In the Murine and Bovine Maternal Mammary Gland Signal Transducer and Activator of Transcription 3 is Activated in Clusters of Epithelial Cells around the Day of Birth.

Signal transducers and activators of transcription (STAT) proteins regulate mammary development. Here we investigate the expression of phosphorylated STAT3 (pSTAT3) in the mouse and cow around the day of birth. We present localised colocation analysis, applicable to other mammary studies requiring identification of spatially congregated events. We demonstrate that pSTAT3-positive events are multifocally clustered in a non-random and statistically significant fashion. Arginase-1 expressing cells, consistent with macrophages, exhibit distinct clustering within the periparturient mammary gland. These findings represent a new facet of mammary STAT3 biology, and point to the presence of mammary sub-microenvironments.

Deductive automated pollen classification in environmental samples via exploratory deep learning and imaging flow cytometry.

Pollen and tracheophyte spores are ubiquitous environmental indicators at local and global scales. Palynology is typically performed manually by microscopic analysis; a specialised and time-consuming task limited in taxonomical precision and sampling frequency, therefore restricting data quality used to inform climate change and pollen forecasting models. We build on the growing work using AI (artificial intelligence) for automated pollen classification to design a flexible network that can deal with the uncertainty of broad-scale environmental applications. We combined imaging flow cytometry with Guided Deep Learning to identify and accurately categorise pollen in environmental samples; here, pollen grains captured within c. 5500 Cal yr BP old lake sediments. Our network discriminates not only pollen included in training libraries to the species level but, depending on the sample, can classify previously unseen pollen to the likely phylogenetic order, family and even genus. Our approach offers valuable insights into the development of a widely transferable, rapid and accurate exploratory tool for pollen classification in 'real-world' environmental samples with improved accuracy over pure deep learning techniques. This work has the potential to revolutionise many aspects of palynology, allowing a more detailed spatial and temporal understanding of pollen in the environment with improved taxonomical resolution.

Label-free cell segmentation of diverse lymphoid tissues in 2D and 3D.

Unlocking and quantifying fundamental biological processes through tissue microscopy requires accurate, in situ segmentation of all cells imaged. Currently, achieving this is complex and requires exogenous fluorescent labels that occupy significant spectral bandwidth, increasing the duration and complexity of imaging experiments while limiting the number of channels remaining to address the study's objectives. We demonstrate that the excitation light reflected during routine confocal microscopy contains sufficient information to achieve accurate, label-free cell segmentation in 2D and 3D. This is achieved using a simple convolutional neural network trained to predict the probability that reflected light pixels belong to either nucleus, cytoskeleton, or background classifications. We demonstrate the approach across diverse lymphoid tissues and provide video tutorials demonstrating deployment in Python and MATLAB or via standalone software for Windows.

Spatial statistics is a comprehensive tool for quantifying cell neighbor relationships and biological processes via tissue image analysis.

Automated microscopy and computational image analysis has transformed cell biology, providing quantitative, spatially resolved information on cells and their constituent molecules from the sub-micron to the whole-organ scale. Here we explore the application of spatial statistics to the cellular relationships within tissue microscopy data and discuss how spatial statistics offers cytometry a powerful yet underused mathematical tool set for which the required data are readily captured using standard protocols and microscopy equipment. We also highlight the often-overlooked need to carefully consider the structural heterogeneity of tissues in terms of the applicability of different statistical measures and their accuracy and demonstrate how spatial analyses offer a great deal more than just basic quantification of biological variance. Ultimately, we highlight how statistical modeling can help reveal the hierarchical spatial processes that connect the properties of individual cells to the establishment of biological function.

Imaging flow cytometry: a primer.

