Live-cell imaging and analysis reveal cell phenotypic transition dynamics inherently missing in snapshot data.

Recent advances in single-cell techniques catalyze an emerging field of studying how cells convert from one phenotype to another, in a step-by-step process. Two grand technical challenges, however, impede further development of the field. Fixed cell-based approaches can provide snapshots of high-dimensional expression profiles but have fundamental limits on revealing temporal information, and fluorescence-based live-cell imaging approaches provide temporal information but are technically challenging for multiplex long-term imaging. We first developed a live-cell imaging platform that tracks cellular status change through combining endogenous fluorescent labeling that minimizes perturbation to cell physiology and/or live-cell imaging of high-dimensional cell morphological and texture features. With our platform and an A549 VIM-RFP epithelial-to-mesenchymal transition (EMT) reporter cell line, live-cell trajectories reveal parallel paths of EMT missing from snapshot data due to cell-cell dynamic heterogeneity. Our results emphasize the necessity of extracting dynamical information of phenotypic transitions from multiplex live-cell imaging.