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

Quantifying chromatin-state dynamics in living cells remains challenging, in part because most methods require fixation or cell lysis. Here, we introduce two simple DNA image-derived metrics - Diffuse Signal Index (DSI)and 1-Gini - and benchmark them against the coefficient of variation (CV) as fixation-free readouts of chromatin reorganization. Leveraging the pronounced compact-to-decompact chromatin transition of NETosis, we show that all three metrics track progressive chromatin reorganization, with DSI providing the strongest trajectory-level discrimination between NETing and non-NETing cells. All three metrics also correlate with ATAC-see-based chromatin accessibility measurement in fixed cells, supporting their biological interpretability. In dividing cells, the metrics capture mitotic chromatin compaction and post-mitotic decompaction, demonstrating applicability in diverse biological processes. Together, these results provide a practical framework for extracting readouts of chromatin reorganization from routine live-cell DNA staining. We also provide NucMetrics, an open-source ImageJ/Fiji macro toolset for easily computing CV, DSI and 1-Gini.