Imaging flow cytometry combines the high throughput nature of flow cytometry with the advantages of single cell image acquisition associated with microscopy. The measurement of large numbers of features from the resulting images provides rich datasets which have resulted in a wide range of novel biomedical applications. In this primer we discuss the typical imaging flow instrumentation, the form of data acquired and the typical analysis tools that can be applied to this data. Using examples from the literature we discuss the progression of the analysis methods that have been applied to imaging flow cytometry data. These methods start from the use of simple single image features and multiple channel gating strategies, followed by the design and use of custom features for phenotype classification, through to powerful machine and deep learning methods. For each of these methods, we outline the processes involved in analyzing typical datasets and provide details of example applications. Finally we discuss the current limitations of imaging flow cytometry and the innovations which are addressing these challenges.

Data-Driven Modeling of the Cellular Pharmacokinetics of Degradable Chitosan-Based Nanoparticles.

Nanoparticle drug delivery vehicles introduce multiple pharmacokinetic processes, with the delivery, accumulation, and stability of the therapeutic molecule influenced by nanoscale processes. Therefore, considering the complexity of the multiple interactions, the use of data-driven models has critical importance in understanding the interplay between controlling processes. We demonstrate data simulation techniques to reproduce the time-dependent dose of trimethyl chitosan nanoparticles in an ND7/23 neuronal cell line, used as an in vitro model of native peripheral sensory neurons. Derived analytical expressions of the mean dose per cell accurately capture the pharmacokinetics by including a declining delivery rate and an intracellular particle degradation process. Comparison with experiment indicates a supply time constant, τ = 2 h. and a degradation rate constant, b = 0.71 h-1. Modeling the dose heterogeneity uses simulated data distributions, with time dependence incorporated by transforming data-bin values. The simulations mimic the dynamic nature of cell-to-cell dose variation and explain the observed trend of increasing numbers of high-dose cells at early time points, followed by a shift in distribution peak to lower dose between 4 to 8 h and a static dose profile beyond 8 h.

Inter-laboratory automation of the in vitro micronucleus assay using imaging flow cytometry and deep learning.

The in vitro micronucleus assay is a globally significant method for DNA damage quantification used for regulatory compound safety testing in addition to inter-individual monitoring of environmental, lifestyle and occupational factors. However, it relies on time-consuming and user-subjective manual scoring. Here we show that imaging flow cytometry and deep learning image classification represents a capable platform for automated, inter-laboratory operation. Images were captured for the cytokinesis-block micronucleus (CBMN) assay across three laboratories using methyl methanesulphonate (1.25-5.0 µg/mL) and/or carbendazim (0.8-1.6 µg/mL) exposures to TK6 cells. Human-scored image sets were assembled and used to train and test the classification abilities of the "DeepFlow" neural network in both intra- and inter-laboratory contexts. Harnessing image diversity across laboratories yielded a network able to score unseen data from an entirely new laboratory without any user configuration. Image classification accuracies of 98%, 95%, 82% and 85% were achieved for 'mononucleates', 'binucleates', 'mononucleates with MN' and 'binucleates with MN', respectively. Successful classifications of 'trinucleates' (90%) and 'tetranucleates' (88%) in addition to 'other or unscorable' phenotypes (96%) were also achieved. Attempts to classify extremely rare, tri- and tetranucleated cells with micronuclei into their own categories were less successful (≤ 57%). Benchmark dose analyses of human or automatically scored micronucleus frequency data yielded quantitation of the same equipotent concentration regardless of scoring method. We conclude that this automated approach offers significant potential to broaden the practical utility of the CBMN method across industry, research and clinical domains. We share our strategy using openly-accessible frameworks.

Empirical comparison of genotoxic potency estimations: the in vitro DNA-damage ToxTracker endpoints versus the in vivo micronucleus assay.

