Quantifying nuclear wide chromatin compaction by phasor analysis of histone Förster resonance energy transfer (FRET) in frequency domain fluorescence lifetime imaging microscopy (FLIM) data.

The nanometer spacing between nucleosomes throughout global chromatin organisation modulates local DNA template access, and through continuous dynamic rearrangements, regulates genome function [1]. However, given that nucleosome packaging occurs on a spatial scale well below the diffraction limit, real time observation of chromatin structure in live cells by optical microscopy has proved technically difficult, despite recent advances in live cell super resolution imaging [2]. One alternative solution to quantify chromatin structure in a living cell at the level of nucleosome proximity is to measure and spatially map Förster resonance energy transfer (FRET) between fluorescently labelled histones - the core protein of a nucleosome [3]. In recent work we established that the phasor approach to fluorescence lifetime imaging microscopy (FLIM) is a robust method for the detection of histone FRET which can quantify nuclear wide chromatin compaction in the presence of cellular autofluorescence [4]. Here we share FLIM data recording histone FRET in live cells co-expressing H2B-eGFP and H2B-mCherry. The data was acquired in the frequency domain [5] and processed by the phasor approach to lifetime analysis [6]. The data can be valuable to researchers interested in using the histone FRET assay since it highlights the impact of cellular autofluorescence and acceptor-donor ratio on quantifying chromatin compaction. The data is related to the research article "Phasor histone FLIM-FRET microscopy quantifies spatiotemporal rearrangement of chromatin architecture during the DNA damage response" [4].

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