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

Abstract

@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.

Nanoscale Distribution of Nuclear Sites by Super-Resolved Image Cross-Correlation Spectroscopy

“…For this reason, alternative strategies have been developed to investigate cellular processes at the nanoscale. For instance, a popular method to probe molecular distances in the nanometer range is Förster resonance energy transfer (FRET) ( 7 , 8 , 9 ), but unfortunately, FRET is not sensitive when distances are larger than ∼10 nm. Similarly, in situ proximity ligation assay (PLA) is a powerful method to visualize proximity of two labeled species, but its sensitivity does not exceed 40 nm ( 10 , 11 ).…”

Optical nanoscopy

“…where E is the FRET efficiency and β is the brightness of the respective fluorophores [51] tal limit. The limit is set by concepts of information theory [20].…”

Single-Molecular Förster Resonance Energy Transfer Measurement on Structures and Interactions of Biomolecules

“…Förster resonance energy transfer (FRET) describes the physical phenomenon of energy transfer between two photosensitive molecules, rendering convenience for real-time dynamic research of molecules under various physiological conditions [ 1 , 2 , 3 , 4 ]. FRET was first proposed in 1948 by Theodor Förster [ 5 ].…”