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Plant anatomical and histochemical studies are concerned with the structural organization of tissues as well as localization of various metabolites and enzyme activity inside cells and tissues. Traditionally, rotary microtomes are used for paraffin and resin-embedded samples which provide excellent preservation of tissue morphology but removes enzymes, lipid components, and various specialized metabolites. Freeze sectioning apparently remained unexplored in plant histology because of the presence of rigid cell walls and highly vacuolated cytoplasm in plant tissues. In this study, we have described a simple cryostat-based sectioning technique using polyethylene glycol (PEG) as embedding medium after glycerol infiltration that protects the plant tissues from freezing and thawing damage. We have also compared the suitability of inexpensive aqueous PEG solution as compared to commercially available optimal cutting temperature (OCT) medium and obtained identical microscopic images. Diverse plant organs from different genera were sectioned to check the application of this method in plant anatomical studies. In all the cases, cross sections were shown to be well preserved similar to paraffin-embedded plant tissues. In addition, histochemical analyses showed retention of metabolites and even enzymes in the tissues, which can make this method an alternate choice in cryo-microtomy replacing the expensive OCT medium., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Slicing tissue samples into thin pieces is commonly used in histology analysis and more recently for organotypic culture when tissue samples are sliced alive. Currently available devices for slicing tissue samples are either designed for fixed tissue samples at low cryogenic temperatures ( e.g. , Cryostats), or bulky and expensive ( e.g. , vibratome), preventing them from routine lab usage. Here we report a cost-effective device designed to section live tissues for subsequent culture. This device consists of components crafted from 3D-printed Nylon-12- a material suitable for autoclaving to ensure sterility. Its small footprint enhances portability, allowing for convenient placement within a biosafety cabinet for an added layer of sterility assurance. Using human pluripotent stem cells derived brain organoids as an example, we demonstrated that the device both precisely and accurately makes slices. We further validate its suitability for long-term culture by extended tissue culture following slicing. Our results indicate that brain organoid slices are viable and show improved proliferation rate compared with unsliced organoids., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Like other volume electron microscopy approaches, automated tape-collecting ultramicrotomy (ATUM) enables imaging of serial sections deposited on thick plastic tapes by scanning electron microscopy (SEM). ATUM is unique in enabling hierarchical imaging and thus efficient screening for target structures, as needed for correlative light and electron microscopy. However, SEM of sections on tape can only access the section surface, thereby limiting the axial resolution to the typical size of cellular vesicles with an order of magnitude lower than the acquired xy resolution. In contrast, serial-section electron tomography (ET), a transmission electron microscopy-based approach, yields isotropic voxels at full EM resolution, but requires deposition of sections on electron-stable thin and fragile films, thus making screening of large section libraries difficult and prone to section loss. To combine the strength of both approaches, we developed 'ATUM-Tomo, a hybrid method, where sections are first reversibly attached to plastic tape via a dissolvable coating, and after screening detached and transferred to the ET-compatible thin films. As a proof-of-principle, we applied correlative ATUM-Tomo to study ultrastructural features of blood-brain barrier (BBB) leakiness around microthrombi in a mouse model of traumatic brain injury. Microthrombi and associated sites of BBB leakiness were identified by confocal imaging of injected fluorescent and electron-dense nanoparticles, then relocalized by ATUM-SEM, and finally interrogated by correlative ATUM-Tomo. Overall, our new ATUM-Tomo approach will substantially advance ultrastructural analysis of biological phenomena that require cell- and tissue-level contextualization of the finest subcellular textures., Competing Interests: GK, GF, AW, LR, HJ, CN, AK, NP, TM, TM, IK, MS No competing interests declared, (© 2023, Kislinger et al.)

Background: Several factors in glass slide (GS) preparation affect the quality and data volume of a digitized histological slide. In particular, reducing contamination and selecting the appropriate coverslip have the potential to significantly reduce scan time and data volume., Goals: To objectify observations from our institute's digitization process to determine the impact of laboratory processes on the quality of digital histology slides., Materials and Methods: Experiment 1: Scanning the GS before and after installation of a central console in the microtomy area to reduce dirt and statistical analysis of the determined parameters. Experiment 2: Re-coverslipping the GS (post diagnostics) with glass and film. Scanning the GS and statistical analysis of the collected parameters., Conclusion: The targeted restructuring in the laboratory process leads to a reduction of GS contamination. This causes a significant reduction in the amount of data generated and scanning time required for the digitized sections. Film as a coverslip material minimizes processing errors in contrast to glass. According to our estimation, all the above-mentioned points lead to considerable cost savings., (© 2024. The Author(s).)

Pathological histology examination involves handling a variety of specimens that are cut according to regulations and placed in cassettes. Tissue fragments in the cassettes are then diagnosed after processing, embedding, thin sectioning, staining and other procedures using a processing machine. Maintaining tissue fragment order and orientation during these processes is important for accurate diagnosis. In this study, we present a method of maintaining tissue fragment order and orientation using a thin film of ultra-high-strength agar and evaluate its usefulness during tissue sectioning.Cassettes were prepared, each containing three pieces of porcine liver, and compared embedding time with and without agar thin films (ATFs). Embedding was performed by three medical laboratory scientists with different levels of experience.To enable one-step tissue sample embedding, ATFs were integrated with samples in the cassettes. This resulted in an average reduction of 6.22 s of embedding time per cassette compared with traditional embedding methods.Through the use of ATFs, tissue fragment order and orientation is maintained, and embedding process time shortened. Additionally, ATFs are easily prepared and stored in 10% neutral buffered formalin over extended periods, allowing for immediate use during sectioning. This method is ideal to implement in busy pathology laboratories., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Here, we present a protocol for immunolabeling of molecules in Arabidopsis tissues. We describe steps for tissue fixation and embedding in resin of microtome-derived sections, immunolabeling using fluorescent and non-fluorescent secondary antibodies, and visualization of cytokinin and auxin molecules. This protocol is suitable to study reproductive structures such as inflorescences, flowers, fruits, and tissue-culture-derived samples. This protocol is useful for studying the distribution of a wide range of molecules including hormones and cell wall components. For complete details on the use and execution of this protocol, please refer to Herrera-Ubaldo et al. (2019). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The correlative imaging workflow is a method of combining information and data across modes (e.g. SEM, X-ray CT, FIB-SEM), scales (cm to nm) and dimensions (2D-3D-4D), providing a more holistic interpretation of the research question. Often, subsurface objects of interest (e.g. inclusions, pores, cracks, defects in multilayered samples) are identified from initial exploratory nondestructive 3D tomographic imaging (e.g. X-ray CT, XRM), and those objects need to be studied using additional techniques to obtain, for example, 2D chemical or crystallographic data. Consequently, an intermediate sample preparation step needs to be completed, where a targeted amount of sample surface material is removed, exposing and revealing the object of interest. At present, there is not one singular technique for removing varied thicknesses at high resolution and on a range of scales from cm to nm. Here, we review the manual and automated options currently available for targeted sample material removal, with a focus on those methods which are readily accessible in most laboratories. We summarise the approaches for manual grinding and polishing, automated grinding and polishing, microtome/ultramicrotome, and broad-beam ion milling (BBIM), with further review of other more specialist techniques including serial block face electron microscopy (SBF-SEM), and ion milling and laser approaches such as FIB-SEM, Xe plasma FIB-SEM, and femtosecond laser/LaserFIB. We also address factors which may influence the decision on a particular technique, including the composition, shape and size of the samples, sample mounting limitations, the amount of surface material to be removed, the accuracy and/or resolution of peripheral parts, the accuracy and/or resolution of the technique/instrumentation, and other more general factors such as accessibility to instrumentation, costs, and the time taken for experimentation. It is hoped that this study will provide researchers with a range of options for removal of specific amounts of sample surface material to reach subsurface objects of interest in both correlative and non-correlative workflows., (© 2022 Royal Microscopical Society.)