Genetic toxicology is an essential component of compound safety assessment. In the face of a barrage of new compounds, higher throughput, less ethically divisive in vitro approaches capable of effective, human-relevant hazard identification and prioritisation are increasingly important. One such approach is the ToxTracker assay, which utilises murine stem cell lines equipped with green fluorescent protein (GFP)-reporter gene constructs that each inform on distinct aspects of cellular perturbation. Encouragingly, ToxTracker has shown improved sensitivity and specificity for the detection of known in vivo genotoxicants when compared to existing 'standard battery' in vitro tests. At the current time however, quantitative genotoxic potency correlations between ToxTracker and well-recognised in vivo tests are not yet available. Here we use dose-response data from the three DNA-damage-focused ToxTracker endpoints and from the in vivo micronucleus assay to carry out quantitative, genotoxic potency estimations for a range of aromatic amine and alkylating agents using the benchmark dose (BMD) approach. This strategy, using both the exponential and the Hill BMD model families, was found to produce robust, visually intuitive and similarly ordered genotoxic potency rankings for 17 compounds across the BSCL2-GFP, RTKN-GFP and BTG2-GFP ToxTracker endpoints. Eleven compounds were similarly assessed using data from the in vivo micronucleus assay. Cross-systems genotoxic potency correlations for the eight matched compounds demonstrated in vitro-in vivo correlation, albeit with marked scatter across compounds. No evidence for distinct differences in the sensitivity of the three ToxTracker endpoints was found. The presented analyses show that quantitative potency determinations from in vitro data enable more than just qualitative screening and hazard identification in genetic toxicology.

Practical machine learning for disease diagnosis.

Deep learning neural networks are a powerful tool in the analytical toolbox of modern microscopy, but they come with an exacting requirement for accurately annotated, ground truth cell images. Otesteanu et al. (2021) elegantly streamline this process, implementing network training by using patient-level rather than cell-level disease classification.

Data Driven Cell Cycle Model to Quantify the Efficacy of Cancer Therapeutics Targeting Specific Cell-Cycle Phases From Flow Cytometry Results.

Many chemotherapeutic drugs target cell processes in specific cell cycle phases. Determining the specific phases targeted is key to understanding drug mechanism of action and efficacy against specific cancer types. Flow cytometry experiments, combined with cell cycle phase and division round specific staining, can be used to quantify the current cell cycle phase and number of mitotic events of each cell within a population. However, quantification of cell interphase times and the efficacy of cytotoxic drugs targeting specific cell cycle phases cannot be determined directly. We present a data driven computational cell population model for interpreting experimental results, where in-silico populations are initialized to match observable results from experimental populations. A two-stage approach is used to determine the efficacy of cytotoxic drugs in blocking cell-cycle phase transitions. In the first stage, our model is fitted to experimental multi-parameter flow cytometry results from untreated cell populations to identify parameters defining probability density functions for phase transitions. In the second stage, we introduce a blocking routine to the model which blocks a percentage of attempted transitions between cell-cycle phases due to therapeutic treatment. The resulting model closely matches the percentage of cells from experiment in each cell-cycle phase and division round. From untreated cell populations, interphase and intermitotic times can be inferred. We then identify the specific cell-cycle phases that cytotoxic compounds target and quantify the percentages of cell transitions that are blocked compared with the untreated population, which will lead to improved understanding of drug efficacy and mechanism of action.

Image-Based Cell Profiling Enables Quantitative Tissue Microscopy in Gastroenterology.

Immunofluorescence microscopy is an essential tool for tissue-based research, yet data reporting is almost always qualitative. Quantification of images, at the per-cell level, enables "flow cytometry-type" analyses with intact locational data but achieving this is complex. Gastrointestinal tissue, for example, is highly diverse: from mixed-cell epithelial layers through to discrete lymphoid patches. Moreover, different species (e.g., rat, mouse, and humans) and tissue preparations (paraffin/frozen) are all commonly studied. Here, using field-relevant examples, we develop open, user-friendly methodology that can encompass these variables to provide quantitative tissue microscopy for the field. Antibody-independent cell labeling approaches, compatible across preparation types and species, were optimized. Per-cell data were extracted from routine confocal micrographs, with semantic machine learning employed to tackle densely packed lymphoid tissues. Data analysis was achieved by flow cytometry-type analyses alongside visualization and statistical definition of cell locations, interactions and established microenvironments. First, quantification of Escherichia coli passage into human small bowel tissue, following Ussing chamber incubations exemplified objective quantification of rare events in the context of lumen-tissue crosstalk. Second, in rat jejenum, precise histological context revealed distinct populations of intraepithelial lymphocytes between and directly below enterocytes enabling quantification in context of total epithelial cell numbers. Finally, mouse mononuclear phagocyte-T cell interactions, cell expression and significant spatial cell congregations were mapped to shed light on cell-cell communication in lymphoid Peyer's patch. Accessible, quantitative tissue microscopy provides a new window-of-insight to diverse questions in gastroenterology. It can also help combat some of the data reproducibility crisis associated with antibody technologies and over-reliance on qualitative microscopy. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.

