Search

Sudden wilt syndrome of chilli, an emerging and destructive disorder, is characterized by an abrupt appearance that has increased in Indo‐Gangetic alluvial plains over the past decade. The primary cause associated with the problem is water stagnation that creates hypoxic conditions in the root zone, and the plant mortality is further aggravated by soil‐borne fungi Fusarium oxysporum. The effects of sudden wilt on chilli roots were studied morphologically and histologically, using root scanning, optical microscopy and electron microscopy. Significant changes with respect to root length, projected area, number of root tips and root segments, average root diameter as well as estimated volume of roots were observed via root scanning of healthy and diseased root samples. Through optical microscopy of sections of the microtome‐cut root junctions displayed normal epidermis in healthy plants, while swollen cells indicated stress in the diseased plants. Cells of laterals and root tips in healthy plants were intact and stained strongly, but because of damaged tissues, cells in diseased plants were macerated and stained weakly. Root tips of healthy plants contained mitotic zones, whereas diseased root tips lacked mitotic zones. Electron microscopy studies revealed that sudden wilt had an adverse impact on xylem diameter, stele diameter, epidermal thickness and cortex thickness as evidenced by significantly lesser values of these parameters. The present study is the first systematic attempt to examine the morphological, histological and ultrastructural changes in chilli in response to sudden wilt syndrome. [ABSTRACT FROM AUTHOR]

Copyright of Die Pathologie is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Summary: In recent years, the popularity of X-ray computed tomography (CT), as a non-destructive imaging technique, has continued to expand in various research domains. In wood research, X-ray CT has proven to be useful for three-dimensional (3D) structural studies investigating the complex tissues of trees. Wood formation (i.e., xylogenesis) initiates in the cambium and a narrow zone of subsequent differentiation, both of which play key roles in plant growth and development. However, the dynamics of xylogenesis in eucalypts remain relatively poorly understood, in large part due to challenges in sampling, imaging, and characterizing the cambium. Therefore, the aim of this study was to present a workflow to evaluate the feasibility of using X-ray CT to characterize and quantify the structural properties of the cambium in eucalypts. The growth responses of Corymbia hybrid seedlings, exposed to either irrigated or droughted conditions, was investigated by monitoring the structural development of the cambium. To track microstructural changes in the cambium, the same seedlings were imaged with X-ray micro-CT (μ CT) one day before the treatments and again six days after the respective treatments. After the last X-ray μ CT scan, X-ray nano-CT was also applied. Using image analysis techniques, the morphological characteristics of the cambium could be determined. X-ray μ CT displayed a larger, thicker cambial zone in irrigated plants, while a much thinner cambium was visible in droughted seedlings. X-ray nano-CT revealed that droughted plants were associated with a significantly (p ⩽ 0.05) smaller cambium volume containing smaller cells, compared to the cambium of irrigated plants. Light microscopy was used to validate the CT results and demonstrated no significant (p > 0.05) difference in the cambium width and cell diameter obtained from the two respective CT techniques. The findings of this study proved X-ray CT to be a valuable tool for examining the effect of changing environmental conditions on the complex cambium structure of Corymbia hybrid seedlings. [ABSTRACT FROM AUTHOR]

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.

Semiconductor quantum dots (QDs) have demonstrated utility in long-term single particle tracking of membrane proteins in live cells in culture. To extend the superior optical properties of QDs to more physiologically relevant cell platforms, such as acute brain slices, we examine the photophysics of compact ligand-conjugated CdSe/CdS QDs using both ensemble and single particle analysis in brain tissue media. We find that symmetric core passivation is critical for both photostability in oxygenated media and for prolonged single particle imaging in brain slices. We then demonstrate the utility of these QDs by imaging single dopamine transporters in acute brain slices, achieving 20 nm localization precision at 10 Hz frame rates. These findings detail design requirements needed for new QD probes in complex living environments, and open the door to physiologically relevant studies that capture the utility of QD probes in acute brain slices.

