Fluorescence lifetime measurements in a flow cytometer by amplitude demodulation using digital data acquisition...

We have developed a method for fluorescence lifetime measurements in a flow cytometer based upon the amplitude demodulation of the fluorescence signals using digital data acquisition techniques. Amplitude demodulation is one of the two methods by which excited state lifetimes may be investigated in the frequency domain. The other method involves the phase-shift measurements. In frequency-domain measurement techniques, the amplitude-demodulation and phase-shift data serve mutually complementary roles to enhance the analytical capabilities of the measurements. The purpose of having amplitude demodulation measurement capability is to obtain information that supplements, rather than replaces, that obtained by the phase-shift method alone. Application of amplitude demodulation measurements has been widely explored in static, cuvette-based, frequency domain systems. However, due to time dependence of the amplitude of the modulated fluorescence signal in a flow cytometer, the amplitude demodulation measurements in flow turns out to be more complicated than similar measurements in a static system. The goal of the present work is to explore the problems involved in amplitude demodulation measurements in flow (using digital method), through detailed theoretical modeling and use the model to develop a practical method that can be incorporated into a flow cytometer to measure amplitude modulation lifetimes. We experimentally verify the amplitude demodulation measurement capability of this method using fluorescent microspheres. The experimental measurements show good agreement with static frequency-domain measurements on microspheres in bulk suspensions.