FLUORESCENCE LIFETIME MEASUREMENTS IN A FLOW CYTOMETER BY AMPLITUDE DEMODULATION USING DIGITAL DATA-ACQUISITION TECHNIQUE

We have developed a method for fluorescence lifetime measurements in a flow cytometer based upon the amplitude demodulation of the fluoresce nce signals using digital data acquisition techniques. Amplitude demod ulation is one of the two methods by which excited state lifetimes may be investigated in the frequency domain. The other method involves th e phase-shift measurements. In frequency-domain measurement techniques , the amplitude-demodulation and phase-shift data serve mutually compl ementary roles to enhance the analytical capabilities of the measureme nts. The purpose of having amplitude demodulation measurement capabili ty is to obtain information that supplements, rather than replaces, th at 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 th e amplitude of the modulated fluorescence signal in a flow cytometer, the amplitude demodulation measurements in flow turns out to be more c omplicated than similar measurements in a static system. The goal of t he present work is to explore the problems involved in amplitude demod ulation measurements in flow (using digital method), through detailed theoretical modeling and use the model to develop a practical method t hat can be incorporated into a flow cytometer to measure amplitude mod ulation lifetimes. We experimentally verify the amplitude demodulation measurement capability of this method using fluorescent microspheres. The experimental measurements show good agreement with static frequen cy-domain measurements on microspheres in bulk suspensions. (C) 1994 W iley-Liss, Inc.