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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.

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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.

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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.

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For delayed crosslinking of waterborne epoxy varnishes, dicyandiamide (DICY) is often used as a latent curing agent. While, for amine-based curing agents such as diaminoethane (DAE), chemical interactions with metal oxides are well described, so far, no studies have been performed for DICY and waterborne epoxy varnishes. Hence, in this work X-ray photoelectron spectroscopy (XPS) was used to investigate reactions of DICY and varnishes with technical surfaces of Al, Zn, and Sn. To directly study the reaction of DICY with metal oxides, immersion tests in a boiling solution of DICY in pure water were performed. A clear indication of the formation of metal-organic complexes was deduced from the change in the N1s peak of DICY. To understand the interfacial interaction and consequently the interphase formation during coating of waterborne epoxy varnishes, advanced cryo ultra-low-angle microtomy (cryo-ULAM) was implemented. Interestingly, a comparable reaction mechanism and the formation of metal complexes were confirmed for varnishes. The coatings exhibited a pronounced enrichment of the DICY hardener at the metal oxide-polymer interface.

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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.

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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.

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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.

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There is a great need for the analysis of the chemical composition, structure, functional groups, and interactions at polymer-metal interfaces in terms of adhesion, corrosion, and insulation. Although atomic force microscopy-based infrared (AFM-IR) spectroscopy can provide chemical analysis with nanoscale spatial resolution, it generally requires to thin a sample to be placed on a substrate that has low absorption of infrared light and high thermal conductivity, which is often difficult for samples that contain hard materials such as metals. This study demonstrates that the combination of AFM-IR with low-angle microtomy (LAM) sample preparation can analyze buried polymer-metal interfaces with higher spatial resolution than that with the conventional sample preparation of a thick vertical cross-section. In the LAM of a polymer layer on a metal substrate, the polymer layer is tapered to be thin in the vicinity of the interface, and thus, sample thinning is not required. An interface between an epoxyacrylate layer and copper wire in a flexible printed circuit cable was measured using this method. A carboxylate interphase layer with a thickness of ∼130 nm was clearly visualized at the interface, and its spectrum was obtained without any signal contamination from the neighboring epoxyacrylate, which was difficult to achieve on a thick vertical cross-section. The combination of AFM-IR with LAM is a simple and useful method for high-spatial-resolution chemical analysis of buried polymer-metal interfaces.

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Objective:To explore the advantages and disadvantages of frozen section versus rapid paraffin section in the evaluations of donor organ.Methods:Five cases of donor liver and 8 cases of discarded donor kidney were collected from 2017 to 2021.Tissues were harvested and prepared by frozen section, rapid paraffin section and normal paraffin section.After hematoxylin-eosin (H&E) staining, the specimens of donor kidney/liver were evaluated by differential histopathological structures and donor quality scoring system.Results:Rapid paraffin section was similar to normal paraffin section in reflecting the proportion of glomerulosclerosis (18.6%±22.3%), arteriolar hyaline degeneration (43.7%±23.8%) and arteriolar stenosis (47.9%±29%). The proportion of glomerulosclerosis (0.8%±2.2%), arteriolar hyaline degeneration (4.9%±7.4%) and arteriolar stenosis (5.3%±7.5%) were lower in frozen sections than those in rapid paraffin sections.The diagnoses of hydropic degeneration and necrosis in donor liver were more accurate in rapid paraffin section.Conclusions:Rapid paraffin section is superior to frozen section in observing histopathological changes under microscope.Scoring of donor organ is more precise according to rapid paraffin section.