A longstanding limitation of imaging with serial block-face scanning electron microscopy is specimen surface charging. This charging is largely due to the difficulties in making biological specimens and the resins in which they are embedded sufficiently conductive. Local accumulation of charge on the specimen surface can result in poor image quality and distortions. Even minor charging can lead to misalignments between sequential images of the block-face due to image jitter. Typically, variable-pressure SEM is used to reduce specimen charging, but this results in a significant reduction to spatial resolution, signal-to-noise ratio and overall image quality. Here we show the development and application of a simple system that effectively mitigates specimen charging by using focal gas injection of nitrogen over the sample block-face during imaging. A standard gas injection valve is paired with a precisely positioned but retractable application nozzle, which is mechanically coupled to the reciprocating action of the serial block-face ultramicrotome. This system enables the application of nitrogen gas precisely over the block-face during imaging while allowing the specimen chamber to be maintained under high vacuum to maximise achievable SEM image resolution. The action of the ultramicrotome drives the nozzle retraction, automatically moving it away from the specimen area during the cutting cycle of the knife. The device described was added to a Gatan 3View system with minimal modifications, allowing high-resolution block-face imaging of even the most charge prone of epoxy-embedded biological samples.

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.)

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 BaF2 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. Graphical Abstract [ABSTRACT FROM AUTHOR]

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.)

The "Crucifixion with the twelve Apostles", a unique Coptic embroidered panel, was on display at the Benaki Museum (Athens, Greece). The representation of the "Crucifixion" with Christ in the center and six Apostles on either side, standing next to each other in frontal poses, is quite a rare one. This rare iconographic image of the twelve Apostles could be linked to the Ascension or the Pentecost. This unique representation of the Crucifixion with the twelve Apostles, which also involves the Ascension, is a one-of-a-kind compositional formula representing Christ's Death as a triumph over Death, emphasizing, along with the other factors, its non-Chalcedonic origin. Moreover, the interpretation of an inscription, written in at least three languages embroidered in black silk thread, is a matter which confuses the issue even more. In the present study, we will attempt a comprehensive investigation, a detailed description, and interpretation of this rare iconography, based on written and iconographic evidence traced in the history of art heritage objects. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Correlative fluorescence light microscopy and electron microscopy allows the imaging of spatial distributions of specific biomolecules in the context of cellular ultrastructure. Recent development of super-resolution fluorescence microscopy allows the location of molecules to be determined with nanometer-scale spatial resolution. However, correlative super-resolution fluorescence microscopy and electron microscopy (EM) still remains challenging because the optimal specimen preparation and imaging conditions for super-resolution fluorescence microscopy and EM are often not compatible. Here, we have developed several experiment protocols for correlative stochastic optical reconstruction microscopy (STORM) and EM methods, both for un-embedded samples by applying EM-specific sample preparations after STORM imaging and for embedded and sectioned samples by optimizing the fluorescence under EM fixation, staining and embedding conditions. We demonstrated these methods using a variety of cellular targets.

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.

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. [ABSTRACT FROM AUTHOR]

The mechanisms generating epileptic neuronal networks following insults such as severe seizures are unknown. We have previously shown that interfering with the function of the neuron-restrictive silencer factor (NRSF/REST), an important transcription factor that influences neuronal phenotype, attenuated development of this disorder. In this study, we found that epilepsy-provoking seizures increased the low NRSF levels in mature hippocampus several fold yet surprisingly, provoked repression of only a subset (∼10%) of potential NRSF target genes. Accordingly, the repressed gene-set was rescued when NRSF binding to chromatin was blocked. Unexpectedly, genes selectively repressed by NRSF had mid-range binding frequencies to the repressor, a property that rendered them sensitive to moderate fluctuations of NRSF levels. Genes selectively regulated by NRSF during epileptogenesis coded for ion channels, receptors, and other crucial contributors to neuronal function. Thus, dynamic, selective regulation of NRSF target genes may play a role in influencing neuronal properties in pathological and physiological contexts.

Researchers have used transmission electron microscopy (TEM) to make contributions to cell biology for well over 50 years, and TEM continues to be an important technology in our field. We briefly present for the neophyte the components of a TEM-based study, beginning with sample preparation through imaging of the samples. We point out the limitations of TEM and issues to be considered during experimental design. Advanced electron microscopy techniques are listed as well. Finally, we point potential new users of TEM to resources to help launch their project.

