Fluorescence lifetime imaging of GFP-PKC expressed in CHO cells. GFP-PKC was transiently expressed in CHO cells as described in the legend in Figure 1 and detailed under Methods. Images were acquired using a Nikon 2000 inverted epifluorescence microscope with a 60× objective and GFP/DsRed filter sets and an ICAM camera. The 2P-FLIM images were collected using the TCSPC fast scanning imaging mode as described under Methods. (a) Conventional epifluorescence image of GFP-PKC distribution in a resting CHO cell, (b) lifetime image of the same cell with the analysis area enclosed by the red line (cytosol) shown (with colour coding) in the inset and giving an average lifetime of ~2.2 ns and (c) with the analysis area enclosed by the red line (nucleus) shown in the inset giving an average lifetime of ~2.0 ns. Cells shown are representative images from replicate experiments.