Large Tissue Sections Research Articles

Nova-ST: Nano-patterned ultra-dense platform for spatial transcriptomics

Spatial transcriptomics workflows using barcoded capture arrays are commonly used for resolving gene expression in tissues. However, existing techniques are either limited by capture array density or are cost prohibitive for large-scale atlasing. We present Nova-ST, a dense nano-patterned spatial transcriptomics technique derived from randomly barcoded Illumina sequencing flow cells. Nova-ST enables customized, low-cost, flexible, and high-resolution spatial profiling of large tissue sections. Benchmarking on mouse brain sections demonstrates significantly higher sensitivity compared to existing methods at a reduced cost.

Distribution of prostatic markers in glands of the female urethra and anterior vaginal wall-a rapid autopsy study.

There are varying reports of immunohistochemically detected prostatic marker protein distribution in glands associated with the female urethra that may be related to tissue integrity at the time of fixation. In this study we used tissue derived from rapid autopsies of female patients to determine the distribution of glandular structures expressing prostate-specific antigen (PSA) and prostate-specific acid phosphatase (PSAP) along the female urethra and in surrounding tissues, including the anterior vaginal wall (AVW). Tissue blocks from 7 donors that contained the entire urethra and adjacent AVW were analyzed. These tissue samples were fixed within 4-12hours of death and divided into 5-mm transverse slices that were paraffin embedded. Sections cut from each slice were immunolabeled for PSA or PSAP and a neighboring section was stained with hematoxylin and eosin. The sections were reviewed by light microscopy and analyzed using QuPath software. In tissue from all donors, glandular structures expressing PSA and/or PSAP were located within the wall of the urethra and were present along its whole length. In the proximal half of the urethra from all donors, small glands expressing PSAP, but not PSA, were observed adjacent to the and emptying into the lumen. In the distal half of the urethra from 5 of the 7 donors, tubuloacinar structures lined by a glandular epithelium expressed both PSA and PSAP. In addition, columnar cells at the surface of structures with a multilayered transitional epithelium in the distal half of the urethra from all donors expressed PSAP. No glands expressing PSA or PSAP were found in tissues surrounding the urethra, including the AVW. Greater understanding of the distribution of urethral glands expressing prostatic proteins in female patients is important because these glands are reported to contribute to the female sexual response and to urethral pathology, including urethral cysts, diverticula, and adenocarcinoma. Strengths of the present study include the use of rapid autopsy to minimize protein degradation and autolysis, and the preparation of large tissue sections to demonstrate precise anatomical relations within all the tissues surrounding the urethral lumen. Limitations include the sample size and that all donors had advanced malignancy and had undergone previous therapy which may have had unknown tissue effects. Proximal and distal glands expressing prostate-specific proteins were observed in tissue from all donors, and these glands were located only within the wall of the urethra.

High-resolution integrated microfluidic probe for mass spectrometry imaging of biological tissues

Nanospray desorption electrospray ionization (nano-DESI) is an ambient ionization technique that enables molecular imaging of biological samples with high spatial resolution. We have recently developed an integrated microfluidic probe (iMFP) for nano-DESI mass spectrometry imaging (MSI) that significantly enhances the robustness of the technique. In this study, we designed a new probe that enables imaging of biological samples with high spatial resolution. The new probe design features smaller primary and spray channels and an entirely new configuration of the sampling port that enables robust imaging of tissues with a spatial resolution of 8–10 μm. We demonstrate the spatial resolution, sensitivity, durability, and throughput of the iMFP by imaging mouse uterine and brain tissue sections. The robustness of the high-resolution iMFP allowed us to perform first imaging experiments with both high spatial resolution and high throughput, which is particularly advantageous for high-resolution imaging of large tissue sections of interest to most MSI applications. Overall, the new probe design opens opportunities for mapping of biomolecules in biological samples with high throughput and cellular resolution, which is important for understanding biological systems.

Quantitative and large-format histochemistry to characterize peripheral artery compositional gradients.

