Automated detection of vascular remodeling in tumor-draining lymph nodes by the deep-learning tool HEV-finder.

Vascular remodeling is common in human cancer and has potential as future biomarkers for prediction of disease progression and tumor immunity status. It can also affect metastatic sites, including the tumor-draining lymph nodes (TDLNs). Dilation of the high endothelial venules (HEVs) within TDLNs has been observed in several types of cancer. We recently demonstrated that it is a premetastatic effect that can be linked to tumor invasiveness in breast cancer. Manual visual assessment of changes in vascular morphology is a tedious and difficult task, limiting high-throughput analysis. Here we present a fully automated approach for detection and classification of HEV dilation. By using 12,524 manually classified HEVs, we trained a deep-learning model and created a graphical user interface for visualization of the results. The tool, named the HEV-finder, selectively analyses HEV dilation in specific regions of the lymph nodes. We evaluated the HEV-finder's ability to detect and classify HEV dilation in different types of breast cancer compared to manual annotations. Our results constitute a successful example of large-scale, fully automated, and user-independent, image-based quantitative assessment of vascular remodeling in human pathology and lay the ground for future exploration of HEV dilation in TDLNs as a biomarker. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Machine learning for cell classification and neighborhood analysis in glioma tissue.

Multiplexed and spatially resolved single-cell analyses that intend to study tissue heterogeneity and cell organization invariably face as a first step the challenge of cell classification. Accuracy and reproducibility are important for the downstream process of counting cells, quantifying cell-cell interactions, and extracting information on disease-specific localized cell niches. Novel staining techniques make it possible to visualize and quantify large numbers of cell-specific molecular markers in parallel. However, due to variations in sample handling and artifacts from staining and scanning, cells of the same type may present different marker profiles both within and across samples. We address multiplexed immunofluorescence data from tissue microarrays of low-grade gliomas and present a methodology using two different machine learning architectures and features insensitive to illumination to perform cell classification. The fully automated cell classification provides a measure of confidence for the decision and requires a comparably small annotated data set for training, which can be created using freely available tools. Using the proposed method, we reached an accuracy of 83.1% on cell classification without the need for standardization of samples. Using our confidence measure, cells with low-confidence classifications could be excluded, pushing the classification accuracy to 94.5%. Next, we used the cell classification results to search for cell niches with an unsupervised learning approach based on graph neural networks. We show that the approach can re-detect specialized tissue niches in previously published data, and that our proposed cell classification leads to niche definitions that may be relevant for sub-groups of glioma, if applied to larger data sets.

Automated identification of the mouse brain's spatial compartments from in situ sequencing data.

BACKGROUND: Neuroanatomical compartments of the mouse brain are identified and outlined mainly based on manual annotations of samples using features related to tissue and cellular morphology, taking advantage of publicly available reference atlases. However, this task is challenging since sliced tissue sections are rarely perfectly parallel or angled with respect to sections in the reference atlas and organs from different individuals may vary in size and shape and requires manual annotation. With the advent of in situ sequencing technologies and automated approaches, it is now possible to profile the gene expression of targeted genes inside preserved tissue samples and thus spatially map biological processes across anatomical compartments. RESULTS: Here, we show how in situ sequencing data combined with dimensionality reduction and clustering can be used to identify spatial compartments that correspond to known anatomical compartments of the brain. We also visualize gradients in gene expression and sharp as well as smooth transitions between different compartments. We apply our method on mouse brain sections and show that a fully unsupervised approach can computationally define anatomical compartments, which are highly reproducible across individuals, using as few as 18 gene markers. We also show that morphological variation does not always follow gene expression, and different spatial compartments can be defined by various cell types with common morphological features but distinct gene expression profiles. CONCLUSION: We show that spatial gene expression data can be used for unsupervised and unbiased annotations of mouse brain spatial compartments based only on molecular markers, without the need of subjective manual annotations based on tissue and cell morphology or matching reference atlases.

Deep Learning in Image Cytometry: A Review.

Artificial intelligence, deep convolutional neural networks, and deep learning are all niche terms that are increasingly appearing in scientific presentations as well as in the general media. In this review, we focus on deep learning and how it is applied to microscopy image data of cells and tissue samples. Starting with an analogy to neuroscience, we aim to give the reader an overview of the key concepts of neural networks, and an understanding of how deep learning differs from more classical approaches for extracting information from image data. We aim to increase the understanding of these methods, while highlighting considerations regarding input data requirements, computational resources, challenges, and limitations. We do not provide a full manual for applying these methods to your own data, but rather review previously published articles on deep learning in image cytometry, and guide the readers toward further reading on specific networks and methods, including new methods not yet applied to cytometry data. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Estimating hemodynamic shear stress in murine peripheral collateral arteries by two-photon line scanning.