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. [ABSTRACT FROM AUTHOR]

Obtaining high-quality sections of the nail plate poses a significant challenge to histopathology technicians world over. Nail is a heavily keratotic hard tissue that tends to split or tear while sectioning when processed and embedded in a routine manner. Many agents such as phenol, alcohol, and thioglycolate have been tried for the purpose of softening a variety of experimental materials. However, there is no clear consensus on any single agent. The study was conducted with the aim of evaluating and comparing the role of various compounds as softening agents for nail biopsies with inflammatory disease. Thirty paraffin-embedded nail biopsies were subjected to four softening agents: distilled water (DIH20), 30% potassium hydroxide (KOH), hair removal cream, and fabric conditioner. The ease of sectioning, the incidence of juddering (i.e. 'venetian blind' effect), and the shattering of tissue were recorded. Hematoxylin and eosin-stained sections were examined microscopically. Sectioning was very easy after using fabric conditioner, with good quality sections, and hair removal cream produced comparable results. The incidence of juddered, shattered sections after using hair removal cream was considerably higher (63.33%) compared to fabric conditioner-treated nails (16.67%). Microtomy of nail biopsies was found to be easiest after using 30% KOH with moderate section quality. DIH2O could neither allow easier sectioning nor obtain good sections for interpretation. Fabric conditioner and hair removal cream proved to be the effective keratin softeners, while 30% KOH worked effectively when the nail plate alone was submitted for histological examination. [ABSTRACT FROM AUTHOR]

The locus nucleus (LC) is a multifunctional nucleus which is also the source of norepinephrine in the brain. To date, there is no simple and easy method to locate the small LC in brain sectioning. Here we report a fast, accurate, and easy-to-follow protocol for the localization of mice LC in frozen sectioning. After fixation and dehydration, the intact brains of adult mice were placed on a horizontal surface and vertically cut along the posterior margin of the bilateral cerebral cortex. In the coronal cutting plane, the aqueduct of midbrain can be seen easily with the naked eyes. After embedding the cerebellum part with optimal cutting temperature (OCT) compound, coronal brain slices were cut from the cutting plane, within 1 mm, the aqueduct of midbrain disappeared and the fourth ventricle appeared, then the brain slices contained LC and were collected. From the first collection, at ~200 μm, the noradrenergic neurons' most enriched brain slices can be collected. The tyrosine hydroxylase immunofluorescence staining confirmed that the localization of LC with this method is accurate and the noradrenergic neuron most abundant slices can be determined with this method. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate bonding penetration into different enamel substrates. Materials and Methods: Ten extracted human molars were mesiodistally sectioned. Buccal and lingual enamel surfaces were divided into four equal areas using sticky wax. The central two areas of each tooth (n = 20) were demineralized for 12 weeks using an acidic gel (pH 4.8). The lateral areas served as controls. After demineralization, ten specimens were remineralized in a saliva substitute for three weeks. An amine fluoride solution (Elmex Fluid) was applied on one half of each specimen before acid etching. After etching for 120 s, an enamel-bonding agent (Heliobond) containing 0.1% rhodamine was applied onto test and control areas, and was light cured for 60 s. Subsequently, the specimens were sectioned and tag length was determined using a confocal laser scanning microscope (CLSM). Results were statistically compared with ANOVA followed by Scheffe's and Bonferroni/Dunn post hoc tests. Results: With a mean penetration depth of 68 ± 22 μm, tags in demineralized enamel were significantly longer than in other groups (p ≤ 0.01). Penetration decreased significantly in remineralized areas or when fluoride was used (p ≤ 0.01), but was still significantly deeper than in control sites (p ≤ 0.01). Conclusion: Penetration of an unfilled resin into enamel was considerably influenced by the degree of dental hard tissue mineralization. Penetration was increased in demineralized enamel; however, remineralized enamel also allowed good penetration of the bonding agent. [ABSTRACT FROM AUTHOR]

