Abstract
<title>Abstract</title> <p>Chromatin compaction is a fundamental determinant of genome function, yet quantitative methods capable of measuring its nanoscale organization while directly relating it to local epigenetic states remain scarce. Fluorescence lifetime imaging microscopy coupled to Förster resonance energy transfer (FLIM-FRET) offers a unique opportunity to quantify chromatin packing in intact cells while being fully compatible with multiplexed fluorescence imaging of chromatin-associated proteins and epigenetic markers. Here we present a quantitative FLIM-FRET framework based on the DNA-binding dyes Hoechst 33342 and Syto13 that enables concentration-independent mapping of chromatin compaction with extended dynamic range and broad experimental applicability. The framework relies on a generalized description of donor–acceptor energy transfer that remains valid across arbitrary dye stoichiometries and demonstrates that chromatin compaction can be accurately recovered from either phasor FLIM or conventional average-lifetime (FastFLIM) measurements, making the method readily transferable to standard commercial FLIM platforms. Application to lung and thyroid cell models revealed biologically meaningful differences in chromatin compaction associated with oncogenic backgrounds and 3D tissue organization. Additionally, multiplexed imaging with histone post-translational modifications, RNA polymerase II and genetically encoded histone reporters enabled direct pixel-wise correlation between local chromatin architecture and epigenetic features, revealing the positive association of nanoscale packing with transcriptionally repressed chromatin but not with local histone abundance. Finally, comparison with conventional texture-based metrics demonstrated that fluorescence entropy and intensity heterogeneity capture structural features largely distinct from nanoscale chromatin packing. This work establishes a broadly applicable platform for quantitatively linking chromatin architecture to gene regulation in fixed and living cells.</p>