Colorimetric histology using plasmonically active microscope slides.

The human eye can distinguish as many as 10,000 different colours but is far less sensitive to variations in intensity1, meaning that colour is highly desirable when interpreting images. However, most biological samples are essentially transparent, and nearly invisible when viewed using a standard optical microscope2. It is therefore highly desirable to be able to produce coloured images without needing to add any stains or dyes, which can alter the sample properties. Here we demonstrate that colorimetric histology images can be generated using full-sized plasmonically active microscope slides. These slides translate subtle changes in the dielectric constant into striking colour contrast when samples are placed upon them. We demonstrate the biomedical potential of this technique, which we term histoplasmonics, by distinguishing neoplastic cells from normal breast epithelium during the earliest stages of tumorigenesis in the mouse MMTV-PyMT mammary tumour model. We then apply this method to human diagnostic tissue and validate its utility in distinguishing normal epithelium, usual ductal hyperplasia, and early-stage breast cancer (ductal carcinoma in situ). The colorimetric output of the image pixels is compared to conventional histopathology. The results we report here support the hypothesis that histoplasmonics can be used as a novel alternative or adjunct to general staining. The widespread availability of this technique and its incorporation into standard laboratory workflows may prove transformative for applications extending well beyond tissue diagnostics. This work also highlights opportunities for improvements to digital pathology that have yet to be explored.

Digital Pathology Initiatives and Experience of a Large Academic Institution During the Coronavirus Disease 2019 (COVID-19) Pandemic.

CONTEXT.­: Pathology practices have begun integrating digital pathology tools into their routine workflow. During 2020, the coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged as a pandemic, causing a global health crisis that significantly affected the world population in several areas, including medical practice, and pathology was no exception. OBJECTIVE.­: To summarize our experience in implementing digital pathology for remote primary diagnosis, education, and research during this pandemic. DESIGN.­: We surveyed our pathologists (all subspecialized) and trainees to gather information about their use of digital pathology tools before and during the pandemic. Quality assurance and slide distribution data were also examined. RESULTS.­: During the pandemic, the widespread use of digital tools in our institution allowed a smooth transition of most clinical and academic activities into remote with no major disruptions. The number of pathologists using whole slide imaging (WSI) for primary diagnosis increased from 20 (62.5%) to 29 (90.6%) of a total of 32 pathologists, excluding renal pathology and hematopathology, during the pandemic. Furthermore, the number of pathologists exclusively using whole slide imaging for primary diagnosis also increased from 2 (6.3%) to 5 (15.6%) during the pandemic. In 35 (100%) survey responses from attending pathologists, 21 (60%) reported using whole slide imaging for remote primary diagnosis following the Centers for Medicare and Medicaid Services waiver. Of these 21 pathologists, 18 (86%) responded that if allowed, they will continue using whole slide imaging for remote primary diagnosis after the pandemic. CONCLUSIONS.­: The pandemic served as a catalyst to pathologists adopting a digital workflow into their daily practice and realizing the logistic and technical advantages of such tools.

Recent developments of novel matrices and on-tissue chemical derivatization reagents for MALDI-MSI.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a fast-growing technique for visualization of the spatial distribution of the small molecular and macromolecular biomolecules in tissue sections. Challenges in MALDI-MSI, such as poor sensitivity for some classes of molecules or limited specificity, for instance resulting from the presence of isobaric molecules or limited resolving power of the instrument, have encouraged the MSI scientific community to improve MALDI-MSI sample preparation workflows with innovations in chemistry. Recent developments of novel small organic MALDI matrices play a part in the improvement of image quality and the expansion of the application areas of MALDI-MSI. This includes rationally designed/synthesized as well as commercially available small organic molecules whose superior matrix properties in comparison with common matrices have only recently been discovered. Furthermore, on-tissue chemical derivatization (OTCD) processes get more focused attention, because of their advantages for localization of poorly ionizable metabolites and their' in several cases' more specific imaging of metabolites in tissue sections. This review will provide an overview about the latest developments of novel small organic matrices and on-tissue chemical derivatization reagents for MALDI-MSI. Graphical abstract.

