduced incubation times and a lead aspartate incubation to increase sample conductivity. Using this procedure, whole brains from adult zebrafish were stained within 4 days. Homogenous high-contrast staining was achieved throughout the brain. High-quality image stacks with voxel sizes of 10 × 10 × 25 nm3 were obtained by serial block-face imaging using an electron dose of ~15 e-/nm2. No obvious reduction in staining quality was observed in comparison to smaller samples stained by other state-of-the-art procedures. Furthermore, high-quality images with minimal charging artifacts were obtained from non-neural tissues with low membrane density. fBROPA is therefore likely to be a versatile and efficient sample preparation protocol for a wide range of applications in volume electron microscopy.

SBEMimage: Versatile Acquisition Control Software for Serial Block-Face Electron Microscopy.

We present SBEMimage, an open-source Python-based application to operate serial block-face electron microscopy (SBEM) systems. SBEMimage is designed for complex, challenging acquisition tasks, such as large-scale volume imaging of neuronal tissue or other biological ultrastructure. Advanced monitoring, process control, and error handling capabilities improve reliability, speed, and quality of acquisitions. Debris detection, autofocus, real-time image inspection, and various other quality control features minimize the risk of data loss during long-term acquisitions. Adaptive tile selection allows for efficient imaging of large tissue volumes of arbitrary shape. The software's graphical user interface is optimized for remote operation. In its user-friendly viewport, tile grids covering the region of interest to be acquired are overlaid on previously acquired overview images of the sample surface. Images from other sources, e.g., light microscopes, can be imported and superimposed. SBEMimage complements existing DigitalMicrograph (Gatan Microscopy Suite) installations on 3View systems but permits higher acquisition rates by interacting directly with the microscope's control software. Its modular architecture and the use of Python/PyQt make SBEMimage highly customizable and extensible, which allows for fast prototyping and will permit adaptation to a wide range of SBEM systems and applications.