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Green infrastructures within sprawling cities provide essential ecosystem services, increasingly undermined by environmental stress. The main objective in this study was to relate the allocation patterns of NaCl contaminants to injury within foliage of lime trees mechanistically and distinguish between the effects of salt and other environmental stressors. Using field material representative of salt contamination levels in the street greenery of Riga, Latvia, the contribution of salt contaminants to structural and ultrastructural injury was analyzed, combining different microscopy techniques. On severely salt-polluted and dystrophic soils, the foliage of street lime trees showed foliar concentrations of Na/Cl up to 13,600/16,750 mg kg-1 but a still balanced nutrient content. The salt contaminants were allocated to all leaf blade tissues and accumulated in priority within mesophyll vacuoles, changing the vacuolar ionic composition at the expense of especially K and Ca. The size of mesophyll cells and vacuoles was increased as a function of NaCl concentration, suggesting impeded transpiration stream. In parallel, the cytoplasm showed degenerative changes, suggesting indirect stress effects. Hence, the lime trees in Riga showed tolerance to the dystrophic environmental conditions enhanced by salt pollution but their leaf physiology appeared directly impacted by the accumulation of contaminants within foliage.

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Raman imaging is a microspectroscopic approach revealing the chemistry and structure of plant cell walls in situ on the micro- and nanoscale. The method is based on the Raman effect (inelastic scattering) that takes place when monochromatic laser light interacts with matter. The scattered light conveys a change in energy that is inherent of the involved molecule vibrations. The Raman spectra are thus characteristic for the chemical structure of the molecules and can be recorded spatially ordered with a lateral resolution of about 300 nm. Based on thousands of acquired Raman spectra, images can be assessed using univariate as well as multivariate data analysis approaches. One advantage compared to staining or labeling techniques is that not only one image is obtained as a result but different components and characteristics can be displayed in several images. Furthermore, as every pixel corresponds to a Raman spectrum, which is a kind of "molecular fingerprint," the imaging results should always be evaluated and further details revealed by analysis (e.g., band assignment) of extracted spectra. In this chapter, the basic theoretical background of the technique and instrumentation are described together with sample preparation requirements and tips for high-quality plant tissue sections and successful Raman measurements. Typical Raman spectra of the different plant cell wall components are shown as well as an exemplified analysis of Raman data acquired on the model plant Arabidopsis. Important preprocessing methods of the spectra are included as well as single component image generation (univariate) and spectral unmixing by means of multivariate approaches (e.g., vertex component analysis).

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A vibrating microtome is widely used to produce good-quality sections of plant organs or tissues. This method allows for an improved preservation of antigenicity and structure and is compatible with most (immuno)cytochemical staining procedures.

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BACKGROUND: Coronary artery stenting has become a common procedure and cardiovascular pathology specimens containing these metallic stents are accordingly becoming common. Histologic examination of stented vessels is imperative, but special techniques are needed due to the presence of metal within the tissue. We describe a rapid and inexpensive method for preparing stented vascular specimens for routine histology suitable for use in almost any histology laboratory. DESIGN: After formalin fixation and decalcification, stented vascular segments were freeze-embedded and sectioned using a handheld power micro cutoff wheel tool into ~1 mm slices. Sections were allowed to thaw and the strut shards removed with fine forceps. No longer containing metal, the sections were processed for routine paraffin embedding, microtomy and staining. RESULTS: Histologic sections showed only minor tissue disruption around the stent struts. In our experience with 25 stented arteries (mean interval from implantation 5.6 years), the mean subjective section quality score was 4.1 out of 5. The position of each strut could easily be determined, along with neointimal in-stent restenosis and thrombosis. Local reaction to each strut could be surmised even if minor tissue disruption occurred. The entire process was completed in 2-3 days. The incremental cost over that of routine histology is nominal. CONCLUSION: This method for examining stented vascular segments histologically could readily be applied in most pathology laboratories and serves as a highly practical solution to dilemma of examining stents histologically.

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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.