Interstitial fibrosis is characterized by the increased deposition of extracellular matrix (ECM) components within the interstitial space of various organs, such as the kidneys, heart, lungs, liver, and skin. The primary component of interstitial fibrosis-related scarring is interstitial collagen. Therefore, the therapeutic application of anti-fibrotic medication hinges on the accurate measurement of interstitial collagen levels within tissue samples. Current histological measurement techniques for interstitial collagen are generally semi-quantitative in nature and only provide a ratio of collagen levels within tissues. However, the Genesis™ 200 imaging system and supplemental image analysis software, FibroIndex™, from HistoIndex™, is a novel, automated platform for imaging and characterizing interstitial collagen deposition and related topographical properties of the collagen structures within an organ, in the absence of any staining. This is achieved by using a property of light known as second harmonic generation (SHG). Using a rigorous optimization protocol, collagen structures in tissue sections can be imaged with a high degree of reproducibility and ensures homogeneity across all samples while minimizing the introduction of any imaging artefacts or photobleaching (decreased tissue fluorescence due to prolonged exposure to the laser). This chapter outlines the protocol that should be undertaken to optimize HistoIndex scanning of tissue sections, and the outputs that can be measured and analyzed using the FibroIndex™ software., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

RNA in situ hybridization offers a means to study the spatial expression of candidate genes by making use of specific, labelled RNA probes on thin tissue sections. Unlike other methods, such as promoter GUS fusions, for which all regulatory sequences should be available and transgenic plants have to be generated, RNA in situ hybridization allows specific and direct detection of even low abundant transcripts at cellular resolution. Although various protocols exist, the results published throughout the literature indicate a very obvious problem of the technique: each step has the potential to affect the outcome, that is, the signal strength, presence or absence of background, and visibility of individual cells. The protocol described here tries to avoid all these problems by addressing each step in detail and providing advice regarding critical steps for a distinct visualization of gene expression on intact tissue sections without any background., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Paraffin histology is one of the most important and commonly used laboratory techniques enabling the study of the microscopic structure of animal and plant tissues. This technique uses paraffin wax, which in liquid form impregnates fixed and dehydrated tissues and allows the preparation of thin sections when solidified in blocks. This protocol on good practices in paraffin histology of Nothobranchius furzeri (Nothobranchiidae) summarizes the authors' current experience in terms of technique, evaluation, and interpretation of sectioned tissues. The steps that precede paraffin block preparation are also presented as they play a key role in maximizing the quality of examined sections. The paraffin technique as described only requires basic laboratory conditions to produce good-quality results. The description of staining methods is limited to Mayer's hematoxylin and eosin (H&E), the routinely used histological dye staining cell nuclei in blue-black (hematein) and cell cytoplasm and connective tissue fibers in shades of pink-red (eosin). Killifish specialists are encouraged to engage in the study of histology and histopathology, taking advantage of interdisciplinary cooperation., (© 2022 Cold Spring Harbor Laboratory Press.)

Cholesterol is essential to all animal life, and its dysregulation is observed in many diseases. For some of these, the precise determination of cholesterol's histological location and absolute abundance at cellular length scales within tissue samples would open the door to a more fundamental understanding of the role of cholesterol in disease onset and progression. We have developed a fast and simple method for absolute quantification of cholesterol within brain samples based on the sensitive detection and mapping of cholesterol by silver-assisted laser desorption ionization mass spectrometry imaging (AgLDI MSI) from thin tissue sections. Reproducible calibration curves were generated by depositing a range of cholesterol-D 7 concentrations on brain homogenate tissue sections combined with the homogeneous spray deposition of a non-animal steroid reference standard detectable by AgLDI MSI to minimize experimental variability. Results obtained from serial brain sections gave consistent cholesterol quantitative values in very good agreement with those obtained with other mass spectrometry-based methods., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

The CelLock™ procedure kit is used to collect and prepare cellular specimens such as fine needle aspirates (FNA), cytology specimens, cultured cells, small tissue biopsies, and samples with scant tissue fragments or cells into a paraffin cell-block. This cell-block can be used for subsequent microtomy and staining using hematoxylin and eosin (H&E), special stains, immunohistochemistry (IHC), and applicable molecular techniques such as in situ hybridization (ISH). CelLock is a standardized method that provides optimal receipt, preservation, preparation, and processing of cell-blocks which, contain virtually all of the submitted specimens and are able to be embedded and sectioned in a reproducible fashion. The specimen contained within the cell-block is preserved such that all the cellular protein and genetic information is available for histological and ancillary testing.

Formalin fixed paraffin embedded tissue occasionally requires reprocessing if the histologic quality of a section is inadequate for clinical diagnosis. The Pat Dry (PD) and the Serial Xylene (SX) methods are two techniques described in the literature to reprocess under-fixed and/or under-processed tissue samples. To date, no study has compared the effects of these methods on the histologic quality of tissue sections, cost, and turnaround times. In the present study, these two methods were evaluated on 129 tissue samples taken from 40 submitted clinical specimens, 3 blocks per sampled location. Before processing, sample Group 1 (Control) was cut at routine 3-5 mm thickness. Sample Groups 2 and 3 were cut at 10 mm to ensure the thicker tissues would be poorly processed. Histotechnicians performed a subjective evaluation of all the samples at the time of embedding and microtomy. Hematoxylin and eosin stained sections from all samples were scored for histologic quality by two pathology residents. Thicker samples (Groups 2 and 3) were then reprocessed using either PD or SX methods, re-sectioned, stained, and then re-scored by the pathology residents. The two reprocessing methods equally improved quality scores and reduced the fraction of slides that were rejected. The PD method average preparation time was 66 minutes as compared to 250 minutes for the SX method. The PD method was easier to perform than the SX method, required less reagent, and was less susceptible to reagent spillage than the SX method.

