Lifetime imaging of GFP-PKC co-expressed with DsRed-cav in CHO cells: effect of phorbol ester. Epifluorescence and 2P-FLIM images were collected as described in the legend to Figure 2. Cells were treated with TPA (100 nM) for 3 min before mounting and fixation as described in Methods. (a) left: GFP-PKC and DsRed-cav co-distribution revealed in a green and red epifluorescence image (red and green filters) and right: DsRed-cav visualised using a red filter, the caveolin was mainly restricted to the perinuclear region with PKC more widely distributed. Three distinct GFP lifetimes are discernable in the lifetime image and were separately analysed. For the lifetime images shown in (b, c and d), representative single point analyses within the regions enclosed by dashed white lines are analysed in (f, g and h) respectively, as follows: (b and f) peripheral regions (τ avg: 1.8 ns; single point 1.87 ns [χ2 1.00]); (c and g) the nuclear region (τ avg: 2.0 ns; single point 2.00 [χ2 1.00]); (d and h) the cytoplasm (τ avg: 1.5 ns; single point 1.48 ns [χ2 1.56]). (e) example of derivation of average lifetime for one of the three areas, shown for the cytoplasm, with the analysis for the area enclosed in red (lifetime colour coding shown in the inset) and other similar areas in the cytoplasm indicated by white arrows. Cells shown are representative images from replicate experiments.