Spatially resolved absolute quantitation in thin tissue by mass spectrometry.

Mass spectrometry (MS) has become the de facto tool for routine quantitative analysis of biomolecules. MS is increasingly being used to reveal the spatial distribution of proteins, metabolites, and pharmaceuticals in tissue and interest in this area has led to a number of novel spatially resolved MS technologies. Most spatially resolved MS measurements are qualitative in nature due to a myriad of potential biases, such as sample heterogeneity, sampling artifacts, and ionization effects. As applications of spatially resolved MS in the pharmacological and clinical fields increase, demand has become high for quantitative MS imaging and profiling data. As a result, several varied technologies now exist that provide differing levels of spatial and quantitative information. This review provides an overview of MS profiling and imaging technologies that have demonstrated quantitative analysis from tissue. Focus is given on the fundamental processes affecting quantitative analysis in an array of MS imaging and profiling technologies and methods to address these biases.Graphical abstract.

Preparing Cell Smears for Staining.

Increasing use is being made of cell smears for cell-staining studies. Suspension cells can be attached to slides by drying, and cell smears can also be prepared from biopsy samples, such as needle aspirates, tissue scrapings, or freshly dissected tissues. In these procedures, a thin layer of cells is deposited on a dry slide by physical methods. The most important factor in obtaining good staining patterns is that the smear be only a single cell thick. Tissue smears do not preserve tissue architecture, but are useful for identifying pathological changes and infectious organisms in tissue samples. Cell smears are easily prepared and can be fixed readily by any of the methods used for attached cells.

4D-Printed Transformable Tube Array for High-Throughput 3D Cell Culture and Histology.

3D cell cultures are rapidly emerging as a promising tool to model various human physiologies and pathologies by closely recapitulating key characteristics and functions of in vivo microenvironment. While high-throughput 3D culture is readily available using multi-well plates, assessing the internal microstructure of 3D cell cultures still remains extremely slow because of the manual, laborious, and time-consuming histological procedures. Here, a 4D-printed transformable tube array (TTA) using a shape-memory polymer that enables massively parallel histological analysis of 3D cultures is presented. The interconnected TTA can be programmed to be expanded by 3.6 times of its printed dimension to match the size of a multi-well plate, with the ability to restore its original dimension for transferring all cultures to a histology cassette in order. Being compatible with microtome sectioning, the TTA allows for parallel histology processing for the entire samples cultured in a multi-well plate. The test result with human neural progenitor cell spheroids suggests a remarkable reduction in histology processing time by an order of magnitude. High-throughput analysis of 3D cultures enabled by this TTA has great potential to further accelerate innovations in various 3D culture applications such as high-throughput/content screening, drug discovery, disease modeling, and personalized medicine.

Accurate assessment of nonalcoholic fatty liver disease lesions in liver allograft biopsies by a smartphone platform: A proof of concept.

Macrovesicular steatosis (MS) is a major risk factor for liver graft failure after transplantation and pathological microscopic examination of a frozen tissue section remains the gold standard for its assessment. However, the latter requires an experienced in-house pathologist for correct and rapid diagnosis as well as specific equipment that is not always available. Smartphones, which are must-have tools for everyone, are very suitable for incorporation into promising technology to generate moveable diagnostic tools as for telepathology. The study aims to compare the microscopic assessment of nonalcoholic fatty liver disease (NAFLD) spectrum in liver allograft biopsies by a smartphone microscopy platform (DIPLE device) to standard light microscopy. Forty-two liver graft biopsies were evaluated in transmitted light, using an iPhone X and the microscopy platform. A significant correlation was reported between the two different approaches for graft MS assessment (Spearman's correlation coefficient: r = .93; p < .001) and for steatohepatitis feature (r = .56; p < .001; r = .45; p < .001). Based on these findings, a smartphone integrated with a cheap microscopy platform can achieve adequate accuracy in the assessment of NAFLD in liver graft and could be used as an alternative to standard light microscopy when the latter is unavailable.

