Benchmarking three simple DNA staining-based image metrics for live-cell tracking of chromatin organization

Abstract

Quantifying chromatin-state dynamics in living cells remains challenging, in part because most methods require fixation or cell lysis. Here, we benchmark and introduce three simple live-cell image-derived metrics computed from routine DNA staining - the coefficient of variation (CV), 1-Gini, and the Diffuse Signal Index (DSI), introduced here - as fixation-free readouts of chromatin organization. Using HL60-derived neutrophils (dHL-60) undergoing NETosis as a model system with a pronounced compact-to-decompact chromatin transition, we show that all three metrics track progressive chromatin reorganization in live-cell trajectories, with DSI providing the strongest trajectory-level discrimination between NETing and non-NETing cells. Comparison with Tn5-based chromatin accessibility measurements in fixed cells shows that all three metrics correlate with chromatin accessibility, supporting their biological interpretability. We further show that all three metrics track chromatin reorganization in a second, mechanistically distinct biological process, capturing mitotic chromatin compaction and post-mitotic chromatin decompaction in U2OS cells. Together, our results provide a practical framework for extracting chromatin-organization readouts from routine live-cell DNA staining and highlight that metric performance is task- and context-dependent. To make our metrics accessible to a broad audience, we built NucMetrics, an open-source ImageJ/Fiji macro toolset that computes all three metrics without requiring programming.