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State-of-the-art tissue-clearing methods provide subcellular-level optical access to intact tissues from individual organs and even to some entire mammals. When combined with light-sheet microscopy and automated approaches to image analysis, existing tissue-clearing methods can speed up and may reduce the cost of conventional histology by several orders of magnitude. In addition, tissue-clearing chemistry allows whole-organ antibody labelling, which can be applied even to thick human tissues. By combining the most powerful labelling, clearing, imaging and data-analysis tools, scientists are extracting structural and functional cellular and subcellular information on complex mammalian bodies and large human specimens at an accelerated pace. The rapid generation of terabyte-scale imaging data furthermore creates a high demand for efficient computational approaches that tackle challenges in large-scale data analysis and management. In this Review, we discuss how tissue-clearing methods could provide an unbiased, system-level view of mammalian bodies and human specimens and discuss future opportunities for the use of these methods in human neuroscience.Tissue-clearing methods are now allowing 3D imaging of intact tissues and some entire mammals. In this Review, Ueda and colleagues discuss the various tissue-clearing methods, related techniques and data analysis and management, as well as the application of these methods in neuroscience.

Abstract Objective Develop a consensus for the nomenclature and definition of adrenal histopathologic features in unilateral primary aldosteronism (PA). Context Unilateral PA is the most common surgically treated form of hypertension. Morphologic examination combined with CYP11B2 (aldosterone synthase) immunostaining reveals diverse histopathologic features of lesions in the resected adrenals. Patients and Methods Surgically removed adrenals (n = 37) from 90 patients operated from 2015 to 2018 in Munich, Germany, were selected to represent the broad histologic spectrum of unilateral PA. Five pathologists (Group 1 from Germany, Italy, and Japan) evaluated the histopathology of hematoxylin-eosin (HE) and CYP11B2 immunostained sections, and a consensus was established to define the identifiable features. The consensus was subsequently used by 6 additional pathologists (Group 2 from Australia, Brazil, Canada, Japan, United Kingdom, United States) for the assessment of all adrenals with disagreement for histopathologic diagnoses among group 1 pathologists. Results Consensus was achieved to define histopathologic features associated with PA. Use of CYP11B2 immunostaining resulted in a change of the original HE morphology-driven diagnosis in 5 (14%) of 37 cases. Using the consensus criteria, group 2 pathologists agreed for the evaluation of 11 of the 12 cases of disagreement among group 1 pathologists. Conclusion The HISTALDO (histopathology of primary aldosteronism) consensus is useful to standardize nomenclature and achieve consistency among pathologists for the histopathologic diagnosis of unilateral PA. CYP11B2 immunohistochemistry should be incorporated into the routine clinical diagnostic workup to localize the likely source of aldosterone production.

To establish a histopathological scoring system for changes in subchondral bone in murine models of knee osteoarthritis (OA), three key parameters, subchondral bone plate (Subcho.BP) consisting of the combination of Subcho.BP.thickness (Subcho.BP.Th) and angiogenesis, bone volume (BV/TV) and osteophytes, were selected. The new grading system was tested in two mouse OA models, (1) senescence accelerated mouse (SAM)-prone 8 (SAMP8) as spontaneous OA model with SAM-resistant 1 (SAMR1) as control; (2) destabilization of the medial meniscus in C57BL/6 mice as surgical OA model. Results of the spontaneous OA model showed that Subcho.BP.Th was significantly wider, angiogenesis was greater, and BV/TV was higher in SAMP8 than SAMR1. Notably, subchondral bone score was dramatically higher in SAMP8 at 6 weeks than SAMR1, while OARSI cartilage scores became higher only at 14 weeks. In the surgical OA model, the results were similar to the spontaneous OA model, but osteophytes appeared earlier. There were strong correlations both in Subcho.BP.Th and BV/TV between this scoring system and µCT (r = 0.89, 0.84, respectively). Inter-rater reliabilities for each parameter using this system were more than 0.943. We conclude that this new histopathological scoring system is readily applicable for evaluating the early changes in aging and OA-affected murine subchondral bone.