A safe and reusable imaging chamber compatible with organic solvents.

High refractive index organic solvents are commonly used as an imaging medium in tissue clearing approaches. While effective, such solvents provide serious concerns for the safety of users and the equipment, especially in a central microscopy unit. To overcome these concerns, we have developed a large and reusable imaging chamber compatible with the universal mounting frame AK (PeCon GmbH). This chamber is easy to assemble and significantly improves the working environment in a central microscopy unit, where hazardous chemicals could negatively affect equipment and people. To encourage the uptake of these chambers, the design is made publicly available for download.

Tissue clearing and its applications in neuroscience.

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.

Introducción al uso de imágenes digitales en formato web en el aprendizaje de la histología humana / Introduction to the use of digital images in a web format in learning human histology

INTRODUCCIÓN: La microscopia virtual se ha convertido en un recurso educativo alternativo para la enseñanza de la organización estructural de células, tejidos y órganos. Su uso mediante el acceso a páginas web de distintas instituciones actúa como refuerzo y sirve como material adicional combinado con el uso de la microscopia óptica convencional en las prácticas de la asignatura de Histología. OBJETIVO: Evaluar el interés de los alumnos de la asignatura Histología de Sistemas del 2.° curso del grado en Medicina por las imágenes virtuales procedentes de páginas web como complemento educativo. MATERIAL Y MÉTODO: El estudio se realizó sobre una muestra de 156 estudiantes del 2.° curso de Medicina mediante un acercamiento cuantitativo para realizar un análisis descriptivo. RESULTADOS: Los conceptos con las valoraciones más altas fueron para utilidad y mejora en comparación con los atlas convencionales. CONCLUSIONES: Los datos presentados en este estudio muestran una preferencia, todavía tímida, de estudio de la Histología mediante aprendizaje electrónico, aunque junto con los recursos tradicionales

INTRODUCTION: Virtual microscopy has become an educational resource alternative for teaching the structural organization of cells, tissues and organs. Its use through access to web pages of different institutions acts as reinforcement and additional material combined with the use of conventional optical microscopy in histology practices. OBJECTIVE: To evaluate the interest of the second-year medical students for virtual images from educational web pages as a complement during histology study. MATERIAL AND METHOD: The study was conducted on 156 second-year medical students through a quantitative approach to perform a descriptive analysis. RESULTS: Usefulness and improvement related to conventional atlas achieved the highest scores. CONCLUSIONS: The data presented in this study demonstrate a preference, still timid, for the study of histology through electronic learning nevertheless along with the traditional educational tools

Generating brain matrices for zebra finch brain sectioning using three-dimensional printing technology.

BACKGROUND: The demand to sample brain regions in non-model species is increasing as more studies are integrating neurological data into behavioural, ecological or evolutionary analysis. However, the sampling operation is difficult for researchers without neuroscience background. It is also a challenge to collect neuroanatomical regions from animals in the field. NEW METHOD: Here we developed a new brain matrix for guiding researchers to section zebra finches' (Taeniopygia guttata) brains more steadily than by freehand trimming. Based on the 3D printing technology, we produced the zebra finch brain matrix from scratch. We also provided a step-by-step protocol to make brain matrices for any species with a brain size between that of shrews and dogs. RESULTS: The brain matrix could guide us to find the zebra finch's neuroanatomical landmarks, such as the hypothalamus, optic chiasm and occulomotor nerve. The matrix's channels near these landmarks could be used to section brains steadily and rapidly. COMPARISON WITH EXISTING METHODS: Standardized brain sectioning often requires expensive machines that may not be available in most laboratories or in the field, such as microtomes. In addition, machine-based trimming is time-consuming. Although commercial brain matrices can overcome these problems, they are only available for rats and mice. The brain matrices we developed are affordable to most laboratories and can be customised for non-model species in both lab and field experiments. CONCLUSIONS: The matrix-guided approach requires a relatively short training period and can allow researchers to properly and quickly sample brains, and thus will facilitate neuroscience-based interdisciplinary research.