Microtomy is a medical laboratory sciences procedure that medical laboratory technologists (MLTs) use to cut tissues for microscopic examination. Due to safety concerns and the potential to destroy tissue samples, learners must perform the procedure correctly. In order to allow for safe and controlled learning, this procedure should be conducted in a simulated setting before attempting with human tissues. The objective of this study is to describe the development and user-based evaluation of a virtual simulation training module. A research group developed the virtual simulation training module's content and design, and a local MLT expert provided the content. Nine students enrolled in a university-based medical laboratory sciences program provided feedback about the module. The results demonstrated that the virtual simulation training module was an effective and user-friendly learning tool for the medical laboratory sciences program. Although more validity and efficacy testing are required in the future, the students indicated a potential to use this module to prepare future students for hands-on exercise in a simulation laboratory setting., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2022, Wahab et al.)

Histopathological visualizations are a pillar of modern medicine and biological research. Surgical oncology relies exclusively on post-operative histology to determine definitive surgical success and guide adjuvant treatments. The current histology workflow is based on bright-field microscopic assessment of histochemical stained tissues and has some major limitations. For example, the preparation of stained specimens for brightfield assessment requires lengthy sample processing, delaying interventions for days or even weeks. Therefore, there is a pressing need for improved histopathology methods. In this paper, we present a deep-learning-based approach for virtual label-free histochemical staining of total-absorption photoacoustic remote sensing (TA-PARS) images of unstained tissue. TA-PARS provides an array of directly measured label-free contrasts such as scattering and total absorption (radiative and non-radiative), ideal for developing H&E colorizations without the need to infer arbitrary tissue structures. We use a Pix2Pix generative adversarial network to develop visualizations analogous to H&E staining from label-free TA-PARS images. Thin sections of human skin tissue were first virtually stained with the TA-PARS, then were chemically stained with H&E producing a one-to-one comparison between the virtual and chemical staining. The one-to-one matched virtually- and chemically- stained images exhibit high concordance validating the digital colorization of the TA-PARS images against the gold standard H&E. TA-PARS images were reviewed by four dermatologic pathologists who confirmed they are of diagnostic quality, and that resolution, contrast, and color permitted interpretation as if they were H&E. The presented approach paves the way for the development of TA-PARS slide-free histological imaging, which promises to dramatically reduce the time from specimen resection to histological imaging., (© 2022. The Author(s).)

Background: Microtomy is a risky procedure that medical laboratory technologists (MLTs) use to cut tissue samples for microscopic examination. Due to the safety concerns and the potential to destroy tissue samples, it is critical for learners to perform the procedure correctly. To allow for safe and controlled learning, this procedure should be acquired in a safe and controlled simulated setting before being attempted on human tissues. The overarching purpose of this work is the development of a virtual training module for undergraduate students to learn from. However, because of the heterogeneity in the steps required to successfully complete the procedure from the MLTs as well as in the literature, the aim of this study was to reach a consensus from a panel of experts about identifying the steps of the procedure using the think-aloud and modified-Delphi methods., Methods: First, we conducted a think-aloud protocol with a single MLT expert trained in microtomy to generate the list of steps of the microtomy procedure objectively. In order to remove any idiosyncratic steps, next, we asked eight experts that were trained in histology to rate the criticalness of each step using a (1-5) Likert scale and provide evaluative feedback., Results: The think-aloud protocol generated 10 steps for the microtomy procedure. During the subsequent two rounds of the Delphi exercise, the experts agreed to modify one step of the 10 steps., Conclusions: Through this work, the 10 steps of the microtomy procedure have been validated by experts in the field. Following that, a virtual simulation training module was built to instruct learners on the microtomy procedure. The virtual simulation training module may be used for further research in microtomy., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2022, Wahab et al.)

Microfibers are one of the most abundant microplastic particle types found in the environment, where they cause negative impacts on organisms and possibly on human health. Microfibers should be included in a wide range of laboratory studies; however, microfibers for scientific studies are not commercially available. Current methods to make microfibers generally create particles with large size ranges and poor precision, and efficient production of particles ≤100 µm is difficult. Laboratory studies of the biological and toxicological effects and chemical interactions of microfibers require uniform, small microfibers in sufficient numbers for environmentally relevant experiments. We developed a novel fiber embedding technique and modified a seminal cryomicrotomy method to produce precise microfibers in quantities suitable for environmentally relevant concentrations. Polyethylene terephthalate (PET) and nylon fibers were strategically wound onto a spindle, embedded in paraffin wax, and sectioned using a standard paraffin microtome. After processing with a suitable organic solvent to remove the wax, microfiber size distributions were assessed. The small microfibers (10-42 µm) were accurate to the target lengths with excellent precision and a production rate ≥13.5 times higher than previous methods. As a proof of application, three lengths of manufactured PET fibers were stained with Nile red and exposed to eastern oyster larvae (Crassostrea virginica) for 24 h. Larvae ingested the smaller fiber lengths (14 and 28 µm), and the Nile red-stained fibers were visible and distinguishable in the guts of the larvae. This experiment was the first to demonstrate ingestion of plastic particles other than microspheres by oyster larvae. The present method facilitates the use of small microfibers in laboratory experiments, allowing for a more complete understanding of microplastic effects in the environment. Environ Toxicol Chem 2022;41:944-953. © 2021 SETAC., (© 2021 SETAC.)

The origin of histology-the study of microscopic anatomy-is intimately connected with the development of the light microscope and improvements in lens design and manufacture.However, knowledge of the ultrastructure of the cell was hampered by the very nature of light microscopy, which, due to the physical properties of the visible electromagnetic spectrum, could never provide the magnification and resolution for study of the granules seen in cells, which we now know as the organelles. When the electron microscope was developed in the 1930s, a beam of electrons replaced light as the source of illumination, and the inner details of the cell could be observed directly. With thin sections obtained by transmission electron microscopy, cell biologists could embark on the task of reconstructing 3D microstructure via the painstaking stacking of the individual slices.The three-dimensional visualization of the mitochondrion was particularly challenging, as its convoluted structure could be interpreted in several ways based on differences observed by George Palade at the Rockefeller Institute for Medical Research (NYC), and Fritiof Sjöstrand at the Karolinska Institutet (Stockholm). Palade's interpretation was eventually accepted as correct due to its alignment with the findings of biochemists investigating the cascade of molecular interactions known as the Krebs cycle, responsible for the production of cellular energy in the form of adenosine triphosphate (ATP). However, it can also be argued that Palade's visualization via a physical model of the mitochondrion, which he built with sheets of wax, photographed, and published in 1953, better enabled colleagues to comprehend its unique inner structures known as cristae.To teach undergraduate science students about this pivotal moment in cell biology and add to their understanding of the reconstruction process, a pedagogical exercise was created in which students are provided with outline drawings of various organic objects cut in random planes of section. Working individually at first, and then in groups, they are tasked with collaborating to devise an accurate description of the shape and texture of the object. After their observations are presented to the class, they are shown a photo of the object prior to its sectioning to determine if their observations were correct., (© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

