All cells were pre-treated with IFNγ for 16 hr prior investigation (a) Left panels. GFP fluorescence intensity images of G-mGBP2 or G-mGBP2/mCh-mGBP(1,2,3,5,6) MEFs highlighted with selections of pixels with different intensities. Bars, 10 µm. Right panels. Two MFIS 2D-histograms of GFP fluorescence lifetimes (<τD>f) on y axes, GFP/mCherry fluorescence intensity ratios (FG/FR) or photon number per pixel (N) on x axes. The pixel populations locating in cytosol (N < 1000: red island) and VLS (N > 1000: green island) were separated according to photon numbers. (b) Schematic 2D MFIS plot detailing the effects of hetero- and/or homo-FRET on a reference data set (green circle). The average GFP <τD>f is plotted on the x axis from short to long, while the average steady-state rD is plotted on the y axis. For detailed explanation refer to results section. (c) Upper panel. For individual G-mGBP2, G-mGBP2/mCh-mGBP2or G-mGBP2/mCh-mGBP6 MEFs, mean values of rD in the cytosol (empty squares) and in the VLS (solid squares) were plotted against <τD>f and G-mGBP2 concentrations (CG-mGBP2). Lower panel. Mean anisotropy <rD>loc values (average over all cells weighted by CG-mGBP2) were plotted against <τD>f or CG-mGBP2. The two left panels contain an overlay calculated according to the Perrin equation: with GFP fundamental anisotropy r0 = 0.38 and rotational correlation time ρglobal= 15 ns. The two right panels are overlaid with function curves plotting which assumes a mGBP2 Langmuir binding model with an apparent dissociation constant KD,app. In all donor-only experiments the formation of mGBP2 homo-multimers could be described by KD,app = 9 μM, rmax = 0.32 and rmin = 0.22 (black curve). If other interaction processes interfere with homo-FRET between G-mGBP2 proteins, this curve is shifted upwards (violet curve) while keeping KD,app invariant (rmax = 0.345 and rmin = 0.245). (d, e) εmix(t) and ε(D,A)(t) diagrams of a representative G-mGBP2/mCh-mGBP2 MEF (d) and G-mGBP2/mCh-mGBP6 MEF (e). The drop in εmix(t) curves, as marked by the arrows, represents the species fractions of FRET-active complexes (xFRET) in the VLS (green) and in the cytosol (red). In (d), the FRET rate constant (kFRET) in the cytosol is 0.09 ns-1 and in the VLS 0.20 ns-1.