In signaling studies, the investigator can compensate for low fluorescent signal by exciting the cells and collecting signal for longer times, limited only by the eventual photobleaching of the XFPs. However, when using flow cytometry, the investigator can acquire XFP signal only during the time the cell passes through the laser beam, but to some extent can compensate for the short signal acquisition time by the brighter excitation light provided by the cytometer’s lasers. In addition to measurement of fluorophore’s fluorescence intensity within a specified wavelength range, it is also possible to measure its fluorescence lifetime. This is the mean time between the fluorophore’s excitation and its decay to the ground state, typically several nanoseconds. This lifetime is comprised of a natural “radiative lifetime,” characteristic of each species of fluorophore, and a contribution brought about by the fluorophore’s environment. For example, a crowded atomic Paclitaxel Microtubule inhibitor environment near the fluorophore shortens the lifetime by providing more paths for non-radiative decay from the excited state. The time that it takes an excited fluorophore of a known species to emit a photon thus contains information about the fluorophore’s immediate cellular environment. Information from fluorescence lifetime measurements can complement information from measurements of fluorescence intensity. For example, FRET occurring during the association of a donor and acceptor XFP pair causes a decrease in the ratio of donor-to-acceptor fluorescence, and a concomitant decrease in the fluorescence lifetime of the donor. In the future, we also hope that fluorescence lifetime information might increase the number of distinguishable XFP signals from individual cells, facilitating the use of Bayesian network methods in live cells to find features of signaling networks specific to different disease states and generate hypotheses about cause and effect relationships among measured variables. One way to measure fluorescence lifetime is by “frequency domain” methods, in which the investigator excites collections of fluorophores using light modulated sinusoidally at radio frequencies. The excited fluorophores emit light modulated at the same frequency as the excitatory light, but the modulation is delayed in phase and reduced in modulation depth, and/or by using the demodulation. Simultaneous measurement of both phase delay and demodulation in frequency domain fluorescence lifetime measurements enables the use of “phasor analysis”. In this, the investigator uses the phase and demodulation measurements to construct phasors– complex numbers with magnitudes equal to the measured demodulation factors, and arguments equal to the measured phase delays.