Interpreting Image‐based Profiles using Similarity Clustering and Single‐Cell Visualization

Abstract Image‐based profiling quantitatively assesses the effects of perturbations on cells by capturing a breadth of changes via microscopy. Here, we provide two complementary protocols to help explore and interpret data from image‐based profiling experiments. In the first protocol, we examine the similarity among perturbed cell samples using data from compounds that cluster by their mechanisms of action. The protocol includes steps to examine feature‐driving differences between samples and to visualize correlations between features and treatments to create interpretable heatmaps using the open‐source web tool Morpheus. In the second protocol, we show how to interactively explore images together with the numerical data, and we provide scripts to create visualizations of representative single cells and image sites to understand how changes in features are reflected in the images. Together, these two tutorials help researchers interpret image‐based data to speed up research. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : Exploratory analysis of profile similarities and driving features Basic Protocol 2 : Image and single‐cell visualization following profile interpretation