The confocal laser scanning microscope allows us to examine tissue sections in greater detail than a widefield fluorescence microscope. However, this requires samples to be better preserved than standard cryostat sections, which are not usually aldehyde-fixed. Thick sections (approximately 70 μm) of formaldehyde-fixed tissue can be cut using a vibrating microtome and subsequently labeled with primary and secondary fluorescent antibodies and/or fluorescent stains. When imaged in the confocal microscope, these samples allow us to collect high-resolution images, detailing the intracellular location of multiple proteins and structures. In this chapter, we describe the technique used to prepare vibrating microtome sections, using porcine tissue infected with African swine fever virus as an example. This technique can easily be applied to any animal tissue with any suitable combination of antibodies, depending on the hypothesis., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Alternate least squares (ALS) reconstructions of the infrared (IR) spectra of the individual layers from original automotive paint were analyzed using machine learning methods to improve both the accuracy and speed of a forensic automotive paint examination. Twenty-six original equipment manufacturer (OEM) paints from vehicles sold in North America between 2000 and 2006 served as a test bed to validate the ALS procedure developed in a previous study for the spectral reconstruction of each layer from IR line maps of cross-sectioned OEM paint samples. An examination of the IR spectra from an in-house library (collected with a high-pressure transmission diamond cell) and the ALS reconstructed IR spectra of the same paint samples (obtained at ambient pressure using an IR transmission microscope equipped with a BaF 2 cell) showed large peak shifts (approximately 10 cm -1 ) with some vibrational modes in many samples comprising the cohort. These peak shifts are attributed to differences in the residual polarization of the IR beam of the transmission IR microscope and the IR spectrometer used to collect the in-house IR spectral library. To solve the problem of frequency shifts encountered with some vibrational modes, IR spectra from the in-house spectral library and the IR microscope were transformed using a correction algorithm previously developed by our laboratory to simulate ATR spectra collected on an iS-50 FT-IR spectrometer. Applying this correction algorithm to both the ALS reconstructed spectra and in-house IR library spectra, the large peak shifts previously encountered with some vibrational modes were successfully mitigated. Using machine learning methods to identify the manufacturer and the assembly plant of the vehicle from which the OEM paint sample originated, each of the twenty-six cross-sectioned automotive paint samples was correctly classified as to the "make" and model of the vehicle and was also matched to the correct paint sample in the in-house IR spectral library.

Plant plasmodesmata (PD) are complex intercellular channels consisting of a thin endoplasmic reticulum (ER) tubule enveloped by the plasma membrane (PM). PD were first observed by electron microscopy about 50 years ago and, since, numerous studies in transmission and scanning electron microscopy have provided important information regarding their overall organization, revealing at the same time their diversity in terms of structure and morphology. However, and despite the fact that PD cell-cell communication is of critical importance for plant growth, development, cellular patterning, and response to biotic and abiotic stresses, linking their structural organization to their functional state has been proven difficult. This is in part due to their small size (20-50 nm in diameter) and the difficulty to resolve these structures in three dimensions at nanometer resolution to provide details of their internal organization.In this protocol, we provide in detail a complete process to produce high-resolution transmission electron tomograms of PD. We describe the preparation of the plant sample using high-pressure cryofixation and cryo-substitution. We also describe how to prepare filmed grids and how to cut and collect the sections using an ultramicrotome. We explain how to acquire a tilt series and how to reconstruct a tomogram from it using the IMOD software. We also give a few guidelines on segmentation of the reconstructed tomogram., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

dropletProbe mass spectrometry is a novel technique for molecular characterization of surfaces. It can be used for rapid ex vivo analysis of therapeutics from thin animal tissue sections and has been shown to improve understanding of a drug's absorption, distribution, metabolism and excretion (ADME) properties. Here, we describe the tissue distribution analysis of diclofenac from a dosed whole-body mouse thin tissue section using a dropletProbe mass spectrometry system., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cytology specimens and biopsy tissues are frequently small and pale, making them difficult to visualize grossly in paraffin. Ten dyes were assayed on small tissues to determine if specimen discernibility could be increased during the embedding and microtomy steps in the histological process. The ideal dye should not remain visible in a tissue section microscopically after subsequent staining and must not interfere with immunohistochemistry (IHC) assays. This study found that Harris hematoxylin and 1% aq. toluidine blue solution were the best labelers for gross tissue visualization and did not adversely affect post-processing staining and IHC assays.

Techniques involving three-dimensional (3D) tissue structure reconstruction and analysis provide a better understanding of changes in molecules and function. We have developed AutoCUTS-LM, an automated system that allows the latest advances in 3D tissue reconstruction and cellular analysis developments using light microscopy on various tissues, including archived tissue. The workflow in this paper involved advanced tissue sampling methods of the human cerebral cortex, an automated serial section collection system, digital tissue library, cell detection using convolution neural network, 3D cell reconstruction, and advanced analysis. Our results demonstrated the detailed structure of pyramidal cells (number, volume, diameter, sphericity and orientation) and their 3D spatial organization are arranged in a columnar structure. The pipeline of these combined techniques provides a detailed analysis of tissues and cells in biology and pathology., (© 2021. The Author(s).)

