FIGURE 6.
FLIM, fD and mfD images of GFP-BD interaction with acetylated mCherry-H4 in the nucleus of HEK293 live cells using the TriM-FLIM system with 11 time-gated images. (A) Intensity, FLIM, fD, and mfD images of GFP-BD expressed alone as control (upper panel) or with mCherry-H4 as cotransfection (lower panel) in HEK293 live cells using the TriM-FLIM system at 950 nm excitation and 535AF45 emission filter with 11 time-gated images. Intensity images were obtained by summing the time-gated stack. FLIM images were obtained by using Eq. 2 in a pixel-by-pixel manner. White arrows show two chromatin domains in which GFP-BD mean lifetime decreases significantly. The mean lifetime averaged throughout the nucleus decreased from 2.41 ns for the control to 2.34 ns for the cotransfection. fD and mfD images were obtained by using Eq. 5 (with τD = 2.41 ns and τF = 0.65 ns) and Eq.6 (with τD = 2.41 ns), respectively. Three-dimensional representations of the corresponding fD and mfD images using a threshold limit given by the control (0.2) are also presented. Scale bar = 2 μm. (B) (Left panel) Corresponding lifetime histograms of control (black curve) and cotransfection (red curve). (Right panel) Comparison of fD (black) and mfD (red) histograms of the cotransfected cell (mean fD of 0.13 and mean mfD of 0.11).