Diffusion MRI is an exquisitely sensitive probe of tissue microstructure, and is currently the only non-invasive measure of the brain's fibre architecture. As this technique becomes more sophisticated and microstructurally informative, there is increasing value in comparing diffusion MRI with microscopic imaging in the same tissue samples. This study compared estimates of fibre orientation dispersion in white matter derived from diffusion MRI to reference measures of dispersion obtained from polarized light imaging and histology. Three post-mortem brain specimens were scanned with diffusion MRI and analyzed with a two-compartment dispersion model. The specimens were then sectioned for microscopy, including polarized light imaging estimates of fibre orientation and histological quantitative estimates of myelin and astrocytes. Dispersion estimates were correlated on region – and voxel-wise levels in the corpus callosum, the centrum semiovale and the corticospinal tract. The region-wise analysis yielded correlation coefficients of r = 0.79 for the diffusion MRI and histology comparison, while r = 0.60 was reported for the comparison with polarized light imaging. In the corpus callosum, we observed a pattern of higher dispersion at the midline compared to its lateral aspects. This pattern was present in all modalities and the dispersion profiles from microscopy and diffusion MRI were highly correlated. The astrocytes appeared to have minor contribution to dispersion observed with diffusion MRI. These results demonstrate that fibre orientation dispersion estimates from diffusion MRI represents the tissue architecture well. Dispersion models might be improved by more faithfully incorporating an informed mapping based on microscopy data.

Chromogenic immunohistochemistry (immunostaining using an enzyme-labeled probe) is an essential histochemical technique for analyzing pathogenesis and making a histopathological diagnosis in routine pathology services. In neoplastic lesions, immunohistochemistry allows the study of specific clinical and biological features such as histogenesis, behavioral characteristics, therapeutic targets, and prognostic biomarkers. The needs for appropriate and reproducible methods of immunostaining are prompted by technical development and refinement, commercial availability of a variety of antibodies, advanced applicability of immunohistochemical markers, accelerated analysis of clinicopathological correlations, progress in molecular targeted therapy, and the expectation of advanced histopathological diagnosis. However, immunostaining does have various pitfalls and caveats. Pathologists should learn from previous mistakes and failures and from results indicating false positivity and false negativity. The present review article describes various devices, technical hints, and trouble-shooting guides to keep in mind when performing immunostaining.

Stain colour estimation is a prominent factor of the analysis pipeline in most of histology image processing algorithms. Providing a reliable and efficient stain colour deconvolution approach is fundamental for robust algorithm. In this paper, we propose a novel method for stain colour deconvolution of histology images. This approach statistically analyses the multi-resolutional representation of the image to separate the independent observations out of the correlated ones. We then estimate the stain mixing matrix using filtered uncorrelated data. We conducted an extensive set of experiments to compare the proposed method to the recent state of the art methods and demonstrate the robustness of this approach using three different datasets of scanned slides, prepared in different labs using different scanners.

The secondary tissues of woody plants consist of fragile cells and rigid cell walls. However, the structures are easily damaged during mechanical cross-sectioning for electron microscopy analysis. Broad argon ion beam (BIB) milling is commonly employed for scanning electron microscopy (SEM) of hard materials to generate a large and distortion-free cross-section. However, BIB milling has rarely been used in plant science. In the present study, SEM combined with BIB milling was validated as an accurate tool for structural observation of secondary woody tissues of two samples, living pine ( Pinus densiflora ) and high-density oak wood ( Quercus phillyraeoides ), and compared with classical microtome cross-sectioning. The BIB milling method does not require epoxy resin embedding because of prior chemical fixation and critical point drying of the sample, thus producing a three-dimensional image. The results showed that xylem structures were well-preserved in their natural state in the BIB-milled cross-section compared with the microtome cross-section. The observations using SEM combined with BIB milling were useful for wide-area imaging of both hard and soft plant tissues, which are difficult to observe with transmitted electron microscopy because it is difficult to obtain sections of such tissues, particularly those of fragile reaction woods.