Changes in wall shear stress of blood vessels are assumed to be an important component of many physiological and pathophysiological processes. However, due to technical limitations experimental in vivo data are rarely available. Here, we investigated two-photon excitation fluorescence microscopy as an option to measure vessel diameter as well as blood flow velocities in a murine hindlimb model of arteriogenesis (collateral artery growth). Using line scanning at high frequencies, we measured the movement of blood cells along the vessel axis. We found that peak systolic blood flow velocity averaged 9 mm/s and vessel diameter 42 µm in resting collaterals. Induction of arteriogenesis by femoral artery ligation resulted in a significant increase in centerline peak systolic velocity after 1 day with an average of 51 mm/s, whereas the averaged luminal diameter of collaterals (52 µm) changed much less. Thereof calculations revealed a significant fourfold increase in hemodynamic wall shear rate. Our results indicate that two-photon line scanning is a suitable tool to estimate wall shear stress e.g., in experimental animal models, such as of arteriogenesis, which may not only help to understand the relevance of mechanical forces in vivo, but also to adjust wall shear stress in ex vivo investigations on isolated vessels as well as cell culture experiments.

Tracking Microscope Performance: A Workflow to Compare Point Spread Function Evaluations Over Time.

Routine system checks are essential for supervising the performance of an advanced light microscope. Recording and evaluating the point spread function (PSF) of a given system provides information about the resolution and imaging. We compared the performance of fluorescent and gold beads for PSF recordings. We then combined the open-source evaluation software PSFj with a newly developed KNIME pipeline named PSFtracker to create a standardized workflow to track a system's performance over several measurements and thus over long time periods. PSFtracker produces example images of recorded PSFs, plots full-width-half-maximum (FWHM) measurements over time and creates an html file which embeds the images and plots, together with a table of results. Changes of the PSF over time are thus easily spotted, either in FWHM plots or in the time series of bead images which allows recognition of aberrations in the shape of the PSF. The html file, viewed in a local browser or uploaded on the web, therefore provides intuitive visualization of the state of the PSF over time. In addition, uploading of the html file on the web allows other microscopists to compare such data with their own.

Cell-type-specific expression pattern of ceramide synthase 2 protein in mouse tissues.

Ceramide synthase 2 (CerS2) catalyzes the synthesis of dihydroceramides from dihydrosphingosine and very long fatty acyl (C22-C24)-CoAs. CerS2-deficient (gene trap) mice were reported to exhibit myelin and behavioral abnormalities, associated with the expression of CerS2 in oligodendrocytes and neurons based on expression of lacZ reporter cDNA instead of the cers2 gene in these mice. In order to clarify the cell-type-specific expression of CerS2 protein, we have raised antibodies that specifically recognize the glycosylated and non-glycosylated CerS2 protein in wild-type but not in CerS2-deficient mouse tissues. In early postnatal, juvenile and adult mouse brain, the new antibodies detect CerS2 protein only in oligodendrocytes but not in neurons, suggesting that the gene trap vector in CerS2-deficient mice led to ectopic expression of the lacZ reporter gene in neurons. In liver, the CerS2 protein is expressed in hepatocytes but not in Ito cells or Kupffer cells. We conclude that the behavioral abnormalities observed in CerS2-deficient mice originate primarily in oligodendrocytes and not in neurons. The identification of specific cell types in which CerS2 protein is expressed is prerequisite to further mechanistic characterization of phenotypic abnormalities exhibited by CerS2-deficient mice. The amount of CerS2 protein detected in different tissues by immunoblot analyses does not strictly correspond to the activity of the CerS2 enzyme. Disproportional results are likely due to post-translational regulation of the CerS2 protein.

Visualization and quality control tools for large-scale multiplex tissue analysis in TissUUmaps3.