Label-Free Leukemia Monitoring by Computer Vision.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. While there are a number of well-recognized prognostic biomarkers at diagnosis, the most powerful independent prognostic factor is the response of the leukemia to induction chemotherapy (Campana and Pui: Blood 129 (2017) 1913-1918). Given the potential for machine learning to improve precision medicine, we tested its capacity to monitor disease in children undergoing ALL treatment. Diagnostic and on-treatment bone marrow samples were labeled with an ALL-discriminating antibody combination and analyzed by imaging flow cytometry. Ignoring the fluorescent markers and using only features extracted from bright-field and dark-field cell images, a deep learning model was able to identify ALL cells at an accuracy of >88%. This antibody-free, single cell method is cheap, quick, and could be adapted to a simple, laser-free cytometer to allow automated, point-of-care testing to detect slow early responders. Adaptation to other types of leukemia is feasible, which would revolutionize residual disease monitoring. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Activity Mapping of Children in Play Using Multivariate Analysis of Movement Events.

PURPOSE: (i) To develop an automated measurement technique for the assessment of both the form and intensity of physical activity undertaken by children during play. (ii) To profile the varying activity across a cohort of children using a multivariate analysis of their movement patterns. METHODS: Ankle-worn accelerometers were used to record 40 min of activity during a school recess, for 24 children over five consecutive days. Activity events of 1.1 s duration were identified within the acceleration time trace and compared with a reference motif, consisting of a single walking stride acceleration trace, obtained on a treadmill operating at a speed of 4 km h. Dynamic time warping of motif and activity events provided metrics of comparative movement duration and intensity, which formed the data set for multivariate mapping of the cohort activity using a principal component analysis (PCA). RESULTS: The two-dimensional PCA plot provided clear differentiation of children displaying diverse activity profiles and clustering of those with similar movement patterns. The first component of the PCA correlated to the integrated intensity of movement over the 40-min period, whereas the second component informed on the temporal phasing of activity. CONCLUSIONS: By defining movement events and then quantifying them by reference to a motion-standard, meaningful assessment of highly varied activity within free play can be obtained. This allows detailed profiling of individual children's activity and provides an insight on social aspects of play through identification of matched activity time profiles for children participating in conjoined play.

Diffusion Mapping of Eosinophil-Activation State.

Eosinophils are granular leukocytes that play a role in mediating inflammatory responses linked to infection and allergic disease. Their activation during an immune response triggers spatial reorganization and eventual cargo release from intracellular granules. Understanding this process is important in diagnosing eosinophilic disorders and in assessing treatment efficacy; however, current protocols are limited to simply quantifying the number of eosinophils within a blood sample. Given that high optical absorption and scattering by the granular structure of these cells lead to marked image features, the physical changes that occur during activation should be trackable using image analysis. Here, we present a study in which imaging flow cytometry is used to quantify eosinophil activation state, based on the extraction of 85 distinct spatial features from dark-field images formed by light scattered orthogonally to the illuminating beam. We apply diffusion mapping, a time inference method that orders cells on a trajectory based on similar image features. Analysis of exogenous cell activation using eotaxin and endogenous activation in donor samples with elevated eosinophil counts shows that cell position along the diffusion-path line correlates with activation level (99% confidence level). Thus, the diffusion mapping provides an activation metric for each cell. Assessment of activated and control populations using both this spatial image-based, activation score and the integrated side-scatter intensity shows an improved Fisher discriminant ratio rd = 0.7 for the multivariate technique compared with an rd = 0.47 for the traditional whole-cell scatter metric. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Physical activity, motor competence and movement and gait quality: A principal component analysis.

