The ability of cells to maintain genome integrity depends not only on the recruitment of repair proteins but also on the dynamic reorganization of chromatin. Thus, it is important to develop methods for probing simultaneously the mobility of molecules and chromatin architecture at different spatial and temporal scales. To probe molecular mobility with microsecond resolution, we recently established segmented fluorescence correlation spectroscopy (FCS), a powerful and widely accessible method to measure diffusion on commercially available confocal microscopes, removing the need for specialized hardware (Longo et al, Sci Rep 2024). This strategy enables accurate mapping of diffusion across nuclear compartments and provides quantitative insights into protein mobility during DNA damage response. Applied to PARP1, segmented FCS revealed its change in mobility during the earliest steps of chromatin repair (Longo et al, Biophys J 2025). To probe chromatin compaction from very noisy images, we developed the coefficient of variation by image correlation spectroscopy (CV-ICS). Unlike conventional CV, which is strongly affected by noise, CV-ICS extracts the spatial autocorrelation of fluorescence fluctuations, filtering out uncorrelated noise and yielding a robust, biologically meaningful measure of chromatin structure in live cells, even under low-photon conditions. To investigate chromatin environment at the nanoscale, we are now developing a live-cell imaging approach that integrates FLIM-FRET of vital DNA dyes with environmental-sensitive probes to directly quantify chromatin compaction and plasticity during DNA repair. By combining this strategy with image scanning microscopy (ISM), we will map nanoscale chromatin properties at the improved spatial resolution provided by ISM. This multimodal fluorescence-based approach aims to uncover the dynamic regulation of chromatin during DNA repair and provides a powerful strategy to understand how nuclear architecture safeguards genome stability. Partially funded by: AIRC-MFAG 2018 ID.21931; PHARMA-HUB T4-AN-04 (E63C22001680001); PRIN-PNRR 2022 Project: LLIPS P20228CCLL.
Longo et al. (Sun,) studied this question.