The femoropopliteal artery (FPA) is a long, flexible vessel that travels down the anteromedial compartment of the thigh as the femoral artery andthen behind the kneecap as the popliteal artery. This artery undergoes various degrees of flexion, extension, and torsion during normal walking movements. The FPA is also the most susceptible peripheral artery to atherosclerosis and is where peripheral artery disease manifests in 80% of cases. The connection between peripheral artery location, its mechanical flexion, and its physiological or pathological biochemistry has been investigated for decades; however, histochemical methods remain poorly leveraged in their ability to spatially correlate normal or abnormal extracellular matrix and cells with regions of mechanical flexion. This study generates new histological image processing pipelines to quantitate tissue composition across high-resolution FPA regions-of-interest or low-resolution whole-section cross-sections in relation to their anatomical locations and flexions during normal movement. Comparing healthy ovine femoral, popliteal, and cranial-tibial artery sections as a pilot, substantial arterial contortion was observed in the distal popliteal and cranial tibial regions of the FPA which correlated with increased vascular smooth muscle cells and decreased elastin content. These methods aim to aid in the quantitative characterization of the spatial distribution of extracellular matrix and cells in large heterogeneous tissue sections such as the FPA. RESEARCH HIGHLIGHTS: Large-format histology preserves artery architecture. Elastin and smooth muscle content is correlated with distance from heart and contortion during flexion. Cell and protein analyses are sensitive to sectioning plane and image magnification.

Automation of slide staining for large tissue sections.

Staining is a critical step in tissue analysis as it enhances the visibility and contrast of tissue structures for microscopic examination. Large tissue sections such as the human brain, heart, and liver are becoming increasingly important in studying complex tissue structures, providing critical information about the tissue's normal or abnormal development, function, and disease processes. Manual staining methods are still widely used and are prone to inconsistencies and inaccuracies, leading to unreliable results. Commercially available automated staining systems offer a more efficient alternative, but currently, these systems are only available for smaller 1" x 3" slides which are ill-suited for examining larger tissue sections. To address this challenge, we present a custom-designed Large format Automated Slide Stainer that can handle various glass slides, from the standard 1" x 3" slides to the custom-sized 2" x 3", 5" x 7", and 6" x 8" glass slides. The system uses a Cartesian robotic arm to stain the slides and has a user-friendly and intuitive interface for creating and modifying custom staining protocols. Safety features include chemical isolation, a ventilation system, an emergency shutdown, and a protective shield to minimize hazards from handling chemicals and biological materials. The automated stainer showed little variability in positioning with a mean offset error of 1.65 ± 0.65 mm and 1.73 ± 0.76 mm in the X and Y axes, respectively. In addition, the automated staining process showed better uniformity than manual staining. A pairwise distance was used to evaluate how well image histograms matched within a batch. The automated staining had a mean pairwise distance of 0.0070 ± 0.0017 (Nissl) and 0.0060 ± 0.0003 (Hematoxylin and Eosin(H&E)), which were far superior to the manual staining distances (Nissl: 0.0173 ± 0.0107 and H&E: 0.0185 ± 0.0067). This system represents a substantial advancement in tissue staining and has the potential to improve the reliability of tissue analysis significantly.Clinical relevance - Automated system for providing accurate, reproducible, and high-throughput staining of large tissue sections for use in histopathology and research.

Automation of slide coverslipping for large tissue sections.

Coverslipping is the process of placing a cover glass or coverslip over a glass slide mounted with a stained tissue specimen without forming air bubbles, which can negatively impact the microscopic examination. While manual coverslipping is still widely used, automated systems have made the process easier and more consistent. Commercially available automated cover-slippers are limited to handling only slides that are 1" x 3", suitable for processing smaller tissue specimens. However, for larger tissue specimens sectioned from organs like the brain, liver, etc., slides can reach sizes up to 6" x 8", exceeding the capabilities of these systems. We present SLIDE PROTEKT, a fully automated large format coverslipping system designed to efficiently coverslip large format slides. This system has multiple zones for slide and coverslip transportation, dispensing of mounting medium, and precise placement of the coverslip without air bubbles. The ability of the system to output quality coverslipped slides was validated by processing 50 large-format brain tissue slides. The results were found to be comparable to manual coverslipping. The system achieved a coverslip placement accuracy of 80% with a mean positional offset that was within a tolerance of ±3 millimeters. Additionally, 75% of the slides had no air bubbles, while the remaining slides had air bubbles that were less than 120 micrometers in size. These results demonstrate the potential impact of SLIDE PROTEKT in the field of histology.

