Plastic Embedding Research Articles

Optimized Methyl methacrylate embedding of small and large undecalcified bones.

Methyl methacrylate (MMA) plastic embedding has been long established as a technique for the processing and histological assessment of bones. It provides the added benefit over paraffin in that it does not require decalcification of the tissue in order visualize the cellular detail, thus preserving vital information about the amount of unmineralized osteoid present in addition to the degree of mineralization in the bone. It also allows for the incorporation of dynamic histomorphometric analysis through the retention of fluorescent labels incorporated into the bone. Efficient infiltration of hard tissue is essential to the processing of bones and producing quality slides suitable for achieving usable quantifiable histology out the other end. This technique:• Updates previously published MMA embedding protocols to reflect utilization of stabilized acrylamides (over the unstabilized reagents of the past)• Outlines the techniques that are important for embedding both small (mus), medium (rattus), and large (porcine, lagomorph, human) histological samples.• Updates the clearing and infiltration processes utilized and validates quality of the sample preparation though histological staining to confirm preservation of cellular detail, mineralization information, and enzymatic activity

A preliminary study of polymer optical fiber’s knittability for smart wear applications

PurposeThe purpose of this research is to ascertain the feasibility of fabricating polymer optical fibers (POFs) based textile structures by knitting with Polymethylmethacrylate (PMMA) based optical fibers for textile sensor application. It has long been established that by using the principles of physics, POFs have the capability to function as sensors, detecting strain, temperature and other variables. However, POF applications such as strain and pressure sensing using knitting techniques has since not been very successful due to a number of reasons. Commercially available PMMA-based optical fibers tend to be fragile and susceptible to breakages when subjected to stress during the knitting processes. Also light transmitted within these fibers is prone to leakage due to the curvature that results when optical fibers are interlaced or interlooped within fabric structures.Design/methodology/approachUsing Stoll’s multi-gauge CMS 350 HP knitting machine, five fabric structures namely, 1 × 4 float knit structure, tunnel inlay knit structure, 3:1 fleece fabric and 2:1 fleece fabric structure respectively were used to knit sensor samples. The samples were subsequently tested for length of illumination and sensitivity relative to applied pressure.FindingsThe results of this preliminary study establish that embedding plastic optical fibers into a knitted structure during the fabric formation process for soft strain sensor application possible. The best illumination performance was recorded for tunnel inlay structure which had an average of 94 cm course length of POF being illuminated. Sensor sensitivity experiments also establish that the relative spectral intensity of the fiber is sensitive to both light and pressure. Problems encountered and recommendations for further research have also been discussed and proffered.Research limitations/implicationsDue to resource limitations, an innovative technique (use of precision weight set) was used to apply pressure to the sensors. Consequently, information regarding the extent of corresponding sensor deformation has not been used in this initial analysis.Practical implicationsBecause the fundamental step toward finding a solution to any engineering problem is the acquisition of reliable data, and considering the fact that most of the popular technologies used for soft textile sensors are still bedeviled with the problem of signal instability and noise, the success of this application thus has the tendency to promote the wide spread adoption of POF sensors for smart apparel applications.Originality/valueAs far as research on soft strain sensors is concerned, to the best of the authors’ knowledge, this is the first study to have attempted to knit deformable sensors using commercially available POFs.

Improved Visualization of Oral Microbial Consortia.