Background: In the neurosciences, the physical disector method represents an established quantitative stereological method for unbiased sampling and counting of cells in histological tissue sections of known thickness. Physical disector analyses are conventionally performed using plastic-embedded tissue samples, because plastic-embedding causes a comparably low and definable shrinkage of the embedded tissue, and the thickness of thin plastic sections can be determined adequately. However, immunohistochemistry protocols often don't work satisfactorily in sections of plastic-embedded tissue., New Method: Here, a new methodological approach is presented, allowing for physical disector analyses of immunohistochemically labeled cells in paraffin sections. The embedding-related tissue shrinkage is standardized by using defined tissue sample volumes and paraffin volumes, and the extent of tissue shrinkage can be determined accurately from the sample volumes prior to and after embedding. Co-embedding of polyethylene section thickness standards together with the tissue samples allows the precise determination of individual paraffin section thicknesses by spectral reflectance measurements., Results and Comparison With Existing Method(s): The applicability of the new method is demonstrated by physical disector analysis of immunohistochemically identified somatotroph cells in paraffin sections of porcine pituitary gland tissue. With consideration of individual shrinkage factors and section thicknesses, the cell numbers and mean volumes estimated in paraffin disector sections do not significantly differ from the results obtained by analyses of plastic-embedded pituitary tissue samples of the identical animals (2.4% average difference)., Conclusions: The featured method enables combination of paraffin section immunohistochemistry and physical disector analyses for unbiased quantitative stereological analyses of different cell types., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Understanding tree growth and carbon sequestration are of crucial interest to forecast the feedback of forests to climate change. To have a global understanding of the wood formation, it is necessary to develop new methodologies for xylogenesis measurements, valid across diverse wood structures and applicable to both angiosperms and gymnosperms. In this study, the authors present a new workflow to study xylogenesis using high-resolution X-ray computed tomography (HRXCT), which is generic and offers high potential for automatization. The HXRCT-based approach was benchmarked with the current classical approach (microtomy) on three tree species with contrasted wood anatomy ( Pinus nigra, Fagus sylvatica , and Quercus robur ). HRXCT proved to estimate the relevant xylogenesis parameters (timing, duration, and growth rates) across species with high accuracy. HRXCT showed to be an efficient avenue to investigate tree xylogenesis for a wide range of wood anatomies, structures, and species. HRXCT also showed its potential to provide quantification of intra-annual dynamics of biomass production through high-resolution 3D mapping of wood biomass within the forming growth ring., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lehnebach, Campioli, Gričar, Prislan, Mariën, Beeckman and Van den Bulcke.)

The standard histological processing procedure, which produces excellent staining of sections for most tissues, fails to yield satisfactory results in adult mouse orbits or eyeballs. Here, we show that a protocol using tissue block staining and domestic adhesive tapes resulted in qualified integral serial cryo-sections of whole orbits or eyeballs, and the fine structures were well preserved. The histological processing protocol comprises paraformaldehyde fixation, ethylenediaminetetraacetic acid decalcification, tissue block staining with hematoxylin and eosin, embedding, adhesive tape aided sectioning, and water-soluble mounting. This protocol was proved to be the best in comparison with seven other related existing histological traditional or non-traditional processing methods, according to the staining slice quality. We observed a hundred percent success rate in sectioning, collection, and mounting with this method. The reproducibility tested on qualified section success rates and slice quality scores confirmed that the technique is reliable. The feasibility of the method to detect target molecules in orbits was verified by successful trial tests on block immunostaining and adhesive tape-aided sectioning. Application of this protocol in joints, brains, and so on,-the challenging integral sectioning tissues, also generated high-quality histological staining sections., Competing Interests: The authors have read the journal’s policy and have the following competing interests: Julia Faßbender, Clara Wenhart, Hans-Peter Holthoff, and Goetz Muench are paid employees of Advancecor GmbH of Germany. There are no patents, products in development or marketed products associated with this research to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Postsynaptic structures on excitatory neurons, dendritic spines, are actin-rich. It is well known that actin-binding proteins regulate actin dynamics and by this means orchestrate structural plasticity during the development of the brain, as well as synaptic plasticity mediating learning and memory processes. The actin-binding protein cortactin is localized to pre- and postsynaptic structures and translocates in a stimulus-dependent manner between spines and the dendritic compartment, thereby indicating a crucial role for synaptic plasticity and neuronal function. While it is known that cortactin directly binds F-actin, the Arp2/3 complex important for actin nucleation and branching as well as other factors involved in synaptic plasticity processes, its precise role in modulating actin remodeling in neurons needs to be deciphered. In this study, we characterized the general neuronal function of cortactin in knockout mice. Interestingly, we found that the loss of cortactin leads to deficits in hippocampus-dependent spatial memory formation. This impairment is correlated with a prominent dysregulation of functional and structural plasticity. Additional evidence shows impaired long-term potentiation in cortactin knockout mice together with a complete absence of structural spine plasticity. These phenotypes might at least in part be explained by alterations in the activity-dependent modulation of synaptic actin in cortactin-deficient neurons.

Cerebral ischemia and its sequelae, which include memory impairment, constitute a leading cause of disability worldwide. Micro-RNAs (miRNA) are evolutionarily conserved short-length/noncoding RNA molecules recently implicated in adaptive/maladaptive neuronal responses to ischemia. Previous research independently implicated the miRNA-132/212 cluster in cholinergic signaling and synaptic transmission, and in adaptive/protective mechanisms of neuronal responses to hypoxia. However, the putative role of miRNA-132/212 in the response of synaptic transmission to ischemia remained unexplored. Using hippocampal slices from female miRNA-132/212 double-knockout mice in an established electrophysiological model of ischemia, we here describe that miRNA-132/212 gene-deletion aggravated the deleterious effect of repeated oxygen-glucose deprivation insults on synaptic transmission in the dentate gyrus, a brain region crucial for learning and memory functions. We also examined the effect of miRNA-132/212 gene-deletion on the expression of key mediators in cholinergic signaling that are implicated in both adaptive responses to ischemia and hippocampal neural signaling. miRNA-132/212 gene-deletion significantly altered hippocampal AChE and mAChR-M1, but not α7-nAChR or MeCP2 expression. The effects of miRNA-132/212 gene-deletion on hippocampal synaptic transmission and levels of cholinergic-signaling elements suggest the existence of a miRNA-132/212-dependent adaptive mechanism safeguarding the functional integrity of synaptic functions in the acute phase of cerebral ischemia.

This protocol describes how to prepare intact mouse cochleae for serial block-face scanning electron microscopy (SBEM). The detailed workflow includes cochlea fixation, en bloc staining, resin embedding, X-ray microscopy-guided trimming and SBEM data acquisition. This protocol allows large-scale, nanometer-resolution three-dimensional imaging of subcellular structures in a targeted tonotopic range of the cochlea and enables fast volumetric scan at submicron resolution using a compact X-ray microscope. For complete details on the use and execution of this protocol, please refer to Hua et al. (2021)., Competing Interests: P.B. is employed by Zeiss Research Microscopy Solutions. All other authors have no conflicts of interest to declare., (© 2021 The Author(s).)