MR Imaging-Histology Correlation by Tailored 3D-Printed Slicer in Oncological Assessment.

3D printing and reverse engineering are innovative technologies that are revolutionizing scientific research in the health sciences and related clinical practice. Such technologies are able to improve the development of various custom-made medical devices while also lowering design and production costs. Recent advances allow the printing of particularly complex prototypes whose geometry is drawn from precise computer models designed on in vivo imaging data. This review summarizes a new method for histological sample processing (applicable to e.g., the brain, prostate, liver, and renal mass) which employs a personalized mold developed from diagnostic images through computer-aided design software and 3D printing. Through positioning the custom mold in a coherent manner with respect to the organ of interest (as delineated by in vivo imaging data), the cutting instrument can be precisely guided in order to obtain blocks of tissue which correspond with high accuracy to the slices imaged. This approach appeared crucial for validation of new quantitative imaging tools, for an accurate imaging-histopathological correlation and for the assessment of radiogenomic features extracted from oncological lesions. The aim of this review is to define and describe 3D printing technologies which are applicable to oncological assessment and slicer design, highlighting the radiological and pathological perspective as well as recent applications of this approach for the histological validation of and correlation with MR images.

Antecedents, development, adoption, and application of Duchenne's trocar for histopathologic studies of neuromuscular disorders in the nineteenth century.

Duchenne de Boulogne was one of the founders of clinical neurology. His name has been eponymically linked to the most common form of muscular dystrophy, originally described by him as pseudo-hypertrophic muscular paralysis or myo-sclerotic paralysis. Obtaining muscle biopsy specimens was essential to gain insight about the etiopathogenensis of the disease. Duchenne invented a novel instrument: l'emporte-pièce histologique, also known as "Duchenne's trocar," to perform muscle biopsies. Following Duchenne's design and instructions, a Parisian company, Charrière, constructed the first instrument probably in 1864. That instrument was essential for Duchenne's description of the histopathological abnormalities typical of pseudo-hypertrophic muscular paralysis. The innovative needle-biopsy technique enabled physicians to analyze the spectrum of pathological changes at varying stages of different neuromuscular diseases. Duchenne's trocar was a forerunner of several types of modern muscle-biopsy needles. His invention was instrumental in the development of the disciplines of muscle pathology and clinical myology.

Automated and Reproducible Detection of Vascular Endothelial Growth Factor (VEGF) in Renal Tissue Sections.

BACKGROUND: Manual analysis of tissue sections, such as for pathological diagnosis, requires an analyst with substantial knowledge and experience. Reproducible image analysis of biological samples is steadily gaining scientific importance. The aim of the present study was to employ image analysis followed by machine learning to identify vascular endothelial growth factor (VEGF) in kidney tissue that had been subjected to hypoxia. METHODS: Light microscopy images of renal tissue sections stained for VEGF were analyzed. Subsequently, machine learning classified the cells as VEGF+ and VEGF- cells. RESULTS: VEGF was detected and cells were counted with high sensitivity and specificity. CONCLUSION: With great clinical, diagnostic, and research potential, automatic image analysis offers a new quantitative capability, thereby adding numerical information to a mostly qualitative diagnostic approach.

Randomized cross-over study and a qualitative analysis comparing virtual microscopy and light microscopy for learning undergraduate histopathology.