Bacteria on the tongue dorsum (TD) form consortia tens to hundreds of microns in diameter organized around a core of epithelial cells. Whole-mount preparations have been instrumental in revealing their organization and specific microbial associations. However, their thickness and intricate 3-dimensional complexity present challenges for a comprehensive spatial analysis. To overcome these challenges, we employed a complementary approach: embedding in hydrophilic plastic followed by sectioning and postsectioning labeling. Samples were labeled by hybridization with multiplexed fluorescent oligonucleotide probes and visualized by spectral imaging and linear unmixing. Application of this strategy to TD biofilms improved the visualization of bacteria that were difficult to resolve in whole-mount imaging. Actinomyces, previously detected as patches, became resolved at the single-cell level. The filamentous taxa Leptotrichia and Lachnospiraceae, located at the core of the consortium, were regularly visualized whereas previously they were rarely detected when using whole mounts. Streptococcus salivarius, heterogeneously detected in whole mounts, were regularly and homogenously observed. Two-dimensional images provide valuable information about the organization of bacterial biofilms. However, they offer only a single plane of view for objects that can extend to hundreds of microns in thickness, and information obtained from such images may not always reflect the complexity of a 3-dimensional object. We combined serial physical sectioning with optical sectioning to facilitate the 3-dimensional reconstruction of consortia, spanning over 100 µm in thickness. Our work showcases the use of hydrophilic plastic embedding and sectioning for examining the structure of TD biofilms through spectral imaging fluorescence in situ hybridization. The result was improved visualization of important members of the human oral microbiome. This technique serves as a complementary method to the previously employed whole-mount analysis, offering its own set of advantages and limitations. Addressing the spatial complexity of bacterial consortia demands a multifaceted approach for a comprehensive and effective analysis.

Modeling of human placental capillaries by using a unique 3d reconstruction program

Purpose: Visualization of the three-dimensional structures is performed by examining two-dimensional sections in histology. It is not always possible to comprehend structure-function integrity. Therefore, it was planned to obtain three-dimensional (3D) models of the capillaries in the placenta terminal villus by using the program developed in the Başkent University’s laboratory and used in studies in different disciplines. The program’s suitability for histology studies will also be assessed.
 Materials and Methods: A term placenta tissue sample was obtained from a 33-year-old healthy woman. Following the plastic embedding procedure, semi-thin serial sections were taken from these blocks. The sections were stained using toluidine blue and examined and photographed by using a light microscope. The 3D modeling procedure was conducted on the light micrographs of serial sections in order to reconstruct villous capillaries. 
 Results: 3D images were obtained from two-dimensional light micrographs by using the same program. It was determined that the vessels running parallel to each other branched harmoniously, forming a capillary network in the terminal villus. Additionally, challenges encountered during the utilization of this program in histological studies are also presented in this study. 
 Conclusion: Fetal blood reaches every part of the terminal villus through the branching capillary network. This feature would increase the surface area of vasculo-syncytial membrane and facilitate feto-maternal exchange. This can be clearly visualized by using 3D models. Three-dimensional modeling helps to understand the structure of all organs and tissues. Given that the current program is freely accessible, it will not incur additional costs for researchers and is open to further improvement by the programmer (erhankiziltan@gmail.com).

Large Growth Deformations of Thin Tissue using Solid-Shells.

Simulating large scale expansion of thin structures, such as in growing leaves, is challenging. Solid-shells have a number of potential advantages over conventional thin-shell methods, but have thus far only been investigated for small plastic deformation cases. In response, we present a new general-purpose FEM growth framework for handling a wide range of challenging growth scenarios using the solid-shell element. Solid-shells are a middle-ground between traditional volume and thin-shell elements where volumetric characteristics are retained while being treatable as a 2D manifold much like thin-shells. These elements are adaptable to accommodate the many techniques that are required for simulating large and intricate plastic deformations, including morphogen diffusion, plastic embedding, strain-aware adaptive remeshing, and collision handling. We demonstrate the capabilities of growing solid-shells in reproducing buckling, rippling, curling, and collision deformations, relevant towards animating growing leaves, flowers, and other thin structures. Solid-shells are compared side-by-side with thin-shells to examine their bending behavior and runtime performance. The experiments demonstrate that solid-shells are a viable alternative to thin-shells for simulating large and intricate growth deformations.

High-Pressure Freezing Followed by Freeze Substitution: An Optimal Electron Microscope Technique to Study Golgi Apparatus Organization and Membrane Trafficking.

