CellProfiler has enabled the scientific research community to create flexible, modular image analysis pipelines since its release in 2005. Here, we describe CellProfiler 3.0, a new version of the software supporting both whole-volume and plane-wise analysis of three-dimensional (3D) image stacks, increasingly common in biomedical research. CellProfiler’s infrastructure is greatly improved, and we provide a protocol for cloud-based, large-scale image processing. New plugins enable running pretrained deep learning models on images. Designed by and for biologists, CellProfiler equips researchers with powerful computational tools via a well-documented user interface, empowering biologists in all fields to create quantitative, reproducible image analysis workflows.

Segmenting the nuclei of cells in microscopy images is often the first step in the quantitative analysis of imaging data for biological and biomedical applications. Many bioimage analysis tools can segment nuclei in images but need to be selected and configured for every experiment. The 2018 Data Science Bowl attracted 3,891 teams worldwide to make the first attempt to build a segmentation method that could be applied to any two-dimensional light microscopy image of stained nuclei across experiments, with no human interaction. Top participants in the challenge succeeded in this task, developing deep-learning-based models that identified cell nuclei across many image types and experimental conditions without the need to manually adjust segmentation parameters. This represents an important step toward configuration-free bioimage analysis software tools.

Stored red blood cells (RBCs) are needed for life-saving blood transfusions, but they undergo continuous degradation. RBC storage lesions are often assessed by microscopic examination or biochemical and biophysical assays, which are complex, time-consuming, and destructive to fragile cells. Here we demonstrate the use of label-free imaging flow cytometry and deep learning to characterize RBC lesions. Using brightfield images, a trained neural network achieved 76.7% agreement with experts in classifying seven clinically relevant RBC morphologies associated with storage lesions, comparable to 82.5% agreement between different experts. Given that human observation and classification may not optimally discern RBC quality, we went further and eliminated subjective human annotation in the training step by training a weakly supervised neural network using only storage duration times. The feature space extracted by this network revealed a chronological progression of morphological changes that better predicted blood quality, as measured by physiological hemolytic assay readouts, than the conventional expert-assessed morphology classification system. With further training and clinical testing across multiple sites, protocols, and instruments, deep learning and label-free imaging flow cytometry might be used to routinely and objectively assess RBC storage lesions. This would automate a complex protocol, minimize laboratory sample handling and preparation, and reduce the impact of procedural errors and discrepancies between facilities and blood donors. The chronology-based machine-learning approach may also improve upon humans’ assessment of morphological changes in other biomedically important progressions, such as differentiation and metastasis.

Imaging flow cytometry (IFC) captures multichannel images of hundreds of thousands of single cells within minutes. IFC is seeing a paradigm shift from low- to high-information-content analysis, driven partly by deep learning algorithms. We predict a wealth of applications with potential translation into clinical practice.

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.