Purpose: This study investigated the ultimate tensile strength (UTS) of human dentin as a function of tubule orientation and density. Materials and Methods: Slabs of dentin (ca 0.7 mm thick) were obtained from human third molars by either transversally or longitudinally sectioning the crowns with an Isomet saw. The slabs were gently trimmed to reduce the central area of the coronal dentin to a cross-sectional area of approximately 0.5 mm2. The longitudinally sectioned slabs were either trimmed from the mesial and distal sides or from the occlusal and pulpal aspects to permit the tensile load to be applied either parallel or perpendicular to the tubule orientation. The transversally sectioned specimens were obtained at several distances from the pulp and were used to evaluate the effects of tubule density. The trimmed specimens were tested in tension on a Kratos testing machine at 0 5 mm/min. After failure, the UTS of each was calculated and expressed in MPa. The fractured ends of the transversally sectioned specimens were viewed under SEM to calculate the number of tubules per mm2 at the site of fracture, and its relation with the UTS was investigated by regression analysis. Results: The UTS of dentin is higher when the load is applied perpendicular to the tubule orientation (80 ± 13 MPa) than when applied parallel to tubule orientation (58 ± 11 MPa, p < 0.05) There was a tendency for dentin to be weaker as the number of tubules at the site of fracture increased, although this relationship was not statistically significant (R2 = 0.051, P > 0.05). Conclusion: The UTS of dentin is dependent on the tubule direction. Dentin tends to be weaker as the number of tubules per area increases. [ABSTRACT FROM AUTHOR]

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).)

BackgroundThe purpose of this investigation was to quantify the amount of intralesional calcium detected by intravascular ultrasound (IVUS) compared with undecalcified histology in human arteries. This method preserves intralesional calcium and reduces sectioning artifacts, thereby providing an accurate measure of calcium plaque morphology.Methods and resultsTen arterial segments (5 coronary, 5 iliac) were obtained at autopsy. IVUS imaging was performed with a 4.9F catheter at an automated pullback rate of 1.0 mm/s. The undecalcified arteries were dehydrated in ascending alcohol and polymerized in glycol methylmethacrylate. The arteries were cut into 200-microm sections with an Isomet low-speed saw and stained with Goldner's trichrome. The lumen cross-sectional area, the calcium plaque cross-sectional area, the calcium plaque depth, length, and angle of arc of calcified plaque were measured from the IVUS images and histologic sections. In 24 selected cross sections, there were 38 separate calcium plaques. An independent observer correctly identified 34 of 38 calcified plaques for a sensitivity of 89% and specificity of 97%. The total mean calcified plaque cross-sectional area measured from histology was 4.6 +/- 4.1 mm2 compared with 2.8 +/- 2.3 mm2 by IVUS (P =.002). Plaque depth measured by histology was 1.2 +/- 0.4 mm versus 0.7 +/- 0.2 mm by IVUS (P =.001). The length of calcium plaques measured by histology was 3.6 +/- 1.78 mm versus 3.6 +/- 1.5 mm for IVUS (r = 0.79).ConclusionsIVUS accurately depicts circumferential calcified lesions with high sensitivity (89%) and specificity (97%). However, IVUS underestimates the total calcified plaque cross-sectional area by 39%. This is mainly because of the inability of the ultrasound to penetrate intralesional calcium, which leads to an underestimation of the depth of calcium by 45%.

Humans and machines both have an inherent error rate. As long as this is true, sub-optimal events will occur in the histology laboratory. The best approach to troubleshooting and remedying these events is to (1) understand the various theories of action behind histology procedures and stains then (2) apply a problem-solving mentality to develop a corrective action. These theories and problem solving strategies are presented in this review article. [ABSTRACT FROM AUTHOR]

The three-dimensional ultra-structure is the comprehensive structure that cannot be observed from a two-dimensional electron micrograph. Array tomography is one method for three-dimensional electron microscopy. In this method, to obtain consecutive cross sections of tissue, connected consecutive sections of a resin block are mounted on a flat substrate, and these are observed with scanning electron microscopy. Although array tomography requires some bothersome manual procedures to prepare specimens, a recent study has introduced some techniques to ease specimen preparation. In addition, array tomography has some advantages compared with other three-dimensional electron microscopy techniques. For example, sections on the substrate are stored semi-eternally, so they can be observed at different magnifications. Furthermore, various staining methods, including post-embedding immunocytochemistry, can be adopted. In the present review, the preparation of specimens for array tomography, including ribbon collection and the staining method, and the adaptability for correlative light and electron microscopy are discussed. [ABSTRACT FROM AUTHOR]