Large-scale multiplex tissue analysis aims to understand processes such as development and tumor formation by studying the occurrence and interaction of cells in local environments in, for example, tissue samples from patient cohorts. A typical procedure in the analysis is to delineate individual cells, classify them into cell types, and analyze their spatial relationships. All steps come with a number of challenges, and to address them and identify the bottlenecks of the analysis, it is necessary to include quality control tools in the analysis workflow. This makes it possible to optimize the steps and adjust settings in order to get better and more precise results. Additionally, the development of automated approaches for tissue analysis requires visual verification to reduce skepticism with regard to the accuracy of the results. Quality control tools could be used to build users' trust in automated approaches. In this paper, we present three plugins for visualization and quality control in large-scale multiplex tissue analysis of microscopy images. The first plugin focuses on the quality of cell staining, the second one was made for interactive evaluation and comparison of different cell classification results, and the third one serves for reviewing interactions of different cell types.

TissUUmaps 3: Improvements in interactive visualization, exploration, and quality assessment of large-scale spatial omics data.

Background and objectives: Spatially resolved techniques for exploring the molecular landscape of tissue samples, such as spatial transcriptomics, often result in millions of data points and images too large to view on a regular desktop computer, limiting the possibilities in visual interactive data exploration. TissUUmaps is a free, open-source browser-based tool for GPU-accelerated visualization and interactive exploration of 107+ data points overlaying tissue samples. Methods: Herein we describe how TissUUmaps 3 provides instant multiresolution image viewing and can be customized, shared, and also integrated into Jupyter Notebooks. We introduce new modules where users can visualize markers and regions, explore spatial statistics, perform quantitative analyses of tissue morphology, and assess the quality of decoding in situ transcriptomics data. Results: We show that thanks to targeted optimizations the time and cost associated with interactive data exploration were reduced, enabling TissUUmaps 3 to handle the scale of today's spatial transcriptomics methods. Conclusion: TissUUmaps 3 provides significantly improved performance for large multiplex datasets as compared to previous versions. We envision TissUUmaps to contribute to broader dissemination and flexible sharing of largescale spatial omics data.

Spatial Statistics for Understanding Tissue Organization.

Interpreting tissue architecture plays an important role in gaining a better understanding of healthy tissue development and disease. Novel molecular detection and imaging techniques make it possible to locate many different types of objects, such as cells and/or mRNAs, and map their location across the tissue space. In this review, we present several methods that provide quantification and statistical verification of observed patterns in the tissue architecture. We categorize these methods into three main groups: Spatial statistics on a single type of object, two types of objects, and multiple types of objects. We discuss the methods in relation to four hypotheses regarding the methods' capability to distinguish random and non-random distributions of objects across a tissue sample, and present a number of openly available tools where these methods are provided. We also discuss other spatial statistics methods compatible with other types of input data.

SLC38A10 Regulate Glutamate Homeostasis and Modulate the AKT/TSC2/mTOR Pathway in Mouse Primary Cortex Cells.

Glutamate acts as a critical regulator of neurotransmitter balance, recycling, synaptic function and homeostasis in the brain and glutamate transporters control glutamate levels in the brain. SLC38A10 is a member of the SLC38 family and regulates protein synthesis and cellular stress responses. Here, we uncover the role of SLC38A10 as a transceptor involved in glutamate-sensing signaling pathways that control both the glutamate homeostasis and mTOR-signaling. The culture of primary cortex cells from SLC38A10 knockout mice had increased intracellular glutamate. In addition, under nutrient starvation, KO cells had an impaired response in amino acid-dependent mTORC1 signaling. Combined studies from transcriptomics, protein arrays and metabolomics established that SLC38A10 is involved in mTOR signaling and that SLC38A10 deficient primary cortex cells have increased protein synthesis. Metabolomic data showed decreased cholesterol levels, changed fatty acid synthesis, and altered levels of fumaric acid, citrate, 2-oxoglutarate and succinate in the TCA cycle. These data suggests that SLC38A10 may act as a modulator of glutamate homeostasis, and mTOR-sensing and loss of this transceptor result in lower cholesterol, which could have implications in neurodegenerative diseases.

A Hitchhiker's guide through the bio-image analysis software universe.

Modern research in the life sciences is unthinkable without computational methods for extracting, quantifying and visualising information derived from microscopy imaging data of biological samples. In the past decade, we observed a dramatic increase in available software packages for these purposes. As it is increasingly difficult to keep track of the number of available image analysis platforms, tool collections, components and emerging technologies, we provide a conservative overview of software that we use in daily routine and give insights into emerging new tools. We give guidance on which aspects to consider when choosing the platform that best suits the user's needs, including aspects such as image data type, skills of the team, infrastructure and community at the institute and availability of time and budget.