Background: Virtual microscopy (VM) use in teaching and learning is increasing worldwide. However, there is a paucity of information comparing it to light microscopy (LM) in learning undergraduate histopathology. We investigated whether VM or LM had a higher impact on student learning and performance in histopathology. In addition, we investigated whether students preferred VM over LM, and whether VM use provided a platform to fulfill the Accreditation Council for Graduate Medical Education core competencies. Materials and Methods: We used a sequential exploratory mixed method study design. A qualitative phase inquiring about student preference for VM or LM was followed by a randomized cross-over study. Student preference was measured by an online survey based on a Likert scale. In the cross-over study, students were randomized to either the VM or the LM arm, and their mean scores in standardized exams were compared after using VM and LM. Results: A total of 152 students completed the qualitative study and a total of 64 students participated in the cross-over study. Eighty-three percent (83%) of the students preferred to use VM over LM. Students who used VM scored significantly (P < 0.001) higher [(87.1% vs. 72.4%) and (85.3% vs. 76.1%)], respectively, in both phases of the cross-over study compared to those who used LM. Conclusions: Using VM to learn histopathology has significantly increased student learning and performance compared to using LM.

Histological comparison between laser microtome sections and ground specimens of implant-containing tissues.

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.

Method for Combined Observation of Serial Sections of Stented Arteries Embedded in Resin by Light Microscopy and Transmission Electron Microscopy.

We have developed a new method for obtaining information on whole tissues by light microscopy (LM) and ultrastructural features by transmission electron microscopy (TEM). This method uses serial sections of a stented artery embedded in resin. Stents were implanted in porcine coronary arteries in this study. The heart was perfusion fixed in a 2% paraformaldehyde and 1.25% glutaraldehyde mixed solution. The stented artery was then removed, fixed in 1% osmium, embedded in Quetol 651 resin, and sectioned serially. For LM, the black color of osmium was removed from the section by immersion in periodic acid and hydrogen peroxide after deplasticization. These sections were stained with hematoxylin and eosin and Elastica-Masson trichrome stain. For TEM, thin sections were re-embedded in Quetol 812 resin by the resupinate method and cut into ultrathin sections. A clear, fine structure was obtained, and organelles, microvilli, and cell junctions in the endothelium were easily observed. The combined observation of adjacent specimens by LM and TEM enabled us to relate histopathological changes in the millimeter scale to those in the nanometer scale.

Automatic deformable registration of histological slides to µCT volume data.

Localizing a histological section in the three-dimensional dataset of a different imaging modality is a challenging 2D-3D registration problem. In the literature, several approaches have been proposed to solve this problem; however, they cannot be considered as fully automatic. Recently, we developed an automatic algorithm that could successfully find the position of a histological section in a micro computed tomography (µCT) volume. For the majority of the datasets, the result of localization corresponded to the manual results. However, for some datasets, the matching µCT slice was off the ground-truth position. Furthermore, elastic distortions, due to histological preparation, could not be accounted for in this framework. In the current study, we introduce two optimization frameworks based on normalized mutual information, which enabled us to accurately register histology slides to volume data. The rigid approach allocated 81 % of histological sections with a median position error of 8.4 µm in jaw bone datasets, and the deformable approach improved registration by 33 µm with respect to the median distance error for four histological slides in the cerebellum dataset.

A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy.

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin sections of the specimen. Although preparing serial ultrathin sections is considered to be very difficult, it is rather easy if the proper method is used. In this paper, we show a step-by-step procedure for safely obtaining serial ultrathin sections of microorganisms. The key points of this method are: 1) to use the large part of the specimen and adjust the specimen surface and knife edge so that they are parallel to each other; 2) to cut serial sections in groups and avoid difficulty in separating sections using a pair of hair strands when retrieving a group of serial sections onto the slit grids; 3) to use a 'Section-holding loop' and avoid mixing up the order of the section groups; 4) to use a 'Water-surface-raising loop' and make sure the sections are positioned on the apex of the water and that they touch the grid first, in order to place them in the desired position on the grids; 5) to use the support film on an aluminum rack and make it easier to recover the sections on the grids and to avoid wrinkling of the support film; and 6) to use a staining tube and avoid accidentally breaking the support films with tweezers. This new method enables obtaining serial ultrathin sections without difficulty. The method makes it possible to analyze cell structures of microorganisms at high resolution in 3D, which cannot be achieved by using the automatic tape-collecting ultramicrotome method and serial block-face or focused ion beam scanning electron microscopy.