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Assessing the internal morphology of Caenorhabditis elegans by a topographical technique like atomic force microscopy (AFM) is a challenging process. As a prerequisite for a successful image acquisition, direct contact between the structure of interest and the AFM probe needs to be established. To gain this insight into the morphology of cuticle and intestine in C. elegans before and after treatment with a tannin-enriched hydro-ethanolic extract from Combretum mucronatum, we developed an approach based on polyethylene glycol embedding, ultra-sectioning, de-embedding and hexamethyldisilazane-dehydration prior to measuring in ambient conditions by intermittent contact mode AFM. The used experimental protocol allowed a facile and fast insight into the ultrastructure of treated versus untreated C. elegans individuals, directly leading to the identification of treatment-associated morphological alterations in the cuticle but not the intestine of C. elegans. Additionally, the presented ultra-microtomy based protocol could allow future insight into virtually any tissue or organism by AFM.

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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.

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Formalin-fixed, paraffin-embedded tissue (FFPE) still plays an important role in biobanking, since it is comparatively easy to obtain and store in comparison to fresh frozen tissue. They are stored as paraffin or FFPE blocks. Unstained slides derived from FFPE blocks may be used for hematoxylin and eosin histology, special stains, immunohistochemistry, and chromogenic or fluorescent in situ hybridization. In addition, tissue scraped off FFPE slides or from scrolls of FFPE tissue may be used for molecular or proteomic analyses. Hematoxylin and eosin staining of FFPE sections reviewed by a pathologist are highly valuable to ensure the presence of adequate lesional cells for molecular and other analyses. Therefore, proper microtomy technique is essential in the preparation of formalin-fixed, paraffin-embedded tissue for biobanking purposes. Here we describe the process of cutting paraffin embedded sections using a rotary microtome. We also highlight the possible pitfalls that may arise and discuss how to avoid them.

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Evaluation of bone regeneration and peri-implant bone apposition can only be accomplished using laboratory techniques that allow assessment of decalcified hard tissue. It is known that 5-15µm thick sections can be prepared with the cutting-grinding technique, but their production causes a high material loss (≥0.5mm) between two sections and requires years of training and experience. With the development of the laser microtome it has become possible to cut decalcified bone without high sample material loss. Many scientific publications deal with the application possibilities of the individual methods So far, there is no comparison work between the cutting-grinding technique and laser microtomy. For this reason, new tissue sections were prepared by laser microtome and analyzed histologically from samples that had been previously been prepared by the cutting-grinding technique. Using both methods, it could be demonstrated that the different implants were completely surrounded by a connective tissue layer. In sections (50-100µm) produced by the routine cutting-grinding technique, magnifications up to 20× revealed no detailed histological information because cell structures could not be clearly identified. By contrast, laser microtome sections (10µm) revealed these information as e.g. osteocytes are already clearly visible at 10× magnification. Furthermore, the interface between implant and the surrounding bone could be clearly demonstrated due to visible demarcation between a capsule and connective tissue. At the histological level, laser microtome sections were clearly superior at thicknesses ≥30µm compared to sections produced by the cutting-grinding technique. In addition, laser microtomy has the advantages of time saving and markedly reduced sample loss, especially in cases of the production of serial sections.

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In 1927, the German popular science magazine Die Koralle published an article entitled "The Library of Brains." The article was about the Kaiser Wilhelm Institute for Brain Research in Berlin, established in 1914 as the continuation of the "Neurological Central Station" founded by Oskar Vogt (1870-1959) in 1898. The library metaphor plays on the huge collection of human and animal brains Oskar and his wife Cécile (1875-1862) had gathered over several decades. For examination, the brains were cut into paper-thin slices and embedded in paraffin: from one single organ, up to 30,000 slices could be extracted, which were to be "read" and studied like the pages of a book. In this chapter, we take the metaphor at face value, arguing that Vogt's institute actually functioned as a library. Numerous publications have emphasized the role of the Vogts and, in particular, of the brain collection for the constitution of modern neuroscience. The "library," however, has never been closely investigated. How was it designed? How was it filled? According to which criteria were the brains collected and ordered? How did the order and the collection itself reflect the Vogts' research program? Through a detailed investigation of the collection and the Vogt Archive, we will examine this "library" and reconstruct the order of the Vogt brains. The mutual relationship between collecting, sorting, examining and publishing about the brain will be discussed.

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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.