Experimental FLIM measurements with our fast-FLIM prototype for quantifying G protein activation in living cells.

The polar plots of a 3T3 cell co-expressing Rac-GFP and PBD-mCherry (with Nmean = 850 photons) and of a cell co-expressing Rac-GFP+mCherry (with Nmean = 830 photons) as a reference (negative control) are shown in (A). The shift between the two spots is the proof of lifetime modification which is also clearly visible in the mean lifetime images <τ> (scale bar: 10 µm). We have plotted in (B) the temporal variations of the mean lifetime for each cell. We have also calculated fDP, fDM and mfD of the cell co-expressing Rac-GFP and PBD-mCherry and the images are reported respectively in (C1), (C2) and (C3). The evolution of each parameter calculated in the white region of interest is also plotted in (C4). We finally show the images of τFP and τFM in (D1) and (D2) and the corresponding lifetime distributions in (D3).