A method for Boolean analysis of protein interactions at a molecular level.

Determining the levels of protein-protein interactions is essential for the analysis of signaling within the cell, characterization of mutation effects, protein function and activation in health and disease, among others. Herein, we describe MolBoolean - a method to detect interactions between endogenous proteins in various subcellular compartments, utilizing antibody-DNA conjugates for identification and signal amplification. In contrast to proximity ligation assays, MolBoolean simultaneously indicates the relative abundances of protein A and B not interacting with each other, as well as the pool of A and B proteins that are proximal enough to be considered an AB complex. MolBoolean is applicable both in fixed cells and tissue sections. The specific and quantifiable data that the method generates provide opportunities for both diagnostic use and medical research.

Focal adhesion kinase stabilizes the cytoskeleton.

Focal adhesion kinase (FAK) is a central focal adhesion protein that promotes focal adhesion turnover, but the role of FAK for cell mechanical stability is unknown. We measured the mechanical properties of wild-type (FAKwt), FAK-deficient (FAK-/-), FAK-silenced (siFAK), and siControl mouse embryonic fibroblasts by magnetic tweezer, atomic force microscopy, traction microscopy, and nanoscale particle tracking microrheology. FAK-deficient cells showed lower cell stiffness, reduced adhesion strength, and increased cytoskeletal dynamics compared to wild-type cells. These observations imply a reduced stability of the cytoskeleton in FAK-deficient cells. We attribute the reduced cytoskeletal stability to rho-kinase activation in FAK-deficient cells that suppresses the formation of ordered stress fiber bundles, enhances cortical actin distribution, and reduces cell spreading. In agreement with this interpretation is that cell stiffness and cytoskeletal stability in FAK-/- cells is partially restored to wild-type level after rho-kinase inhibition with Y27632.

ImageJ and CellProfiler: Complements in Open-Source Bioimage Analysis.

ImageJ and CellProfiler have long been leading open-source platforms in the field of bioimage analysis. ImageJ's traditional strength is in single-image processing and investigation, while CellProfiler is designed for building large-scale, modular analysis pipelines. Although many image analysis problems can be well solved with one or the other, using these two platforms together in a single workflow can be powerful. Here, we share two pipelines demonstrating mechanisms for productively and conveniently integrating ImageJ and CellProfiler for (1) studying cell morphology and migration via tracking, and (2) advanced stitching techniques for handling large, tiled image sets to improve segmentation. No single platform can provide all the key and most efficient functionality needed for all studies. While both programs can be and are often used separately, these pipelines demonstrate the benefits of using them together for image analysis workflows. ImageJ and CellProfiler are both committed to interoperability between their platforms, with ongoing development to improve how both are leveraged from the other. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Studying cell morphology and cell migration in time-lapse datasets using TrackMate (Fiji) and CellProfiler Basic Protocol 2: Creating whole plate montages to easily assess adaptability of segmentation parameters.

Highlights from the 2016-2020 NEUBIAS training schools for Bioimage Analysts: a success story and key asset for analysts and life scientists.

NEUBIAS, the European Network of Bioimage Analysts, was created in 2016 with the goal of improving the communication and the knowledge transfer among the various stakeholders involved in the acquisition, processing and analysis of biological image data, and to promote the establishment and recognition of the profession of Bioimage Analyst. One of the most successful initiatives of the NEUBIAS programme was its series of 15 training schools, which trained over 400 new Bioimage Analysts, coming from over 40 countries. Here we outline the rationale behind the innovative three-level program of the schools, the curriculum, the trainer recruitment and turnover strategy, the outcomes for the community and the career path of analysts, including some success stories. We discuss the future of the materials created during this programme and some of the new initiatives emanating from the community of NEUBIAS-trained analysts, such as the NEUBIAS Academy. Overall, we elaborate on how this training programme played a key role in collectively leveraging Bioimaging and Life Science research by bringing the latest innovations into structured, frequent and intensive training activities, and on why we believe this should become a model to further develop in Life Sciences.

Bioimage analysis workflows: community resources to navigate through a complex ecosystem.

