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

Abstract

We report a novel method to quantify fluorescence intensity and lifetime of free (solution) and particle-bound fluorophore in a flow stream. The technique combines flow cytometry and frequency-domain lifetime spectroscopy principles to make unique fluorescence measurements on free fluorophore and fluorophore-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 sinusoidal signal (fluorophore solution) and a sinusoidally modulated Gaussian-shaped signal pulse (fluorophore-labeled particles) are processed electronically to quantify intensities and lifetimes. The cw-excited, particle-associated fluorophore (pulse) and steady-state (dc) solution fluorescence intensity signals are measured using low-pass filtering to remove the high-frequency signal components and an ac-coupled and a gated dc amplifier to process the respective particle-bound and free fluorophore signals. The high-frequency excited, particle-bound fluorophore and free fluorophore lifetimes are individually measured using two pairs of phase-sensitive detectors to provide signals proportional to the sine and cosine of the respective phase shifts, which are ratioed to determine the respective lifetimes. The fluorescence signal intensity and lifetime detection channel outputs are displayed as frequency distribution histograms using a computer-based data acquisition system.