Analysis of Near-Miss Events and Errors in Handling Thyroid Specimens; A Gross Room Experience from a Pathology Lab

<p>Background and Objective: Thyroid specimens constitute a major bulk of the head and neck surgical specimens received at the Pathology Departments. Careful handling of the specimens in the grossing room will result in an efficient reporting of useful histological parameters required for patient management and prognosis. The objective of this study was to analyze different errors and near-miss events in the grossing of thyroid specimens in the surgical pathology gross room of a Tertiary Care Hospital in Lahore, Pakistan.</p>
 <p>Methods: A cross-sectional analysis was done on different thyroid specimens received at the Pathology reception of Lahore General Hospital, Lahore, Pakistan, from January 2022 to April 2023. Different types of errors involved in standard gross examinations were studied. Data were entered using SPSS version 23.0.</p>
 <p>Results: Errors observed in the preanalytical phase included the use of inappropriate fixative (15.2%), insufficient clinical information (76.5), mislabeled jar/request form (7.6%), and loss of specimen (8%). Grossing errors included insufficient grossing notes (12.2%), cutting thick slices (10.7%), missing lesions on gross examination (9.9%), inappropriate inking (6.9%), overfilling cassette with large tissue sections (6.9%), mislabeled cassettes (93.8%), and incomplete submission of capsule (2.3%).</p>
 <p>Conclusion: This study concludes that errors and near-miss events in handling thyroid specimens can be avoided by effective training of the handling staff and grossing residents. Collaboration between clinical wards and histopathology laboratories can also be helpful in this regard.</p>

Artificial intelligence-augmented histopathologic review using image analysis to optimize DNA yield from formalin-fixed paraffin-embedded slides

To achieve minimum DNA input requirements for next-generation sequencing (NGS), pathologists visually estimate macrodissection and slide count decisions. Unfortunately, misestimation may cause tissue waste and increased laboratory costs. We developed an artificial intelligence (AI)-augmented smart pathology review system (SmartPath) to empower pathologists with quantitative metrics for accurately determining tissue extraction parameters. SmartPath uses two deep learning architectures, a U-Net based network for cell segmentation and a multi-field-of-view convolutional network for tumor area segmentation, to extract features from digitized H&E-stained formalin-fixed paraffin-embedded slides. From the segmented tumor area, SmartPath suggests a macrodissection area. To predict DNA yield per slide, the extracted features from within the macrodissection area are correlated with known DNA yields to fit a regularized linear model (R = 0.85). Then, a pathologist-defined target yield divided by the predicted DNA yield per slide gives the number of slides to scrape. Following model development, an internal validation trial was conducted within the Tempus Labs molecular sequencing laboratory. We evaluated our system on 501 clinical colorectal cancer slides, where half received SmartPath-augmented review and half traditional pathologist review. The SmartPath cohort had 25% more DNA yields within a desired target range of 100–2000 ng. The number of extraction attempts was statistically unchanged between cohorts. The SmartPath system recommended fewer slides to scrape for large tissue sections, saving tissue in these cases. Conversely, SmartPath recommended more slides to scrape for samples with scant tissue sections, especially those with degraded DNA, helping prevent costly re-extraction due to insufficient extraction yield. A statistical analysis was performed to measure the impact of covariates on the results, offering insights on how to improve future applications of SmartPath. With these improvements, AI-augmented histopathologic review has the potential to decrease tissue waste, sequencing time, and laboratory costs by optimizing DNA yields, especially for samples with scant tissue and/or degraded DNA.

Lamina-specific immunohistochemical signatures in the olfactory bulb of healthy, Alzheimer\u2019s and Parkinson\u2019s disease patients

Traditional neuroanatomy immunohistology studies involve low-content analyses of a few antibodies of interest, typically applied and compared across sequential tissue sections. The efficiency, consistency, and ultimate insights of these studies can be substantially improved using high-plex immunofluorescence labelling on a single tissue section to allow direct comparison of many markers. Here we present an expanded and efficient multiplexed fluorescence-based immunohistochemistry (MP-IHC) approach that improves throughput with sequential labelling of up to 10 antibodies per cycle, with no limitation on the number of cycles, and maintains versatility and accessibility by using readily available commercial reagents and standard epifluorescence microscopy imaging. We demonstrate this approach by cumulatively screening up to 100 markers on formalin-fixed paraffin-embedded sections of human olfactory bulb sourced from neurologically normal (no significant pathology), Alzheimer’s (AD), and Parkinson’s disease (PD) patients. This brain region is involved early in the symptomology and pathophysiology of AD and PD. We also developed a spatial pixel bin analysis approach for unsupervised analysis of the high-content anatomical information from large tissue sections. Here, we present a comprehensive immunohistological characterisation of human olfactory bulb anatomy and a summary of differentially expressed biomarkers in AD and PD using the MP-IHC labelling and spatial protein analysis pipeline.