OBJECTIVE: While novel analytical methods have been used to examine movement behaviours, to date, no studies have examined whether a frequency-based measure, such a spectral purity, is useful in explaining key facets of human movement. The aim of this study was to investigate movement and gait quality, physical activity and motor competence using principal component analysis. METHODS: Sixty-five children (38 boys, 4.3 ±â€¯0.7y, 1.04 ±â€¯0.05 m, 17.8 ±â€¯3.2 kg, BMI; 16.2 ±â€¯1.9 kg∙m2) took part in this study. Measures included accelerometer-derived physical activity and movement quality (spectral purity), motor competence (Movement Assessment Battery for Children 2nd edition; MABC2), height, weight and waist circumference. All data were subjected to a principal component analysis, and the internal consistency of resultant components were assessed using Cronbach's alpha. RESULTS: Two principal components, with excellent internal consistency (Cronbach α >0.9) were found; the 1st principal component, termed "movement component", contained spectral purity, traffic light MABC2 score, fine motor% and gross motor% (α = 0.93); the 2nd principal component, termed "anthropometric component", contained weight, BMI, BMI% and body fat% (α = 0.91). CONCLUSION: The results of the present study demonstrate that accelerometric analyses can be used to assess motor competence in an automated manner, and that spectral purity is a meaningful, indicative, metric related to children's movement quality.

The origin of heterogeneous nanoparticle uptake by cells.

Understanding nanoparticle uptake by biological cells is fundamentally important to wide-ranging fields from nanotoxicology to drug delivery. It is now accepted that the arrival of nanoparticles at the cell is an extremely complicated process, shaped by many factors including unique nanoparticle physico-chemical characteristics, protein-particle interactions and subsequent agglomeration, diffusion and sedimentation. Sequentially, the nanoparticle internalisation process itself is also complex, and controlled by multiple aspects of a cell's state. Despite this multitude of factors, here we demonstrate that the statistical distribution of the nanoparticle dose per endosome is independent of the initial administered dose and exposure duration. Rather, it is the number of nanoparticle containing endosomes that are dependent on these initial dosing conditions. These observations explain the heterogeneity of nanoparticle delivery at the cellular level and allow the derivation of simple, yet powerful probabilistic distributions that accurately predict the nanoparticle dose delivered to individual cells across a population.

Label-Free Identification of White Blood Cells Using Machine Learning.

White blood cell (WBC) differential counting is an established clinical routine to assess patient immune system status. Fluorescent markers and a flow cytometer are required for the current state-of-the-art method for determining WBC differential counts. However, this process requires several sample preparation steps and may adversely disturb the cells. We present a novel label-free approach using an imaging flow cytometer and machine learning algorithms, where live, unstained WBCs were classified. It achieved an average F1-score of 97% and two subtypes of WBCs, B and T lymphocytes, were distinguished from each other with an average F1-score of 78%, a task previously considered impossible for unlabeled samples. We provide an open-source workflow to carry out the procedure. We validated the WBC analysis with unstained samples from 85 donors. The presented method enables robust and highly accurate identification of WBCs, minimizing the disturbance to the cells and leaving marker channels free to answer other biological questions. It also opens the door to employing machine learning for liquid biopsy, here, using the rich information in cell morphology for a wide range of diagnostics of primary blood. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Protein-Corona-by-Design in 2D: A Reliable Platform to Decode Bio-Nano Interactions for the Next-Generation Quality-by-Design Nanomedicines.

