FLIM–FRET measurements spatially discriminate differential chromatin compaction levels in vivo. (A) HeLa^H2B-GFP stable cell line transiently transfected with mCherry-C1 empty vector (top) and HeLa^H2B-2FP stable cell line (bottom) were imaged using MPLSM. In both cases, the mean fluorescence lifetime τ (nanoseconds) is detected, and its spatial distribution at each pixel of the ROI (τ map) is shown as described in Materials and methods. Fluorescence lifetimes are presented in a continuous pseudocolor scale representing time values ranging from 2.1 to 2.35 ns. The mean lifetime distribution curves of the donor (H2B-GFP) are shown on the right. Dashed lines mark the position of the peaks of the lifetime distribution curves. (B) The FRET efficiency (percentage) and its spatial distribution (FRET (%) map) are depicted in the ROI using discrete colors (see Materials and methods). The ROI comprised the interphase nuclei; the right panel shows a higher magnification of a nucleus (boxed area) revealing high FRET areas at the nuclear periphery (arrows). (C) FRET distributions graph showing three distinct populations distinguished using discrete pseudocolors: blue (low), FRET efficiency up to 3%; green (medium), between 3–6%; and red (high), 6–12%. The statistical analysis of at least 10 cells per condition is presented as box and whisker plots on the right. The three distinct FRET efficiency populations detected in interphase HeLa^H2B-2FP cells are represented by colored boxes (i.e., low [blue], medium [green], and high [red] FRET populations, respectively). The mean FRET efficiency value is indicated on top of each box. Bars, 10 µm.