Dephosphorylation of target proteins at serine/threonine residues is one of the most crucial mechanisms regulating their activity and, consequently, the cellular functions. The role of phosphatases in synaptic plasticity, especially in long-term depression or depotentiation, has been reported. We studied serine/threonine phosphatase activity during the protein synthesis blocker (PSB)-induced impairment of long-term potentiation (LTP). Established protein phosphatase 2B (PP2B, calcineurin) inhibitor cyclosporin A prevented the LTP early phase (E-LTP) decline produced by pretreatment of hippocampal slices with cycloheximide or anisomycin. For the first time, we directly measured serine/threonine phosphatase activity during E-LTP, and its significant increase in PSB-treated slices was demonstrated. Nitric oxide (NO) donor SNAP also heightened phosphatase activity in the same manner as PSB, and simultaneous application of anisomycin + SNAP had no synergistic effect. Direct measurement of the NO production in hippocampal slices by the NO-specific fluorescent probe DAF-FM revealed that PSBs strongly stimulate the NO concentration in all studied brain areas: CA1, CA3, and dentate gyrus (DG). Cyclosporin A fully abolished the PSB-induced NO production in the hippocampus, suggesting a close relationship between nNOS and PP2B activity. Surprisingly, cyclosporin A alone impaired short-term plasticity in CA1 by decreasing paired-pulse facilitation, which suggests bi-directionality of the influences of PP2B in the hippocampus. In conclusion, we proposed a minimal model of signaling events that occur during LTP induction in normal conditions and the PSB-treated slices.

Sharp wave-ripples (SWRs) represent synchronous discharges of hippocampal neurons and are believed to play a major role in memory consolidation. A large body of evidence suggests that SWRs are exclusively generated in the CA3-CA2 network. In contrast, here, we provide several lines of evidence showing that the subiculum can function as a secondary SWRs generator. SWRs with subicular origin propagate forward into the entorhinal cortex as well as backward into the hippocampus proper. Our findings suggest that the output structures of the hippocampus are not only passively facilitating the transfer of SWRs to the cortex, but they also can actively contribute to the genesis of SWRs. We hypothesize that SWRs with a subicular origin may be important for the consolidation of information conveyed to the hippocampus via the temporoammonic pathway., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

We report a multiplexed imaging mass spectrometry method which spatially localizes and selectively accesses the extracellular matrix on formalin-fixed paraffin-embedded tissue sections. The extracellular matrix (ECM) consists of (1) fibrous proteins, post-translationally modified (PTM) via N- and O-linked glycosylation, as well as hydroxylation on prolines and lysines, and (2) glycosaminoglycan-decorated proteoglycans. Accessing all these components poses a unique analytical challenge. Conventional peptide analysis via trypsin inefficiently captures ECM peptides due to their low abundance, intra- and intermolecular cross-linking, and PTMs. In previous studies, we have developed matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) techniques to capture collagen peptides via collagenase type III digestion, both alone and after N-glycan removal via PNGaseF digest. However, in fibrotic tissues, the buildup of ECM components other than collagen-type proteins, including elastin and glycosaminoglycans, limits efficacy of any single enzyme to access the complex ECM. Here, we have developed a novel serial enzyme strategy to define the extracellular matrix, including PTMs, from a single tissue section for MALDI-IMS applications. Graphical Abstract.

The human pulmonary environment is complex, containing a matrix of cells, including fibroblasts, epithelial cells, interstitial macrophages, alveolar macrophages and neutrophils. When confronted with foreign material or invading pathogens, these cells mount a robust response. Nevertheless, many bacterial pathogens with an intracellular lifecycle stage exploit this environment for replication and survival. These include, but are not limited to, Coxiella burnetii, Legionella pneumophila, Yersinia pestis, Mycobacterium tuberculosis and Staphylococcus aureus. Currently, few human disease-relevant model systems exist for studying host-pathogen interactions during these bacterial infections in the lung. Here, we present two novel infection platforms, human alveolar macrophages (hAMs) and human precision-cut lung slices (hPCLS), along with an up-to-date synopsis of research using said models. Additionally, alternative uses for these systems in the absence of pathogen involvement are presented, such as tissue banking and further characterization of the human lung environment. Overall, hAMs and hPCLS allow novel human disease-relevant investigations that other models, such as cell lines and animal models, cannot completely provide., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

Transmission electron microscopy of cell sample sections is a popular technique in microbiology. Currently, ultrathin sectioning is done on resin-embedded cell pellets, which consumes milli- to deciliters of culture and results in sections of randomly orientated cells. This is problematic for rod-shaped bacteria and often precludes large-scale quantification of morphological phenotypes due to the lack of sufficient numbers of longitudinally cut cells. Here we report a flat embedding method that enables observation of thousands of longitudinally cut cells per single section and only requires microliter culture volumes. We successfully applied this technique to Bacillus subtilis, Escherichia coli, Mycobacterium bovis, and Acholeplasma laidlawii. To assess the potential of the technique to quantify morphological phenotypes, we monitored antibiotic-induced changes in B. subtilis cells. Surprisingly, we found that the ribosome inhibitor tetracycline causes membrane deformations. Further investigations showed that tetracycline disturbs membrane organization and localization of the peripheral membrane proteins MinD, MinC, and MreB. These observations are not the result of ribosome inhibition but constitute a secondary antibacterial activity of tetracycline that so far has defied discovery.

Objective: To investigate the association between specimen length and number of sections evaluated and the diagnostic yield of temporal artery biopsy (TAB) for giant cell arteritis (GCA)., Methods: A pathologist reviewed all TABs performed for suspected GCA between January 1991 and December 2012. The blocks of all the inadequate and negative biopsy specimens were recut, and further slides at deeper levels were stained with hematoxylin and eosin in order to avoid missing inflammatory changes., Results: In total, findings from 662 TABs were included in the study (71% female; mean age 73.2 years). A total of 427 TAB specimens (65%) were classified as negative, and 235 (35%) were classified as positive for GCA. Compared to those with negative TAB results, patients with positive TAB results were older and more frequently female. There was no difference in postfixation TAB specimen length between TAB specimens negative and positive for GCA (mean 6.5 mm versus 6.9 mm; P = 0.068). Cuts of additional biopsy sections revealed inflammation at deeper levels in 26 of 408 TAB specimens (6.4%) originally reported as uninflamed. The inflamed section was the second in 14 TAB specimens, the third in 9 specimens, and the fourth in 3 specimens. Piecewise logistic regression identified 5 mm as the TAB specimen length change point for diagnostic sensitivity. Compared to a TAB specimen length of <5 mm, the age- and sex-adjusted odds ratio for positive TAB results in samples ≥5 mm long was 1.5 (95% confidence interval 1.0-2.0), P = 0.032., Conclusion: A postfixation TAB specimen length of at least 5 mm should be sufficient to make a histologic diagnosis of GCA. In order not to miss inflammatory changes, at least 3 further sections at deeper levels should be evaluated in all negative TAB specimens., (© 2020, American College of Rheumatology.)