Hard corona (HC) protein, i.e., the environmental proteins of the biological medium that are bound to a nanosurface, is known to affect the biological fate of a nanomedicine. Due to the size, curvature, and specific surface area (SSA) 3-factor interactions inherited in the traditional 3D nanoparticle, HC-dependent bio-nano interactions are often poorly probed and interpreted. Here, the first HC-by-design case study in 2D is demonstrated that sequentially and linearly changes the HC quantity using functionalized graphene oxide (GO) nanosheets. The HC quantity and HC quality are analyzed using NanoDrop and label-free liquid chromatography-mass spectrometry (LC-MS) followed by principal component analysis (PCA). Cellular responses (uptake and cytotoxicity in J774 cell model) are compared using imaging cytometry and the modified lactate dehydrogenase assays, respectively. Cellular uptake linearly and solely correlates with HC quantity (R2 = 0.99634). The nanotoxicity, analyzed by retrospective design of experiment (DoE), is found to be dependent on the nanomaterial uptake (primary), HC composition (secondary), and nanomaterial exposure dose (tertiary). This unique 2D design eliminates the size-curvature-SSA multifactor interactions and can serve as a reliable screening platform to uncover HC-dependent bio-nano interactions to enable the next-generation quality-by-design (QbD) nanomedicines for better clinical translation.

Objective profiling of varied human motion based on normative assessment of magnetometer time series data.

OBJECTIVE: To quantify varied human motion and obtain an objective assessment of relative performance across a cohort. APPROACH: A wrist-worn magnetometer was used to record and quantify the complex motion patterns of 55 children aged 10 to 12 years old, generated during a fundamental movement skills programme. Sensor-based quantification of the physical activity used dynamic time warping of the magnetometer time series data for pairs of children. Pairwise comparison across the whole cohort produced a similarity matrix of all child to child correlations. Normative assessment scores were based on the Euclidean distance between n participants within an n - 1 multi-variate space, created from multi-dimensional scaling of the similarity matrix. The sensor-based scores were compared to the current standardised assessment which involves binary scoring of technique, outcome and time components by trained assessors. MAIN RESULTS: Visualisation of the relative performance using the first three axes of the multi-dimensional matrix, shows a 'performance sphere' in which children sit on concentric shells of increasing radius as performance deteriorates. Good agreement between standard and sensor scores is found, with Spearman rank correlation coefficients of the overall activity score in the range of 0.62-0.71 for different cohorts and a kappa statistic of 0.34 for categorisation of all 55 children into lower, middle, upper tertile and top 5% bands. SIGNIFICANCE: By using multi-dimensional analysis of similarity measures between participants rather than direct parameterisation of the physiological data, complex and varied patterns of physical motion can be quantified, allowing objective and robust profiling of relative function across participant groups.

Profiling Movement and Gait Quality Characteristics in Pre-School Children.

There is a dearth of suitable metrics capable of objectively quantifying motor competence. Further, objective movement quality characteristics during free play have not been investigated in pre-school children. The aims of this study were to characterize children's free play physical activity and investigate how gait quality characteristics cluster with free play in pre-school children (3-5 years old). Sixty-one children (39 boys; 4.3 ± 0.7 years, 1.04 ± 0.05 m, 17.8 ± 3.2 kg) completed the movement assessment battery for children and took part in free play while wearing an ankle- and hip-mounted accelerometer. Characteristics of movement quality were profiled using a clustering algorithm. Spearman's rho and the Mann-Whitney U tests were used to assess relationships between movement quality characteristics and motor competence classification differences in integrated acceleration and spectral purity, respectively. Significant differences were found between motor competency classifications for spectral purity and integrated acceleration (p < .001). Spectral purity was hierarchically clustered with motor competence and integrated acceleration. Significant positive correlations were found between spectral purity, integrated acceleration and motor competence (p < .001). This is the first study to report spectral purity in pre-school children and the results suggest that the underlying frequency component of movement is clustered with motor competence.