Mesothelin is Commonly Expressed in Pancreatic Adenocarcinoma but Unrelated to Cancer Aggressiveness

Data on Mesothelin (MSLN) expression in human normal and cancerous tissues is controversial. We employed immunohistochemistry (IHC) on a tissue microarray (TMA) from 599 pancreatic cancers and 12 large tissue sections of pancreatitis. MSLN expression was highest in pancreatic adenocarcinomas (89%) and adenocarcinomas of the ampulla Vateri (79%), infrequent in pancreatitis and absent in 6 acinus cell carcinomas and normal pancreas. MSLN expression was unrelated to pathological tumor stage, grade, metastasis, and tumor-infiltrating CD8+ lymphocytes. In conclusion, pancreatic cancer may be ideally suited for putative anti- MSLN therapies, and MSLN may represent a suitable biomarker for pancreatic cancer diagnosis, especially on small biopsies.

DOG1 is commonly expressed in pancreatic adenocarcinoma but unrelated to cancer aggressiveness.

BackgroundDOG1 (ANO1; TMEM16A) is a voltage-gated calcium-activated chloride and bicarbonate channel. DOG1 is physiologically expressed in Cajal cells, where it plays an important role in regulating intestinal motility and its expression is a diagnostic hallmark of gastrointestinal stromal tumors (GIST). Data on a possible role of DOG1 in pancreatic cancer are rare and controversial. The aim of our study was to clarify the prevalence of DOG1 expression in pancreatic cancer and to study its association with parameters of cancer aggressiveness.MethodsDOG1 expression was analyzed by immunohistochemistry in 599 pancreatic cancers in a tissue microarray format and in 12 cases of pancreatitis on large tissue sections.ResultsDOG1 expression was always absent in normal pancreas but a focal weak expression was seen in four of 12 cases of pancreatitis. DOG1 expression was, however, common in pancreatic cancer. Membranous and cytoplasmic DOG1 expression in tumor cells was highest in pancreatic ductal adenocarcinomas (61% of 444 interpretable cases), followed by cancers of the ampulla Vateri (43% of 51 interpretable cases), and absent in 6 acinus cell carcinomas. DOG1 expression in tumor associated stroma cells was seen in 76 of 444 (17%) pancreatic ductal adenocarcinomas and in seven of 51 (14%) cancers of the ampulla Vateri. Both tumoral and stromal DOG1 expression were unrelated to tumor stage, grade, lymph node and distant metastasis, mismatch repair protein deficiency and the density of CD8 positive cytotoxic T-lymphocytes in the subgroups of ductal adenocarcinomas and cancers of ampulla Vateri. Overall, the results of our study indicate that DOG1 may represent a potential biomarker for pancreatic cancer diagnosis and a putative therapeutic target in pancreatic cancer. However, DOG1 expression is unrelated to pancreatic cancer aggressiveness.

Frequency and Significance of Pathologic Pulmonary Findings in Postmortem Examinations-A Single Center Experience before COVID-19.

Coronavirus disease 2019 (COVID-19) has shown the importance of postmortem investigation of deceased patients. For a correct interpretation of the pulmonary findings in this new era, it is, however, crucial to be familiar with pathologic pulmonary conditions observed in postmortem investigations in general. Adequate postmortem histopathological evaluation of the lungs may be affected by suboptimal gross work up, autolysis or poor fixation. Using a standardized preparation approach which consisted in instillation of 4% buffered formaldehyde through the large bronchi for proper fixation and preparing large frontal tissue sections of 1–2 cm thickness after at least 24 h fixation, we comprehensively analyzed postmortem pulmonary findings from consecutive adult autopsies of a two-year period before the occurrence of COVID-19 (2016–2017). In total, significant pathological findings were observed in 97/189 patients (51%), with 28 patients showing more than one pathologic condition. Acute pneumonia was diagnosed 33/128 times (26%), embolism 24 times (19%), primary pulmonary neoplasms 18 times (14%), organizing pneumonia and other fibrosing conditions 14 times (11%), pulmonary metastases 13 times (10%), diffuse alveolar damage 12 times (9%), severe emphysema 9 times (7%) and other pathologies, e.g., amyloidosis 5/128 times (4%). Pulmonary/cardiopulmonary disease was the cause of death in 60 patients (32%). Clinical and pathological diagnoses regarding lung findings correlated completely in 75 patients (40%). Autopsy led to confirmation of a clinically suspected pulmonary diagnosis in 57 patients (39%) and clarification of an unclear clinical lung finding in 16 patients (8%). Major discrepant findings regarding the lungs (N = 31; 16%) comprised cases with clinical suspicions that could not be confirmed or new findings not diagnosed intra vitam. A significant proportion of acute pneumonias (N = 8; 24% of all cases with this diagnosis; p = 0.011) was not diagnosed clinically. We confirmed the frequent occurrence of pulmonary pathologies in autopsies, including inflammatory and neoplastic lesions as the most frequent pathological findings. Acute pneumonia was an important cause for discrepancy between clinical and postmortem diagnostics