Analysis of the xylem vessels of Populus euphratica Oliv. (P. euphratica) branch anatomical characteristics is usually performed using microtomy. It is time-consuming to obtain an abundance of cross-sections using microtomy, and the sample was dried and sliced. The more effective technology for the analysis of the anatomical characteristics of P. euphratica fresh branch would be of significance for the study of the tolerance mechanism. In this paper, the xylem vessels structure of P. euphratica branches were investigated by high-resolution computed tomography(HRCT) and scanning electron microscope (SEM) to establish an efficient method to research the tolerance mechanism of P. euphratica. Analysis of the images allowed characterisation of the internal morphological and histology structural features of the fresh and dried branches of P. euphratica. Drying process caused distinct changes in the microstructure of the branches with shrinking in the more external regions and the interspace between vessels compared with fresh branches. The SEM slice of the same sample that clearly has a higher resolution than the HRCT slice, and the structure of the xylem vessels is distinct in the SEM. However, SEM is a destructive process that requires complex sample preparation, and the quality of the sample preparation and staining directly affect the resulting images. It can be concluded that the xylem vessel of P. euphratica branches can be observed using HRCT, which offers an effective means of obtaining the needed quantitative data on the tolerance mechanism of P. euphratica. [ABSTRACT FROM AUTHOR]

In the field of histopathology, the microtomy procedure yields thin sections of tissue embedded in paraffin blocks, then to be further processed for diagnostic purposes. Within microtomy, trimming is an initial but critical process in which the excess paraffin covering the tissue of interest is removed by continuous cutting routines, until the tissue is suitably exposed and ready for sectioning. Trimming is currently a time-consuming process that is manually held by technicians. In this paper, we present a method to automatize this process, by analyzing tissue block surface images resulting from each cyclic cutting routine. Two types of Convolutional Neural Networks (CNNs) were fine-tuned: one for binary segmentation, the other for multi-class classification tasks, by exploring and optimizing lightweight architectures to provide fast analytical results on cost-effective edge computing. Two sequential online conditions followed the CNNs to output the current stage of the block surface and rule if the trimming-end cutting routine was reached. We compared the results obtained through our method with the ones obtained by three skilled technicians processing 75 tissue blocks. The proposed method identified the trimming-end cutting routine approximately as accurate as the technicians did, yielding up to 90% of trimmed blocks of optimal quality and evidencing the potential of this tool in future automated trimming instruments. We deployed our method to an Edge TPU hardware accelerator to showcase its capability to provide immediate and objective results at every microtomy station applied with an ad hoc hardware, potentially guaranteeing a throughput 50% higher than manual trimming. [ABSTRACT FROM AUTHOR]

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.)

The histopathologic assessment of placenta is often marred by poorly fixed and processed sections. An audit conducted in the department resulted in 21% of placental blocks showing features of poor processing. A microwave-assisted fixation procedure was introduced using the Micromed KOS microwave oven (ABACUS-ALS, Brisbane, Australia) for 2 h at 45°C. This resulted in an improvement in the quality of the sections with a zero failure rate on a subsequent audit. [ABSTRACT FROM AUTHOR]

Non-scanning, single-shot, 3D integral microscopy with optical sectioning is presented. The method is based on the combination of Fourier-mode integral microscopy with a 3D deconvolution technique. Specifically, the refocused volume provided by a regular back-projection algorithm is 3D deconvolved with a synthetic 3D impulse response function that takes into account the number and positions of the elemental images. The use of this hybrid technique provides a stack of true-color depth-refocused images with significant gain of optical sectioning. The stack can be used, among other applications, to inspect inside the thick microscope specimen, to calculate collections of perspective views with fine angular resolution and extended full parallax, and also to display 3D images in an integral monitor. The method here presented is validated with both simulation and experimental data. [ABSTRACT FROM AUTHOR]

Background Parasitoid wasps of the genus Pteromalus play an important role in biological pest control, however, the genus includes a large number of cryptic species, which makes reliable identification difficult. The latest identification key dates back to Graham (1969) and since then many new species have been described and nomenclatural changes proposed. New information Here we present an interactive and fully illustrated identification key in Xper3 for 27 species of the Pteromalus albipennis species group as well as for 18 similar species. In addition to qualitative traits, a large set of body measurements is incorporated in the key. We also explored a new set of qualitative features on the propodeum and metasternum. During field work, a new species of the P. albipennis species group, P. capito Baur sp. n., could be reared from flower heads of Asteraceae, which is described here. It looks very similar to P. albipennis and P. cingulipes, however, several qualitative characters and body ratios distinguish it clearly from the most similar species. [ABSTRACT FROM AUTHOR]