A major goal of structural biologists is to preserve samples as close to their living state as possible. High-pressure freezing (HPF) is a state-of-art technique that freezes the samples at high pressure (~2100bar) and low temperature (-196°C) within milliseconds. This ultrarapid fixation enables simultaneous immobilization of all cellular components and preserves the samples in a near-native state. This facilitates the study of dynamic processes in Golgi apparatus organization and membrane trafficking. The work in our laboratory shows that high-pressure freezing followed by freeze substitution (FS), the introduction of organic solvents at low temperature prior to plastic embedding, can better preserve the structure of Golgi apparatus and Golgi-associated vesicles. Here, we present a protocol for freezing monolayer cell cultures on sapphire disks followed by freeze substitution. We were able to use this protocol to successfully study Golgi organization and membrane trafficking in HeLa cells. The protocol gives decidedly better preservation of Golgi apparatus and associated vesicles than conventional chemically fixed preparation and as a plastic embedded preparation can be readily extended to 3D electron microscopy imaging through sequential block face-scanning electron microscopy. The 3D imaging of a multi-micron thick organelle such as the Golgi apparatus located near the cell nucleus is greatly facilitated relative to hydrated sample imaging techniques such as cryo-electron microscopy.

Histological Study of Glandular Variability in the Skin of the Natterjack Toad-Epidalea calamita (Laurenti, 1768)-Used in Spanish Historical Ethnoveterinary Medicine and Ethnomedicine.

Simple SummaryCommon toads, including the natterjack toad (Epidalea calamita), have been used since ancient times for remedies, and thus constitute excellent biological material for pharmacological and natural product research. After a previous analysis of the historical-folk therapeutic use of amphibians in Spain, a histological study was carried out to provide a complementary ethnopharmacological view through the analysis of the integumentary heterogeneity of the serous (venom) and mucous glands from two adult specimens. Plastic-embedded semi-thin sections showed that serous/venom glands are cytologically homogeneous in spite of their genetic and biochemical complexity, leading to a cocktail that remains stored until extrusion. On the contrary, mucous glands, working continuously, show a more complex cytological variation and regional heterogeneity, which suggests an adaptive variability, leading to an invisible topographic map of skin toxicity. Natterjack toad-based folk remedies are usually extracted from the whole animal as a therapeutic unit in ethnoveterinary practice. However, a new ethnopharmacological vision could emerge from the study of tegumentary regional variation.Common toads have been used since ancient times for remedies and thus constitute excellent biological material for pharmacological and natural product research. According to the results of a previous analysis of the therapeutic use of amphibians in Spain, we decided to carry out a histological study that provides a complementary view of their ethnopharmacology, through the natterjack toad (Epidalea calamita). This species possesses a characteristic integument, where the parotoid glands stand out, and it has been used in different ethnoveterinary and ethnomedical practices. This histological study of their glandular variability allow us to understand the stages through which the animal synthesises and stores a heterogeneous glandular content according to the areas of the body and the functional moment of the glands. To study tegumentary cytology, a high-resolution, plastic embedding, semi-thin (1 micron) section method was applied. Up to 20 skin patches sampled from the dorsal and ventral sides were processed from the two adult specimens collected, which were roadkill. Serous/venom glands display a genetic and biochemical complexity, leading to a cocktail that remains stored (and perhaps changes over time) until extrusion, but mucous glands, working continuously to produce a surface protection layer, also produce a set of active protein (and other) substances that dissolve into mucous material, making a biologically active covering. This study provides a better understanding of the use of traditional remedies in ethnoveterinary medicine.

