Chromatin nanoscale compaction in live cells visualized by acceptor-to-donor ratio corrected Förster resonance energy transfer between DNA dyes

@Chromatin nanoscale architecture in live cells can be studied by Förster resonance energy transfer (FRET) between fluorescently labeled chromatin components, such as histones. A higher degree of nanoscale compaction is detected as a higher FRET level, since this corresponds to a higher degree of proximity between donor and acceptor molecules. However, in such a system, the stoichiometry of the donors and acceptors engaged in the FRET process is not well defined and, in principle, FRET variations could be caused by variations in the acceptor-to-donor ratio rather than distance. Here, to get a FRET level independent of the acceptor-to-donor ratio, we combine fluorescence lifetime imaging detection of FRET with a normalization of the FRET level to a pixel-wise estimation of the acceptor-to-donor ratio. We use this method to study FRET between two DNA binding dyes staining the nuclei of live cells. We show that this acceptor-to-donor ratio corrected FRET imaging reveals variations of nanoscale compaction in different chromatin environments. As an application, we monitor the rearrangement of chromatin in response to laser-induced microirradiation and reveal that DNA is rapidly decompacted, at the nanoscale, in response to DNA damage induction.