Using Volumetric 3D Microscopy Techniques to Characterize Astrocyte Morphologies over Large Spatial Scales

Astrocytes are a type of glia found in the central nervous system with many roles, and have recently been reported to be directly involved in modulating and scaling synaptic transmission. Neurotransmitters released in synaptic activity induce astrocyte calcium signaling, which then induces the release gliotransmitters that can act on neighbouring pre- and postsynaptic neuronal elements. Astrocytes are highly 3-dimensional and form large syncytium of diffusion-coupled cells via their many long protrusions. It has been proposed that astrocytes may use these large networks to communicate and correlate to distant synapses. When studying astrocyte network interactions, it is important to establish astrocyte morphology at the the level of single cells and of populations, as this structure is directly related to the astrocytes’ ability for intercellular communication, and speed and frequency of calcium signal propagation. On-going advances in volumetric 3D imaging techniques, such as light-sheet microscopy, now allow for imaging complete murine brain sections, and obtaining a better understanding of how astrocytes are organized and interact in situ on a larger spatial scale. Here, a pipeline for characterizing morphology for both single astrocytes and their networks using volumetric 3D microscopy techniques was optimized using 500 µm and 1 mm thick mouse cortex slices. Various structural features were measured and quantified including territorial volume, cell orientations, number of connections, process arborisations and lengths. This characterization pipeline can be extended to cortical organoids, larger tissue sections, or other brain regions, and is a helpful tool for studying astrocyte network structure and functionality.

A computer-aided diagnosis system for differentiation and delineation of malignant regions on whole-slide prostate histopathology image using spatial statistics and multidimensional DenseNet.

Prostate cancer (PCa) is a major health concern in aging males, and proper management of the disease depends on accurately interpreting pathology specimens. However, reading prostatectomy histopathology slides, which is basically for staging, is usually time consuming and differs from reading small biopsy specimens, which is mainly used for diagnosis. Generally, each prostatectomy specimen generates tens of large tissue sections and for each section, the malignant region needs to be delineated to assess the amount of tumor and its burden. With the aim of reducing the workload of pathologists, in this study, we focus on developing a computer-aided diagnosis (CAD) system based on a densely connected convolutional neural network (DenseNet) for whole-slide histopathology images to outline the malignant regions. We use an efficient color normalization process based on ranklet transformation to automatically correct the intensity of the images. Additionally, we use spatial probability to segment the tissue structure regions for different tissue recognition patterns. Based on the segmentation, we incorporate a multidimensional structure into DenseNet to determine if a particular prostatic region is benign or malignant. As demonstrated by the experimental results with a test set of 2,663 images from 32 whole-slide prostate histopathology images, our proposed system achieved 0.726, 0.6306, and 0.5209 in the average of the Dice coefficient, Jaccard similarity coefficient, and Boundary F1 score measures, respectively. Then, the accuracy, sensitivity, specificity, and the area under the ROC curve (AUC) of the proposed classification method were observed to be 95.0% (2544/2663), 96.7% (1210/1251), 93.9% (1334/1412), and 0.9831, respectively. We provide a detailed discussion on how our proposed system demonstrates considerable improvement compared with similar methods considered in previous researches as well as how it can be used for delineating malignant regions.