Workflows are the keystone of bioimage analysis, and the NEUBIAS (Network of European BioImage AnalystS) community is trying to gather the actors of this field and organize the information around them.  One of its most recent outputs is the opening of the F1000Research NEUBIAS gateway, whose main objective is to offer a channel of publication for bioimage analysis workflows and associated resources. In this paper we want to express some personal opinions and recommendations related to finding, handling and developing bioimage analysis workflows.  The emergence of "big data" in bioimaging and resource-intensive analysis algorithms make local data storage and computing solutions a limiting factor. At the same time, the need for data sharing with collaborators and a general shift towards remote work, have created new challenges and avenues for the execution and sharing of bioimage analysis workflows. These challenges are to reproducibly run workflows in remote environments, in particular when their components come from different software packages, but also to document them and link their parameters and results by following the FAIR principles (Findable, Accessible, Interoperable, Reusable) to foster open and reproducible science. In this opinion paper, we focus on giving some directions to the reader to tackle these challenges and navigate through this complex ecosystem, in order to find and use workflows, and to compare workflows addressing the same problem. We also discuss tools to run workflows in the cloud and on High Performance Computing resources, and suggest ways to make these workflows FAIR.

The influence of Pyk2 on the mechanical properties in fibroblasts.

The cell surface receptor integrin is involved in signaling mechanical stresses via the focal adhesion complex (FAC) into the cell. Within FAC, the focal adhesion kinase (FAK) and Pyk2 are believed to act as important scaffolding proteins. Based on the knowledge that many signal transducing molecules are transiently immobilized within FAC connecting the cytoskeleton with integrins, we applied magnetic tweezer and atomic force microscopic measurements to determine the influence of FAK and Pyk2 in cells mechanically. Using mouse embryonic fibroblasts (MEF; FAK(+/+), FAK(-/-), and siRNA-Pyk2 treated FAK(-/-) cells) provided a unique opportunity to describe the function of FAK and Pyk2 in more detail and to define their influence on FAC and actin distribution.

Comparing the mechanical influence of vinculin, focal adhesion kinase and p53 in mouse embryonic fibroblasts.

Cytoskeletal reorganization is an ongoing process when cells adhere, move or invade extracellular substrates. The cellular force generation and transmission are determined by the intactness of the actomyosin-(focal adhesion complex)-integrin connection. We investigated the intracellular course of action in mouse embryonic fibroblasts deficient in the focal adhesion proteins vinculin and focal adhesion kinase (FAK) and the nuclear matrix protein p53 using magnetic tweezer and nanoparticle tracking techniques. Results show that the lack of these proteins decrease cellular stiffness and affect cell rheological behavior. The decrease in cellular binding strength was higher in FAK- to vinculin-deficient cells, whilst p53-deficient cells showed no effect compared to wildtype cells. The intracellular cytoskeletal activity was lowest in wildtype cells, but increased in the following order when cells lacked FAK+p53>p53>vinculin. In summary, cell mechanical processes are differently affected by the focal adhesion proteins vinculin and FAK than by the nuclear matrix protein, p53.

Thermodynamic evidence of non-muscle myosin II-lipid-membrane interaction.

A unique feature of protein networks in living cells is that they can generate their own force. Proteins such as non-muscle myosin II are an integral part of the cytoskeleton and have the capacity to convert the energy of ATP hydrolysis into directional movement. Non-muscle myosin II can move actin filaments against each other, and depending on the orientation of the filaments and the way in which they are linked together, it can produce contraction, bending, extension, and stiffening. Our measurements with differential scanning calorimetry showed that non-muscle myosin II inserts into negatively charged phospholipid membranes. Using lipid vesicles made of DMPG/DMPC at a molar ratio of 1:1 at 10mg/ml in the presence of different non-muscle myosin II concentrations showed a variation of the main phase transition of the lipid vesicle at around 23 degrees C. With increasing concentrations of non-muscle myosin II the thermotropic properties of the lipid vesicle changed, which is indicative of protein-lipid interaction/insertion. We hypothesize that myosin tail binds to acidic phospholipids through an electrostatic interaction using the basic side groups of positive residues; the flexible, amphipathic helix then may partially penetrate into the bilayer to form an anchor. Using the stopped-flow method, we determined the binding affinity of non-muscle myosin II when anchored to lipid vesicles with actin, which was similar to a pure actin-non-muscle myosin II system. Insertion of myosin tail into the hydrophobic region of lipid membranes, a model known as the lever arm mechanism, might explain how its interaction with actin generates cellular movement.