Mechanical property and structure of polycarbonate /aluminum alloy hybrid prepared by ultrasound-assisted hot-pressing technology

ABSTRACT Polycarbonate/aluminum alloy hybrids were prepared using a homemade ultrasound-assisted hot pressing molding (UAHPM) technique, and their properties and structures at the plastic-metal interface were also investigated. The aluminum alloy surface was chemically etched and anodized to form micron and nano-holes structures. The joint strength of 21.58 MPa was achieved at the optimum molding process by optimizing the process parameters of ultrasonic hot compression molding. The specific structure and morphology of the polycarbonate/aluminum hybrid material bonding layer were observed by field emission scanning electron microscopy (FE-SEM) using the ultrathin sectioning technique, and the plastic embedding depth reached about 3.8 µm under the optimal process conditions. X-ray photoelectron spectroscopy (XPS) analysis revealed that the bonding layer not only has a micro-nano interlocking structure but also forms an Al-O-C chemical bonding interaction. The plastic in the bonding layer was exposed by dissolving the aluminum alloy of the joint, and the filling form of the plastic in the micro-nanoholes was directly observed.

Neuropathology Evaluation in Juvenile Toxicity Studies in Rodents: Comparison of Developmental Neurotoxicity Studies for Chemicals With Juvenile Animal Studies for Pediatric Pharmaceuticals.

The developmental neuropathology examination in juvenile toxicity studies depends on the nature of the product candidate, its intended use, and the exposure scenario (eg, dose, duration, and route). Expectations for sampling, processing, and evaluating neural tissues differ for developmental neurotoxicity studies (DNTS) for chemicals and juvenile animal studies (JAS) for pediatric pharmaceuticals. Juvenile toxicity studies typically include macroscopic observations, brain weights, and light microscopic evaluation of routine hematoxylin and eosin (H&E)-stained sections from major neural tissues (brain, spinal cord, and sciatic nerve) as neuropathology endpoints. The DNTS is a focused evaluation of the nervous system, so the study design incorporates perfusion fixation, plastic embedding of at least one nerve, quantitative analysis of selected brain regions, and sometimes special neurohistological stains. In contrast, the JAS examines multiple systems, so neural tissues undergo conventional tissue processing (eg, immersion fixation, paraffin embedding, H&E staining only). An "expanded neurohistopathology" (or "expanded neuropathology") approach may be performed for JAS if warranted, typically by light microscopic evaluation of more neural tissues (usually additional sections of brain, ganglia, and/or more nerves) or/and special neurohistological stains, to investigate specific questions (eg, a more detailed exploration of a potential neuroactive effect) or to fulfill regulatory requests.

Plastic embedding for precise imaging of large-scale biological tissues labeled with multiple fluorescent dyes and proteins.

Resin embedding of multi-color labeled whole organs is the primary step to preserve structural information for visualization of fine structures in three dimensions. It is essential to study the morphological characteristics, spatial and positional relationships of the millions of neurons, and the intricate network of blood vessels with fluorescent labels in the brain. However, the current resin embedding method is inadequate because of incompatibilities with fluorescent dyes, making it difficult to reconstruct a variety of structures for the interpretation of their complex spatial relationships. We modified the resin embedding method for large biological tissues labeled with multiple fluorescent dyes and proteins through different labeling strategies. With TrueBlack as the background fluorescence inhibitor in the glycol methacrylate (GMA) embedding, we referred to the method as GMA-T (Glycol methacrylate with TB). In the GMA-T embedded mouse brains, structures labeled with fluorescent proteins and dyes were visualized in millimeter-scale networks with sub-cellular resolution, allowing quantitative analysis of different anatomical structures in the same brain, including neurons and blood vessels. In combination with high-resolution whole-brain imaging, it is possible to obtain a variety of fluorescence labeled structures in just a few days. We quantified the distribution and morphology of the tdTomato-labeled vasoactive intestinal polypeptide (VIP) neurons and the BSA-FITC labeled blood vessels in the same brain. These results demonstrated that VIP neurons and blood vessels have their own unique distribution patterns and morphological characteristics among cortical regions and different layers in cerebral cortex, and there was no significant correlation between VIP neurons and vessels. This approach provides a novel approach to study the interaction among different anatomical structures within large-volume biological samples labeled with multiple fluorescent dyes and proteins, which helps elucidating the complex anatomical characteristics of biological organs.

