Spectral response and caspase sensing of CaspeR3. A. Excitation (dashed lines) and emission (solid lines) spectra for TagGFP (green) and TagRFP (red). Spectral overlap is filled with gray. B. Emission spectra of CaspeR3 before (dashed line) and after digestion by Caspase 3 (solid line). C. Green-to-red emission ratio change of CaspeR3 upon staurosporine-induced apoptosis. Approximately 40–50 min after staurosporine infusion, cells demonstrated pronounced changes fluorescence signal ratio. Emission ratio shown for 5 cells, time point aligned to the median of ratio changes, individual for each cell. Excitation at 488 nm, emission was detected at 500–530 nm and 560–600 nm. D. TagGFP fluorescence lifetime τφ (solid lines) and τM (dashed lines) changes for CaspeR3 during staurosporine-induced apoptosis. Excitation was at 488 nm and donor fluorescence emission was passed through a 500–530 nm bandpass filter. E. Two channel fluorescence imaging of CaspeR3 upon staurosporine-induced apoptosis in HeLa cells. Time, in minutes, is shown after staurosporine infusion.