Fluorescence lifetime imaging of GFP-PKC co-expressed with DsRed-cav in CHO cells. 2P-FLIM images were collected as described in the legend to Figure 2. Co-expression of the GFP-PKC with DsRed-cav does not affect the lifetime of the GFP showing that in the unstimulated state PKC is not associated with caveolin. Epifluorescence images for excitation of DsRed (a) and GFP along with DsRed (b) showing that the PKC and caveolin co-distributed in the cytosol. Fluorescence lifetime images with the analysis area enclosed by the red line, (cytosol) (c) or nucleus both essentially showing a lifetime as for Figs 2–3 centred around ~2.2 ns. Cells shown are representative images from replicate experiments.