A new mechanistic model for conductivity of hydraulic fractures with proppants embedment and compaction

To extract unconventional energy more efficiently, the hydraulic fracturing technology is widely used to create fracture network in formations and facilitate the transport of hydrocarbons to production wells. Nevertheless, fracture conductivity decreases sharply and continuously due to the increase of closure pressure (or effective stress). It is a challenging problem to maintain fracture conductivity and reduce the conductivity change rate during extraction. Therefore, it is of great significance to predict the behavior of proppant embedment and compaction within hydraulic fractures. The objective of this work is to establish a reasonable model to determine the essential controls on conductivity of hydraulic fractures (conventional fracturing and channel fracturing) under stress conditions. Compared with other models, our analytical model is not limited to elastic deformation of proppants but takes elastoplastic deformation and fully plastic deformation of proppants into account. In addition, the model also considers proppant compaction (body deformation and structural deformation) besides proppant embedment. The predictions from the proposed model agree well with the available experimental data presented in the literature.The results show that the conductivity curves can be characterized by six stages: elastic embedment and elastic compaction stage, elastoplastic embedment and elastic compaction stage, plastic embedment and elastic compaction stage (including body deformation and structural deformation of proppant packs), plastic embedment and elastic compaction stage (only containing body deformation of proppant packs), plastic embedment and elastoplastic compaction stage, plastic embedment and plastic compaction stage. Although the proppant compaction depth is smaller than the proppant embedment depth, the change of conductivity due to proppant compaction cannot be ignored. Specifically, for the studied case, the change of conductivity due to proppant compaction is larger than that due to proppant embedment during the stages 2–6. Compared to the change of conductivity in “harder” rocks with larger elastic modulus, conductivity changes more dramatically in “softer” rocks with lower hardness. To reduce the rate of conductivity change, proppants with lager elastic modulus and larger size are recommended in the field. This work presents an accurate and efficient analytical model to quantify the conductivity of hydraulic fracture under stress condition. The proposed model reveals more details of the mechanisms that affect the deformation behavior in hydraulic fractures and offer some insights for parameter design and optimization during the execution of hydraulic fracturing.

Histological, Histomorphometrical, and Biomechanical Studies of Bone-Implanted Medical Devices: Hard Resin Embedding

The growing incidence of degenerative musculoskeletal disorders as well as lifestyle changes has led to an increase in the surgical procedures involving implanted medical devices in orthopedics. When studying implant/tissue interface in hard materials (i.e., metals or dense plastics) and/or in large bone segments, the hard plastic embedding of the intact undecalcified tissue envelope with the implant in situ is needed. The aim of this work is to describe the advances and the possibilities of high-temperature methyl methacrylate (MMA) embedding for the histological, histomorphometrical, and biomechanical assessment of bone-implanted medical devices. Unlike routine techniques, undecalcified bone processing histology, using high-temperature MMA, requires a complex and precise sample processing methodology and the availability of sophisticated equipment and software for both sample preparation and analyses. MMA embedding permits the evaluation of biological responses to the presence of implanted medical devices without implant removal, allowing simultaneous qualitative and quantitative histological evaluation, both static and dynamic histomorphometry, and biomechanical analyses not possible with tissue decalcification. MMA embedding, despite being a demanding procedure, is still preferred to other kinds of resin-based embedding because of its peculiar characteristics, which allow the study of samples of big dimensions also implanted with hard materials without reducing the sample or removing the material. Dynamic measurements are allowed together with biomechanical investigations at the bone-biomaterial interface, obtaining a comprehensive and precise evaluation of the safety and effectiveness of medical devices for orthopedic regenerative, reconstructive, and reparative surgery.

Ultrastructural Analysis of Microtubule Ends.