Objective Deliberate C2 nerve root sectioning and its preservation have been described during posterior fusion for atlantoaxial dislocation (AAD). However, the associated outcomes have been less studied, especially in congenital AAD. Our objective was to study the clinical outcomes after C2 nerve root sectioning and the feasibility of C2 preservation in these patients. Methods The data from 190 patients were retrospectively studied. The decision to cut or preserve the C2 nerve root was determined by the preoperative radiologic findings and intraoperative suitability of its preservation. During follow-up, the patients were questioned about C2 nerve-related dysfunction. The pros and cons of sectioning or preservation of the C2 nerve root and the related feasibility factors are also described. Results Complex C1-C2 anatomy that required extensive dissection and drilling was seen in 139 patients. After C2 nerve root sectioning in 178 patients, none reported bothersome hypoesthesia, paresthesia, or dysesthesia that required medication. However, 9 patients (5.1%) developed nonhealing occipital ulcers and most required flap coverage or a skin graft. C2 nerve root preservation is feasible with an adequate inferior C1 lateral mass and normal-size ganglion. In patients with C1–occipital condyle hypoplasia, extremely oblique joints, spondyloptosis, incurved occiput, pseudofacets, and anomalous vertebral artery, preservation of the C2 nerve root is difficult. Conclusions The advantages of sectioning the C2 nerve root are many. However, a subset of patients is prone to morbid occipital neuropathic ulcers. After C2 nerve sectioning, one should be cautious of such complications. C2 nerve root preservation should be strongly considered for patients with favorable anatomy. Highlights • Sectioning or preservation of the C2 ganglion in posterior C1-C2 fusion is controversial. • We studied the clinical outcome after C2 nerve root sectioning in congenital AAD. • A few patients developed nonhealing occipital ulcers that were challenging to treat. • We also discussed the feasibility of C2 nerve root preservation in these patients. [ABSTRACT FROM AUTHOR]

Scanning electron microscopes have longer focal depths than transmission electron microscopes and enable visualization of the three-dimensional (3D) surface structures of specimens. While scanning electron microscopy (SEM) in biological research was generally used for the analysis of bulk specimens until around the year 2000, more recent instrumental advances have broadened the application of SEM; for example, backscattered electron (BSE) signals under low accelerating voltages allow block-face and section-face images of tissues embedded in resin to be acquired. This technical breakthrough has led to the development of novel 3D imaging techniques including focused ion beam SEM, serial-block face SEM and serial section SEM. Using these new techniques, the 3D shapes of cells and cell organelles have been revealed clearly through reconstruction of serial tomographic images. In this review, we address two modern SEM techniques: section-face imaging of resin-embedded tissue samples based on BSE observations, and serial section SEM for reconstruction of the 3D structures of cells and organelles from BSE-mode SEM images of consecutive ultrathin sections on solid substrates. [ABSTRACT FROM AUTHOR]

Abstract: Laser ablation machining or microtomy (LAM) is a relatively new approach to producing slide mounted sections of translucent materials. We evaluated the method with a variety of problems from the bone, joint and dental tissues fields where we require thin undecalcified and undistorted sections for correlative light microscopy (LM) and backscattered electron scanning electron microscopy (BSE SEM). All samples were embedded in poly‐methylmethacrlate (PMMA) and flat block surfaces had been previously studied by BSE‐SEM and confocal scanning light microscopy (CSLM). Most were also studied by X‐yay microtomography (XMT). The block surface is stuck to a glass slide with cyanoacrylate adhesive. Setting the section thickness and levelling uses inbuilt optical coherence tomographic imaging. Tight focusing of near‐infrared laser radiation in the sectioning plane gives extreme intensities causing photodisruption of material at the focal point. The laser beam is moved by a fast scanner to write a cutting line, which is simultaneously moved by an XY positioning unit to create a sectioning plane. The block is thereby released from the slide, leaving the section stuck to the slide. Light, wet polishing on the finest grade (4000 grit) silicon carbide polishing paper is used to remove a 1–2 μm thick damaged layer at the surface of the section. Sections produced by laser cutting are fine in quality and superior to those produced by mechanical cutting and can be thinner than the ‘voxel’ in most laboratory X‐ray microtomography systems. The present extensive pilot studies have shown that it works to produce samples which we can study by both light and electron microscopy. [ABSTRACT FROM AUTHOR]

