Lifetime imaging of GFP-PKC co-expressed with DsRed-cav in CHO cells: effect of Ca2+-ionophore. 2P-FLIM images were collected as described in the legend to Figure 2. Cells were treated with ionophore for 3 min before mounting and fixation as described in Methods. The epifluorescence image in the inset shows the DsRed-cav distribution (cytoplasmic) which was not affected by the Ca2+ ionophore. When the cytoplasmic area was analysed, (a) as shown by the area within the red line, both orange and green/blue areas are seen indicating the presence of both GFP-PKC and quenched GFP-PKC – note that only GFP lifetime can be observed in the lifetime images. This indicates that DsRed-cav was sufficiently close to the PKC-GFP to induce a quenching of the GFP by the DsRed, i.e. the PKC is translocating to caveolin containing areas. By contrast, in the nucleus (b) only GFP-PKC was expressed and the lifetime was unquenched (~2.2 ns). This is the same as the lifetime for GFP-PKC when only the latter is expressed (see Figure 2). Cells shown are representative images from replicate experiments.