Microtubules can be detected in light microscopes, but the limited resolution of these instruments means that the polymers appear as lines whose width is defined by the diffraction of light. Much important work on microtubule dynamics has been accomplished by light microscopy, but the details of microtubule end structure are not accessible in such studies. Slight variations in fluorescence intensity, etc. have been used to comment on the structure of dynamic ends, and the combination of light microscopy with laser tweezers has provided insight into aspects of microtubule elongation. However, for views that reveal structural details of the pathways for microtubule growth and shortening, electron microscopy has been of great value. Here, we describe methods for using electron microscopes to look at the ends of microtubules as they grow and shrink, both in vivo and in vitro. The key problems to be overcome for ultrastructural study of microtubule dynamics are those of reliable sample preparation. Dynamic microtubules are labile and can therefore be modified by preparative methods. Our chapter follows the premise that rapid freezing, which converts sample water into vitreous ice, is the best approach for sample preparation. Therefore, all of the methods described involve finding optimal conditions for sample vitrification, and then getting the frozen sample into a form suitable for electron microscopy. We also posit that the end of a microtubule must be considered in three dimensions, so we employ electron tomography as a way to get the necessary information. The methods described for the study of microtubules in cells employ rapid freezing, freeze-substitution fixation, plastic embedding, serial sectioning, and tomography of stained samples. The methods for following microtubule growth in vitro employ sample preparation on holy grids, blotting, and plunge-freezing, followed by electron cryo-tomography. Quantification of structure from both approaches is accomplished by segmentation and analysis of graphic models.

A microwave method for plastic embedding of nervous tissue for light and electron microscopy

BackgroundFast, effective, and rapid processing of central nervous system (CNS) tissue with good preservation of myelin, especially in tissue from diseased mice, is important to many laboratories studying neurosciences. New methodIn this paper, we describe a new method to process and embed CNS tissue from mice. Spinal cords and optic nerves from naive C57BL/6 mice were used to standardize the microwave protocol following perfusion with fixative. The CNS tissue was processed and embedded using the microwave embedding protocol. ResultsWe observed that the tissue is well preserved and good quality light and electron microscope images were obtained after using the microwave embedding protocol. Comparison with existing methodsTraditional way of embedding CNS tissue in resin is challenging and time consuming. The microwave technology offers an efficient way to quickly embed CNS tissue while preserving morphology and retaining the integrity of the myelin. ConclusionsThis new method is fast, reliable and an effective way to embed CNS tissue in resin.

An optimized method for embedding undecalcified mouse tibias in plastic blocks

To optimize the method for embedding multiple undecalcified mouse tibias in plastic blocks, improve the efficiency and stability of plastic embedding and reduce the detachment rate of plastic slides. Thirty undecalcified tibias from 15 B6 mice were used for plastic embedding after calcein labeling, fixation, dehydration and infiltration. The tibias were embedded in cylindrical plastic blocks with a diameter of 4 mm. For each bone, the 1/4 proximal tibia was cut off, and the remaining 3/4 was used for re-embedding. Five bones were embedded in a single block with each bone standing closely on the surface of a flat plate. The samples were randomized into control and experimental groups in all the processes of embedding, sectioning and staining. In the 3 groups with modified embedment, flowing CO2 was added into the embedding solution, embedding solution was applied to the section surface, and the slides were heated at 95 ℃ for 15 min. The polymerization time, slide detachment rate, bone formation and osteoblast parameters were analyzed. We prepared 6 plastic blocks, each containing 5 tibias, whose cross sections were on the same plane. The blocks were completely polymerized and suitable for sectioning. Flowing CO2 into the embedding solution reduced the polymerization time and increased the rate of complete polymerization. Application of the embedding solution on the section surface significantly reduced the detachment rate of the sections (P < 0.05) without affecting bone formation analysis (P > 0.05). Heating the slides significantly lowered the detachment rate of the sections (P < 0.05) without affecting osteoblast analysis (P > 0.05). The optimized method allows effective embedding of multiple undecalcified mice tibias in the same block and can be an ideal method for histological analysis of undecalcified bones.

