Spatial proteomics in neurons at single-protein resolution.

To understand biological processes, it is necessary to reveal the molecular heterogeneity of cells by gaining access to the location and interaction of all biomolecules. Significant advances were achieved by super-resolution microscopy, but such methods are still far from reaching the multiplexing capacity of proteomics. Here, we introduce secondary label-based unlimited multiplexed DNA-PAINT (SUM-PAINT), a high-throughput imaging method that is capable of achieving virtually unlimited multiplexing at better than 15 nm resolution. Using SUM-PAINT, we generated 30-plex single-molecule resolved datasets in neurons and adapted omics-inspired analysis for data exploration. This allowed us to reveal the complexity of synaptic heterogeneity, leading to the discovery of a distinct synapse type. We not only provide a resource for researchers, but also an integrated acquisition and analysis workflow for comprehensive spatial proteomics at single-protein resolution.

PXPermute reveals staining importance in multichannel imaging flow cytometry.

Imaging flow cytometry (IFC) allows rapid acquisition of numerous single-cell images per second, capturing information from multiple fluorescent channels. However, the traditional process of staining cells with fluorescently labeled conjugated antibodies for IFC analysis is time consuming, expensive, and potentially harmful to cell viability. To streamline experimental workflows and reduce costs, it is crucial to identify the most relevant channels for downstream analysis. In this study, we introduce PXPermute, a user-friendly and powerful method for assessing the significance of IFC channels, particularly for cell profiling. Our approach evaluates channel importance by permuting pixel values within each channel and analyzing the resulting impact on machine learning or deep learning models. Through rigorous evaluation of three multichannel IFC image datasets, we demonstrate PXPermute's potential in accurately identifying the most informative channels, aligning with established biological knowledge. PXPermute can assist biologists with systematic channel analysis, experimental design optimization, and biomarker identification.

Built to Last? Reproducibility and Reusability of Deep Learning Algorithms in Computational Pathology.

Recent progress in computational pathology has been driven by deep learning. While code and data availability are essential to reproduce findings from preceding publications, ensuring a deep learning model's reusability is more challenging. For that, the codebase should be well-documented and easy to integrate into existing workflows and models should be robust toward noise and generalizable toward data from different sources. Strikingly, only a few computational pathology algorithms have been reused by other researchers so far, let alone employed in a clinical setting. To assess the current state of reproducibility and reusability of computational pathology algorithms, we evaluated peer-reviewed articles available in PubMed, published between January 2019 and March 2021, in 5 use cases: stain normalization; tissue type segmentation; evaluation of cell-level features; genetic alteration prediction; and inference of grading, staging, and prognostic information. We compiled criteria for data and code availability and statistical result analysis and assessed them in 160 publications. We found that only one-quarter (41 of 160 publications) made code publicly available. Among these 41 studies, three-quarters (30 of 41) analyzed their results statistically, half of them (20 of 41) released their trained model weights, and approximately a third (16 of 41) used an independent cohort for evaluation. Our review is intended for both pathologists interested in deep learning and researchers applying algorithms to computational pathology challenges. We provide a detailed overview of publications with published code in the field, list reusable data handling tools, and provide criteria for reproducibility and reusability.

Explainable machine learning for profiling the immunological synapse and functional characterization of therapeutic antibodies.

Therapeutic antibodies are widely used to treat severe diseases. Most of them alter immune cells and act within the immunological synapse; an essential cell-to-cell interaction to direct the humoral immune response. Although many antibody designs are generated and evaluated, a high-throughput tool for systematic antibody characterization and prediction of function is lacking. Here, we introduce the first comprehensive open-source framework, scifAI (single-cell imaging flow cytometry AI), for preprocessing, feature engineering, and explainable, predictive machine learning on imaging flow cytometry (IFC) data. Additionally, we generate the largest publicly available IFC dataset of the human immunological synapse containing over 2.8 million images. Using scifAI, we analyze class frequency and morphological changes under different immune stimulation. T cell cytokine production across multiple donors and therapeutic antibodies is quantitatively predicted in vitro, linking morphological features with function and demonstrating the potential to significantly impact antibody design. scifAI is universally applicable to IFC data. Given its modular architecture, it is straightforward to incorporate into existing workflows and analysis pipelines, e.g., for rapid antibody screening and functional characterization.

InstantDL: an easy-to-use deep learning pipeline for image segmentation and classification.

BACKGROUND: Deep learning contributes to uncovering molecular and cellular processes with highly performant algorithms. Convolutional neural networks have become the state-of-the-art tool to provide accurate and fast image data processing. However, published algorithms mostly solve only one specific problem and they typically require a considerable coding effort and machine learning background for their application. RESULTS: We have thus developed InstantDL, a deep learning pipeline for four common image processing tasks: semantic segmentation, instance segmentation, pixel-wise regression and classification. InstantDL enables researchers with a basic computational background to apply debugged and benchmarked state-of-the-art deep learning algorithms to their own data with minimal effort. To make the pipeline robust, we have automated and standardized workflows and extensively tested it in different scenarios. Moreover, it allows assessing the uncertainty of predictions. We have benchmarked InstantDL on seven publicly available datasets achieving competitive performance without any parameter tuning. For customization of the pipeline to specific tasks, all code is easily accessible and well documented. CONCLUSIONS: With InstantDL, we hope to empower biomedical researchers to conduct reproducible image processing with a convenient and easy-to-use pipeline.