Context.--Cell block preparation methods vary substantially across institutions and are frequently suboptimal. The growing importance of biomarker testing in the era of targeted therapies makes optimization of cell block preparation critically important. Objective.--To develop an improved cell block preparation method. Design.--Ex vivo fine-needle aspirates and scrapes from surgically resected tumors were used to develop an improved HistoGel (Thermo Fisher Scientific, Waltham, Massachusetts)-based cell block preparation method. Cellularity yield with the new versus the standard method was assessed in ex vivo split samples and in consecutive clinical fine-needle aspirates processed before (n = 100) and after (n = 100) the new method was implemented in our laboratory. Sufficiency of cell block material for potential molecular studies was estimated by manual cell quantitation. Results.--The key modification in the new method was pretreatment of the pelleted cells with 95% ethanol before the addition of HistoGel (HistoGel þ ethanol method). In addition, we optimized the melting conditions of HistoGel and added a dark, inorganic marker to the cell pellets to highlight the desired level of sectioning during microtomy. Cell blocks from ex vivo split samples showed that the HistoGel þ ethanol method yielded, on average, an 8.3- fold (range, 1--20) greater cellularity compared with the standard HistoGel-only method. After the switch from the standard HistoGel method to the modified method in our clinical practice, sufficiency of positive fine-needle aspirates for some molecular studies increased from 72% to 97% (P = .002). Conclusions.--We describe a simple and readily adopt- able modification of the HistoGel method, which results in substantial improvement in cell capture in cell blocks, leading to a significant increase in sufficiency for potential molecular and other ancillary studies. [ABSTRACT FROM AUTHOR]

The accuracy of quantitative stereological analysis tools such as the (physical) disector method substantially depends on the precise determination of the thickness of the analyzed histological sections. One conventional method for measurement of histological section thickness is to re-embed the section of interest vertically to its original section plane. The section thickness is then measured in a subsequently prepared histological section of this orthogonally re-embedded sample. However, the orthogonal re-embedding (ORE) technique is quite work- and time-intensive and may produce inaccurate section thickness measurement values due to unintentional slightly oblique (non-orthogonal) positioning of the re-embedded sample-section. Here, an improved ORE method is presented, allowing for determination of the factual section plane angle of the re-embedded section, and correction of measured section thickness values for oblique (non-orthogonal) sectioning. For this, the analyzed section is mounted flat on a foil of known thickness (calibration foil) and both the section and the calibration foil are then vertically (re-)embedded. The section angle of the re-embedded section is then calculated from the deviation of the measured section thickness of the calibration foil and its factual thickness, using basic geometry. To find a practicable, fast, and accurate alternative to ORE, the suitability of spectral reflectance (SR) measurement for determination of plastic section thicknesses was evaluated. Using a commercially available optical reflectometer (F20, Filmetrics®, USA), the thicknesses of 0.5 μm thick semi-thin Epon (glycid ether)-sections and of 1–3 μm thick plastic sections (glycolmethacrylate/ methylmethacrylate, GMA/MMA), as regularly used in physical disector analyses, could precisely be measured within few seconds. Compared to the measured section thicknesses determined by ORE, SR measures displayed less than 1% deviation. Our results prove the applicability of SR to efficiently provide accurate section thickness measurements as a prerequisite for reliable estimates of dependent quantitative stereological parameters. [ABSTRACT FROM AUTHOR]

Polarization sensitive optical coherence tomography (PSOCT) with serial sectioning has enabled the investigation of 3D structures in mouse and human brain tissue samples. By using intrinsic optical properties of back-scattering and birefringence, PSOCT reliably images cytoarchitecture, myeloarchitecture and fiber orientations. In this study, we developed a fully automatic serial sectioning polarization sensitive optical coherence tomography ( as -PSOCT) system to enable volumetric reconstruction of human brain samples with unprecedented sample size and resolution. The 3.5 μm in-plane resolution and 50 μm through-plane voxel size allow inspection of cortical layers that are a single-cell in width, as well as small crossing fibers. We show the abilities of as -PSOCT in quantifying layer thicknesses of the cerebellar cortex and creating microscopic tractography of intricate fiber networks in the subcortical nuclei and internal capsule regions, all based on volumetric reconstructions. as -PSOCT provides a viable tool for studying quantitative cytoarchitecture and myeloarchitecture and mapping connectivity with microscopic resolution in the human brain. [ABSTRACT FROM AUTHOR]