Stress relaxation behavior of wood in the plastic region under indoor conditions

The stress relaxation behavior of wood in the plastic region was measured by clamping wood specimens with bolts for up to 5 years under indoor environmental conditions. An initial stress was applied by displacement control, whereby a steel plate or a washer was embedded in the radial direction of the wood. The difference in the stress ratio was small in the plastic region due to the difference in the initial stress level, and the higher the initial stress, the higher the stress that was maintained. By contrast, the clamp force virtually disappeared in the elastic region. The estimated stress ratio after 50 years was 15% in the elastic region and approximately 20–40% in the plastic region. Due to the influence of humidity fluctuation, it was difficult to maintain stress in the elastic region, but it seems to be possible to maintain the stress for a long time using partial plastic embedment with sufficient extra end distance.

A collection of yeast cellular electron cryotomography data.

BackgroundCells are powered by a large set of macromolecular complexes, which work together in a crowded environment. The in situ mechanisms of these complexes are unclear because their 3D distribution, organization, and interactions are largely unknown. Electron cryotomography (cryo-ET) can address these knowledge gaps because it produces cryotomograms—3D images that reveal biological structure at ∼4-nm resolution. Cryo-ET uses no fixation, dehydration, staining, or plastic embedment, so cellular features are visualized in a life-like, frozen-hydrated state. To study chromatin and mitotic machinery in situ, we subjected yeast cells to genetic and chemical perturbations, cryosectioned them, and then imaged the cells by cryo-ET.FindingsHere we share >1,000 cryo-ET raw datasets of cryosectioned budding yeast Saccharomyces cerevisiaecollected as part of previously published studies. These data will be valuable to cell biologists who are interested in the nanoscale organization of yeasts and of eukaryotic cells in general. All the unpublished tilt series and a subset of corresponding cryotomograms have been deposited in the EMPIAR resource for the community to use freely. To improve tilt series discoverability, we have uploaded metadata and preliminary notes to publicly accessible Google Sheets, EMPIAR, and GigaDB.ConclusionsCellular cryo-ET data can be mined to obtain new cell-biological, structural, and 3D statistical insights in situ. These data contain structures not visible in traditional electron-microscopy data. Template matching and subtomogram averaging of known macromolecular complexes can reveal their 3D distributions and low-resolution structures. Furthermore, these data can serve as testbeds for high-throughput image-analysis pipelines, as training sets for feature-recognition software, for feasibility analysis when planning new structural-cell-biology projects, and as practice data for students.

Thin polyester filters as versatile sample substrates for high-pressure freezing of bacterial biofilms, suspended microorganisms and adherent eukaryotic cells.

