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The temporal context of cell death decisions remains generally hidden in ensemble measurements with endpoint readouts. Here, we describe a method to extract event times from fluorescence time traces of cell death-related markers in automated live-cell imaging on single-cell arrays (LISCA) using epithelial A549 lung and Huh7 liver cancer cells as a model system. In pairwise marker combinations, we assess the chronological sequence and delay times of the events lysosomal membrane permeabilization, mitochondrial outer membrane permeabilization and oxidative burst after exposure to 58 nm amino-functionalized polystyrene nanoparticles (PS-NH 2  nanoparticles). From two-dimensional event-time scatter plots we infer a lysosomal signal pathway at a low dose of nanoparticles (25 µg mL −1 ) for both cell lines, while at a higher dose (100 µg mL −1 ) a mitochondrial pathway coexists in A549 cells, but not in Huh7. In general, event-time correlations provide detailed insights into heterogeneity and interdependencies in signal transmission pathways.

A high-throughput microscopy method for single-cell analysis of event-time correlations in nanoparticle-induced cell death