ObjectivePerform a prospective study based on sequential clinical, endoscopic, and histologic evaluations of the foregut late after laparoscopic sleeve gastrectomy (LSG) in obese patients.SummaryAfter LSG, several studies have suggested an increase in the incidence of clinical gastroesophageal reflux (GERD) while others have reported an improvement but based mainly on clinical questionnaires.MethodsProspective study of 104 consecutive patients submitted to LSG. Several postoperative endoscopic and histologic evaluations of the esophagogastric junction (EGJ) and the gastric tube (GT) were performed and correlated with symptomatic findings.ResultsAccording to clinical preoperative findings, patients were divided into non-refluxers (Group I) and refluxers (Group II). Seven patients were unreachable, leaving 97 (93%) for late evaluation. Among Group I, 58.5% developed de novo GERD, while in Group II just 13.6% showed the disappearance of them. Endoscopic evaluations showed progressive deterioration of the EGJ in Group I, with the development of erosive esophagitis (EE), hiatal hernia (HH), and dilated cardia in a large proportion of them. In the GT, the presence of bile was seen in 40%, and an open immobile pylorus was detected in 82%. Short-segment Barrett’s esophagus (BE) appeared in 4%.ConclusionsPatients submitted to LSG showed a significant and progressive increase in the presence of “de novo” GERD. Also, an increased duodenogastric reflux was seen through an open and immobile pylorus. Therefore, based on these results, it seems like LSG is a “pro-reflux” surgical procedure, which should be continuously evaluated late after surgery.

The histological characterization of the intestinal mucus layer is important for many scientific experiments investigating the interaction between intestinal microbiota, mucosal immune response and intestinal mucus production. The aim of this study was to examine and compare different fixation protocols for displaying and quantifying the intestinal mucus layer in piglets and to test which histomorphological parameters may correlate with the determined mucus layer thickness. Jejunal and colonal tissue samples of weaned piglets (n=10) were either frozen in liquid nitrogen or chemically fixed using methacarn solution. The frozen tissue samples were cryosectioned and subsequently postfixed using three different postfixatives: paraformaldehyde vapor, neutrally buffered formalin solution and ethanol solution. After dehydration, methacarn fixed tissues were embedded in paraffin wax. Both sections of cryopreserved and methacarn fixed tissue samples were stained with Alcian blue (AB)-PAS followed by the microscopically determination of the mucus layer thickness. Different pH values of the Alcian Blue staining solution and two mucus layer thickness measuring methods were compared. In addition, various histomorphological parameters of methacarn fixed tissue samples were evaluated including the number of goblet cells and the mucin staining area. Cryopreservation in combination with chemical postfixation led to mucus preservation in the colon of piglets allowing mucus thickness measurements. Mucus could be only partly preserved in cryosections of the jejunum impeding any quantitative description of the mucus layer thickness. The application of different postfixations, varying pH values of the AB solution and different mucus layer measuring methods led to comparable results regarding the mucus layer thickness. Methacarn fixation proved to be unsuitable for mucus depiction as only mucus patches were found in the jejunum or a detachment of the mucus layer from the epithelium was observed in the colon. Correlation analyses revealed that the proportion of the mucin staining area per crypt area (relative mucin staining) measured in methacarn fixed tissue samples corresponded to the colonal mucus layer thickness determined in cryopreserved tissue samples. In conclusion, the results showed that cryopreservation using liquid nitrogen followed by chemical postfixation and AB-PAS staining led to a reliable mucus preservation allowing a mucus thickness determination in the colon of pigs. Moreover, the detected relative mucin staining area may serve as a suitable histomorphological parameter for the assessment of the intestinal mucus layer thickness. The findings obtained in this study can be used for the implementation of an improved standard for the histological description of the mucus layer in the colon of pigs.