High-pressure freezing limits the size of biological samples, because only small samples can be frozen without ice damage. Additionally, these samples must fit into the dimensions of the sample holder provided by the high-pressure freezer. We explored the potential of a 10 μm thin polyester filter membrane (PE-filter) as a versatile sample substrate for high-pressure freezing. Planktonic bacteria, bacterial spores and suspended eukaryotic cells could be concentrated on the PE-filter, whereas biofilm, bacterial microcolonies and HeLa cells were able to grow directly on the PE-filter. These microorganism-loaded PE-filters were used for high-pressure freezing, freeze-substitution and plastic embedding in Epon or Lowicryl. Embedded filters were cross-sectioned so that the interface between microorganism and substrate as well as the overlying medium was revealed. Although the structural preservation was good for thin samples and samples with lower water content, such as biofilms, adherent HeLa-cell cultures were likewise sufficiently preserved for transmission electron microscopy imaging. The fact that microorganism-loaded PE-filters could be also examined with confocal laser scanning fluorescence microscopy under fully hydrated conditions, and freeze-substituted PE-filters samples with scanning electron microscopy, demonstrates the versatility of the PE-filter as a sample substrate for a wide array of microorganisms. LAY DESCRIPTION: In order to investigate biological samples in the transmission electron microscope it is imperative to remove all their water content, or the specimens will be destroyed by boiling in the high vacuum of the microscope. In order to avoid dramatic morphology-changes due to drying artefacts or the impact of chemical stabilisers, high-pressure freezing (HPF) was developed. This protocol allows freezing biological samples in an instant (within a few milliseconds) down to -196°C while applying high pressure at the same time so that the specimen retains all its water in a solidified noncrystalline form. However, the formation of morphology-destroying ice crystals is only avoided, if the cooling of the sample is faster than the ice crystal formation, which is only possible with very thin samples (up to a maximum of 200 μm in optimal cases). High-pressure freezing is regarded as the gold-standard for sample preparation of cells, tissues and small organisms. However, all of these samples must fit into the dimensions of the specific sample holder of the high-pressure freezer and their transfer into the high-pressure freezing machine must be achieved without significant impact on sample physiology. Additionally, it may also necessary to concentrate and immobilise a biological specimen before they can be placed in the HPF sample holder. Although a few number of strategies and sample substrates have been used for different types of biological samples, we explored the potential of a 10 μm thin polyester filter membrane (PE-filter) as a versatile sample substrate for HPF. In culture medium suspended bacteria, suspended bacterial spores and in medium suspended higher cells could be concentrated on the PE-filter, whereas bacterial biofilm or bacterial microcolonies from an agar plate, and surface-adhering higher cells were able to grow directly on the PE-filter. These microorganism-loaded PE-filters could be directly used for high-pressure freezing, and were finally embedded in a plastic resin like Epon or Lowicryl. Embedded filters were cross-sectioned so that the interface between microorganism and substrate or overlying medium was revealed. Although the structural preservation was good for thin samples and samples with lower water content, such as biofilms, adherent HeLa-cell cultures were likewise sufficiently preserved for transmission electron microscopy imaging. The fact that microorganism-loaded PE-filters could be also examined with confocal laser scanning fluorescence microscopy under fully hydrated conditions, and freeze-substituted PE-filters samples with scanning-electron microscopy, demonstrates the versatility of the PE-filter as a sample substrate for a wide array of microorganisms.

Introduction to Currently Applied Device Pathology.

Pathologic evaluation is crucial to the study of medical devices and integral to the Food and Drug Administration and other regulatory entities' assessment of device safety and efficacy. While pathologic analysis is tailored to the type of device, it generally involves at a minimum gross and microscopic evaluation of the medical device and associated tissues. Due to the complex nature of some implanted devices and specific questions posed by sponsors, pathologic evaluation inherently presents many challenges in accurately assessing medical device safety and efficacy. This laboratory's experience in numerous collaborative projects involving veterinary pathologists, biomedical engineers, physicians, and other scientists has led to a set of interrelated assessments to determine pathologic end points as a means to address these challenges and achieve study outcomes. Thorough device evaluation is often accomplished by utilizing traditional paraffin histology, plastic embedding and microground sections, and advanced imaging modalities. Combining these advanced techniques provides an integrative, comprehensive approach to medical device pathology and enhances medical device safety and efficacy assessment.

FIB-SEM of mouse nervous tissue: Fast and slow sample preparation.

Focused ion beam-scanning electron microscopy (FIB-SEM) has become a widely used technique in life sciences. To achieve the best data quality, sample preparation is important and has to be adapted to the specimen and the specific application. Here we illustrate three preparation procedures for mouse nervous tissue: First, the use of high-pressure freezing followed by direct imaging of vitrified tissue without any staining in the FIB-SEM under cryo-conditions as direct and fast procedure. Second, a slow procedure involving freeze substitution of frozen samples combined with additional staining for enhanced contrast and plastic embedding. Third, a fast preparation applying microwave-assisted chemical fixation and processing for resin embedding. All three methods of sample preparation are suitable for obtaining data stacks by FIB-SEM acquisition and 3D reconstruction.