Abnormal intraglandular stromal-epithelial interactions have been known as a main key contributing factor for development of Benign Prostatic Hyperplasia (BPH). However, the underlying mechanism for the dysregulated intercellular communication remains unclear. In this study we compared the proteomic profiles of hyperplastic tissue with adjacent normal tissue of BPH and identified Rab27B small GTPase, a key regulator of exocytosis, as a protein that was overexpressed in the epithelium of BPH tissue. Overexpression of Rab27B in prostatic epithelial cells strongly increased the signaling activities of the PI3K/AKT and ERK1/2 pathways, whereas, downregulation of Rab27B expression in the epithelial cells of BPH reduced the signaling activities and decreased cell proliferation. The elevated Rab27B expression caused an overall increase in cell surface presentation of growth factor receptors without affecting their expression. However, the small GTPase also possesses an inhibitory activity against mTORC1 independent of its role in cell surface presentation of growth factor receptors. Our findings demonstrate a pivotal role of the small GTPase in autocrine and paracrine signaling and suggest that its abnormal expression underlies the dysregulated stromal-epithelial interactions in BPH., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The introduction of cryo-techniques to the focused ion-beam scanning electron microscope (FIB-SEM) has brought new opportunities to study frozen, hydrated samples from the field of Life Sciences. Cryo-techniques have long been employed in electron microscopy. Thin electron transparent sections are produced by cryo-ultramicrotomy for observation in a cryo-transmission electron microscope (TEM). Cryo-TEM is presently reaching the imaging of macromolecular structures. In parallel, cryo-fractured surfaces from bulk materials have been investigated by cryo-SEM. Both cryo-TEM and cryo-SEM have provided a wealth of information, despite being 2D techniques. Cryo-TEM tomography does provide 3D information, but the thickness of the volume has a maximum of 200-300 nm, which limits the 3D information within the context of specific structures. FIB-milling enables imaging additional planes by creating cross-sections (e.g. cross-sectioning or site-specific X-sectioning) perpendicular to the cryo-fracture surface, thus adding a third imaging dimension to the cryo-SEM. This paper discusses how to produce suitable cryo-FIB-SEM cross-section results from frozen, hydrated Life Science samples with emphasis on 'common knowledge' and reoccurring observations. LAY DESCRIPTION: Life Sciences studies life down to the smallest details. Visualising the smallest details requires electron microscopy, which utilises high-vacuum chambers. One method to maintain the integrity of Life Sciences samples under vacuum conditions is freezing. Frozen samples can remain in a suspended state. As a result, research can be carried out without having to change the chemistry or internal physical structure of the samples. Two types of electron microscopes equipped with cryo-sample handling facilities are used to investigate samples: The scanning electron microscope (SEM) which investigates surfaces and the transmission electron microscope (TEM) which investigates thin electron transparent sections (called lamellae). A third method of investigation combines a SEM with a focused ion beam (FIB) to form a cryo-FIB-SEM, which is the basis of this paper. The electron beam images the cryo-sample surface while the ion beam mills into the surface to expose the interior of the sample. The latter is called cross-sectioning and the result provides a way of investigating the 3rd dimension of the sample. This paper looks at the making of cross-sections in this manner originating from knowledge and experience gained with this technique over many years. This information is meant for newcomers, and experienced researchers in cryo-microscopy alike., (© 2020 The Authors. Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.)

Antibodies against Aß amyloid are indispensable research tools and potential therapeutics for Alzheimer's disease. They display several unusual properties, such as specificity for aggregated forms of the peptide, the ability to distinguish polymorphic aggregate structures, and the ability to recognize generic aggregation-related epitopes formed by unrelated amyloid sequences. Understanding the mechanisms underlying these unusual properties and the structures of their corresponding epitopes is crucial for the understanding why antibodies display different therapeutic activities and for the development of more effective therapeutic agents. Here we employed a novel "epitomic" approach to map the fine structure of the epitopes of 28 monoclonal antibodies against amyloid-beta using immunoselection of random sequences from a phage display library, deep sequencing, and pattern analysis to define the critical sequence elements recognized by the antibodies. Although most of the antibodies map to major linear epitopes in the amino terminal 1 to 14 residues of Aß, the antibodies display differences in the target sequence residues that are critical for binding and in their individual preferences for nontarget residues, indicating that the antibodies bind to alternative conformations of the sequence by different mechanisms. Epitomic analysis also identifies discontinuous, nonoverlapping sequence Aß segments that may constitute the conformational epitopes that underlie the aggregation specificity of antibodies. Aggregation-specific antibodies recognize sequences that display a significantly higher predicted propensity for forming amyloid than antibodies that recognize the monomer, indicating that the ability of random sequences to aggregate into amyloid is a critical element of their binding mechanism., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Like any other biological tissue, plant tissue also exhibits optical properties like refraction, transmission, absorption, coloration, scattering and so on. Several studies have been conducted using different parts of plants such as leaves, seedlings, roots, stems and so on, and their optical properties have been analyzed to study plant physiology, influence of environmental cues on plant metabolism, light propagation through plant parts and the like. Thus, it is essential to study in detail the optical properties of several plant parts to determine their structural relationship. In this backdrop, an experimental study was conducted to observe and analyze the optical properties of node and inter-nodal tissue cross-sections of the plant Alternanthera philoxeroides under a polarizing microscope constructed and standardized in the laboratory. The observed optical properties of the microscopic tissue sections have been then studied to determine a significant structural relationship between nodal and inter-nodal tissue arrangement patterns as a whole. Tissue sections that have undergone a sort of biological perturbation like loss of water (dried in air for 15 min) have also been studied to study the change in the pattern of tissue optical property when compared with that of normal plant-tissue cross-sections under a polarizing microscope. This type of biological perturbation was chosen for the study because water plays an important role in maintenance of the normal physiological processes in plants and most other forms of life.

Allergic contact dermatitis (ACD) is a common skin disease with high prevalence in work environments. Human allergic contact dermatitis is triggered by the exposure to haptens that leads to an initial phase known as sensitization. During this phase, hapten-protein complexes presented by antigen-presenting cells activate a T-cell-mediated response, leading to the generation of memory cells against the hapten. Upon re-exposure to the same hapten, the elicitation phase is initiated. This phase is characterized by a quicker acute inflammatory response involving activation and/or infiltration of a variety of immune cell populations. Human ACD can be studied through the use of animal models of contact hypersensitivity (CHS). The 2,4-dinitrofluorobenzene (DNFB)-induced CHS model is a commonly used mouse model that has been helpful in the study of the mechanisms as well as potential therapeutic interventions of ACD. In this chapter I will provide a detailed protocol to develop acute DNFB-induced CHS in mice in a period of 7 days. In addition, I will discuss several key considerations for experimental design including best controls, potential expected outcomes, and sample collection.

