Flow cytometer for resolving signals from heterogeneous fluorescence emissions and quantifying lifetime in fluorochrome-labeled cells/particles by phase-sensitive detection

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Abstract
A phase-sensitive flow cytometer has been developed that combines flow cytometry and fluorescence lifetime spectroscopy measurement principles to provide unique capabilities for making phase-resolved measurements on fluorochrome-labeled cells and particles. Stained cells are analyzed as they intersect a high-frequency intensity-modulated (sinusoid) laser excitation beam. Fluorescence is measured orthogonally using only a single-channel optical detector. The detector output signals, which are phase shifted from a reference signal and amplitude demodulated, are processed by phase-sensitive detection electronics to resolve signals from heterogeneous fluorescence emissions and quantify single-component decay times. Results show signal phase shift and amplitude demodulation on fluorospheres; a detection limit threshold of 300–500 fluorescein molecules equivalence for excitation frequencies 1–30 MHz; a measurement precision (coefficient of variation) of 1.8% on alignment fluorospheres and 3.6% on cells stained for DNA content; the resolution of fluorescence signals from cells stained in combination with two fluorochromes, based on differences in their decay times; and the measurement of fluorescence lifetimes directly by the two-phase ratio method.