Glioblastoma multiforme restructures the topological connectivity of cerebrovascular networks

Glioblastoma multiforme alters healthy tissue vasculature by inducing angiogenesis and vascular remodeling. To fully comprehend the structural and functional properties of the resulting vascular network, it needs to be studied collectively by considering both geometric and topological properties. Utilizing Single Plane Illumination Microscopy (SPIM), the detailed capillary structure in entire healthy and tumor-bearing mouse brains could be resolved in three dimensions. At the scale of the smallest capillaries, the entire vascular systems of bulk U87- and GL261-glioblastoma xenografts, their respective cores, and healthy brain hemispheres were modeled as complex networks and quantified with fundamental topological measures. All individual vessel segments were further quantified geometrically and modular clusters were uncovered and characterized as meta-networks, facilitating an analysis of large-scale connectivity. An inclusive comparison of large tissue sections revealed that geometric properties of individual vessels were altered in glioblastoma in a relatively subtle way, with high intra- and inter-tumor heterogeneity, compared to the impact on the vessel connectivity. A network topology analysis revealed a clear decomposition of large modular structures and hierarchical network organization, while preserving most fundamental topological classifications, in both tumor models with distinct growth patterns. These results augment our understanding of cerebrovascular networks and offer a topological assessment of glioma-induced vascular remodeling. The findings may help understand the emergence of hypoxia and necrosis, and prove valuable for therapeutic interventions such as radiation or antiangiogenic therapy.

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue.

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample's refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Brain maps at the nanoscale.

Large brain tissue sections are imaged with nanoscale resolution using expansion and lattice light sheet microscopy.

From Light Microscopy to Analytical Scanning Electron Microscopy (SEM) and Focused Ion Beam (FIB)/SEM in Biology: Fixed Coordinates, Flat Embedding, Absolute References.

Correlative light and electron microscopy (CLEM) has been in use for several years, however it has remained a costly method with difficult sample preparation. Here, we report a series of technical improvements developed for precise and cost-effective correlative light and scanning electron microscopy (SEM) and focused ion beam (FIB)/SEM microscopy of single cells, as well as large tissue sections. Customized coordinate systems for both slides and coverslips were established for thin and ultra-thin embedding of a wide range of biological specimens. Immobilization of biological samples was examined with a variety of adhesives. For histological sections, a filter system for flat embedding was developed. We validated ultra-thin embedding on laser marked slides for efficient, high-resolution CLEM. Target cells can be re-located within minutes in SEM without protracted searching and correlative investigations were reduced to a minimum of preparation steps, while still reaching highest resolution. The FIB/SEM milling procedure is facilitated and significantly accelerated as: (i) milling a ramp becomes needless, (ii) significant re-deposition of milled material does not occur; and (iii) charging effects are markedly reduced. By optimizing all technical parameters FIB/SEM stacks with 2 nm iso-voxels were achieved over thousands of sections, in a wide range of biological samples.

Automated Slide Scanning and Segmentation in Fluorescently-labeled Tissues Using a Widefield High-content Analysis System

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. Unfortunately, many researchers spend large amounts of time and resources developing and optimizing workflows that are only relevant to their own experiments. In this article, we describe a protocol that can be used by those with access to a widefield high-content analysis system (WHCAS) to image any slide-mounted tissue, with options for customization within pre-built modules found in the associated software. Not originally intended for slide scanning, the steps detailed in this article make it possible to acquire slide scanning images in the WHCAS which can be imported into the associated software. In this example, the automated segmentation of brain tumor slides is demonstrated, but the automated segmentation of any fluorescently-labeled nuclear or cytoplasmic marker is possible. Furthermore, there are a variety of other quantitative software modules including assays for protein localization/translocation, cellular proliferation/viability/apoptosis, and angiogenesis that can be run. This technique will save researchers time and effort and create an automated protocol for slide analysis.

Automated Slide Scanning and Segmentation in Fluorescently-labeled Tissues Using a Widefield High-content Analysis System.

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. Unfortunately, many researchers spend large amounts of time and resources developing and optimizing workflows that are only relevant to their own experiments. In this article, we describe a protocol that can be used by those with access to a widefield high-content analysis system (WHCAS) to image any slide-mounted tissue, with options for customization within pre-built modules found in the associated software. Not originally intended for slide scanning, the steps detailed in this article make it possible to acquire slide scanning images in the WHCAS which can be imported into the associated software. In this example, the automated segmentation of brain tumor slides is demonstrated, but the automated segmentation of any fluorescently-labeled nuclear or cytoplasmic marker is possible. Furthermore, there are a variety of other quantitative software modules including assays for protein localization/translocation, cellular proliferation/viability/apoptosis, and angiogenesis that can be run. This technique will save researchers time and effort and create an automated protocol for slide analysis.