The bone marrow is the major hematopoietic organ, consisting of distinct microenvironmental niches for the production of hematopoietic cells. Advanced visualizing methods are required to define and better understand the interactions between stromal and hematopoietic cells. In this chapter, we describe an ex vivo whole-mount imaging technique of the bone marrow, which allows for a fast, high-quality, and three-dimensional visualization of different bone marrow components. We provide a guide for conducting adoptive transfer experiments of fluorescently labeled leukocytes and visualizing their location in the bone marrow with respect to the bone marrow vasculature. This method presents a quick, easy, and inexpensive approach to image the bone marrow in three dimensions.

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Objective: To assess the feasibility of 3D modelisation of fetal anatomy by using the Computer-assisted anatomic dissection (CAAD) based on immunolabeled histologic slices and MRI slices with a specific 3D software., Study Design: For pelvis and lower limbs, subjects came from legal abortion, medical pregnancy termination, or late miscarriage. Specimens were fixed in 10 % formalin, then embedded in paraffin wax and serially sectioned. The histological slices were stained using HES and Masson Trichrome. Protein S-100 and D2-40 markers were used for immuno-labelling. Serial transverse sections were digitalized and manually aligned. Fetal brain slices were obtained from in utero or post-mortem MRI., Results: CAAD was performed on 10 fetuses: pelvis was modelised with 3 fetuses of 13, 15 and 24 W G, lower limbs with 2 fetuses of 14 and 15 W G and brain with 5 fetuses aged between 19 and 37 W G. Fetal pelvis innervation was analysed after immunolabelling and nerves appeared proportionally bigger than in adults with the same topography. Lower limbs analysis revealed that nerve development was guided by vascular development: the sciatic nerve along the big axial vein, the saphen nerve along the big saphen vein and the sural nerve along the small saphen vein. Fetal brain study allowed to describe the gyration process and the lateral ventricle development., Conclusion: CAAD technique provides an accurate 3D reconstruction of fetal anatomy for lower limbs and pelvis but has to be improved for brain model since midline structures were not amendable for analysis. These results need to be confirmed with larger series of specimens at different stages of development., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Background: The epididymis is a popular research topic in urology and reproduction., Objectives: To explore and identify the anatomical characteristics of the epididymis based on histology, proteomics, and 3D reconstruction of epididymal tubules., Materials and Methods: A 3D reconstruction of epididymal tubules was generated based on 7-μm-thick transverse serial sections of an epididymis. The proteins in the subcompartments of the epididymis were obtained and analyzed by non-labeled sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH MS). Protein function, signaling pathways, protein expression, and the histology in different subcompartments were analyzed., Results: The caput (Cap), corpus (Cor), and cauda (Cau) of the epididymis were divided into 6, 10, and 4 subcompartments, respectively, and the subcompartment between the Cap and Cor is mixed together. A total of 3411 proteins were identified, and 854 proteins were accurately quantified after screening. When the subcompartment Cap 5 transitioned to Cap 6 and Cap 6 to Cap 7, 87 and 52 proteins were upregulated and 14 and 7 proteins were downregulated, respectively. The Cor 9 transition to Cau 1 was marked by 230 proteins that were downregulated, while 74 proteins were upregulated. At the junction of the cauda and the vas deferens, 57 proteins were downregulated, and 410 proteins were upregulated. Cap 6 histology was consistent with that of Cor 1., Discussion and Conclusion: The epididymis contains distinct connective tissue septa that can be identified under a surgical tabletop microscope, enabling it to be divided into 20 subcompartments., (© 2020 American Society of Andrology and European Academy of Andrology.)

Many neurological diseases present with substantial genetic and phenotypic heterogeneity, making assessment of these diseases challenging. This has led to ineffective treatments, significant morbidity, and high mortality rates for patients with neurological diseases, including brain cancers and neurodegenerative disorders. Improved understanding of this heterogeneity is necessary if more effective treatments are to be developed. We describe a new method to measure phenotypic heterogeneity across the whole rodent brain at multiple spatial scales. The method involves co-registration and localized comparison of in vivo radiologic images (e.g. MRI, PET) with ex vivo optical reporter images (e.g. labeled cells, molecular targets, microvasculature) of optically cleared tissue slices. Ex vivo fluorescent images of optically cleared pathology slices are acquired with a preclinical in vivo optical imaging system across the entire rodent brain in under five minutes, making this methodology practical and feasible for most preclinical imaging labs. The methodology is applied in various examples demonstrating how it might be used to cross-validate and compare in vivo radiologic imaging with ex vivo optical imaging techniques for assessing hypoxia, microvasculature, and tumor growth.

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol's critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

Although past decades have witnessed significant reductions in mortality of heart failure together with advances in our understanding of its cellular, molecular, and whole-heart features, a lot of basic cardiac research still fails to translate into clinical practice. In this review we examine myocardial slices, a novel model in the translational arena. Myocardial slices are living ultra-thin sections of heart tissue. Slices maintain the myocardium's native function (contractility, electrophysiology) and structure (multicellularity, extracellular matrix) and can be prepared from animal and human tissue. The discussion begins with the history and current advances in the model, the different interlaboratory methods of preparation and their potential impact on results. We then contextualize slices' advantages and limitations by comparing it with other cardiac models. Recently, sophisticated methods have enabled slices to be cultured chronically in vitro while preserving the functional and structural phenotype. This is more timely now than ever where chronic physiologically relevant in vitro platforms for assessment of therapeutic strategies are urgently needed. We interrogate the technological developments that have permitted this, their limitations, and future directions. Finally, we look into the general obstacles faced by the translational field, and how implementation of research systems utilizing slices could help in resolving these., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Serial section electron microscopy (ssEM), a technique where volumes of tissue can be anatomically reconstructed by imaging consecutive tissue slices, has proven to be a powerful tool for the investigation of brain anatomy. Between the process of cutting the slices, or "sections," and imaging them, however, handling 10°-10 6 delicate sections remains a bottleneck in ssEM, especially for batches in the "mesoscale" regime, i.e., 10 2 -10 3 sections. We present a tissue section handling device that transports and positions sections, accurately and repeatability, for automated, robotic section pick-up and placement onto an imaging substrate. The device interfaces with a conventional ultramicrotomy diamond knife, accomplishing in-line, exact-constraint trapping of sections with 100-μm repeatability. An associated mathematical model includes capillary-based and Stokes-based forces, accurately describing observed behavior and fundamentally extends the modeling of water-air interface forces. Using the device, we demonstrate and describe the limits of reliable handling of hundreds of slices onto a variety of electron and light microscopy substrates without significant defects ( n  = 8 datasets composed of 126 serial sections in an automated fashion with an average loss rate and throughput of 0.50% and 63 s/section, respectively. In total, this work represents an automated mesoscale serial sectioning system for scalable 3D-EM connectomics., (Copyright © 2020 Lee et al.)