Fluorescence intensity and lifetime measurement of free and particle-boundfluorophore in a sample stream by phase-sensitive flow cytometry

We report a novel method to quantify fluorescence intensity and lifetime of free (solution) and particle-bound fluorophore in a flow stream. The techn ique combines flow cytometry and frequency-domain lifetime spectroscopy pri nciples to make unique fluorescence measurements on free fluorophore and fl uorophore-labeled particles. Fluorophore-labeled microspheres suspended in a fluorophore solution are analyzed as they flow through a chamber and pass across an intensity-modulated laser beam consisting of a continuous-wave ( cw) direct-current (dc) and high-frequency (sine wave) excitation component . Fluorescence emission signals consisting of a dc-offset steady-state sinu soidal signal (fluorophore solution) and a sinusoidally modulated Gaussian- shaped signal pulse (fluorophore-labeled particles) are processed electroni cally to quantify intensities and lifetimes. The cw-excited, particle-assoc iated fluorophore (pulse) and steady-state (dc) solution fluorescence inten sity signals are measured using low-pass filtering to remove the high-frequ ency signal components and an ac-coupled and a gated dc amplifier to proces s the respective particle-bound and free fluorophore signals. The high-freq uency excited, particle-bound fluorophore and free fluorophore lifetimes ar e individually measured using two pairs of phase-sensitive detectors to pro vide signals proportional to the sine and cosine of the respective phase sh ifts, which are ratioed to determine the respective lifetimes. The fluoresc ence signal intensity and lifetime detection channel outputs are displayed as frequency distribution histograms using a computer-based data acquisitio n system. [S0034-6748(99)00612-7].