The technology for measuring fluorescence decay lifetimes by flow cytometry in single cells labeled with fluorochromes is under development. The goals are to resolve signals from fluorescent dyes (bound to macromolecular components in cells) that have overlapping emission spectra, but different lifetimes, and to quantify fluorescence lifetime as a parameter. Stained cells are analyzed as they flow through a chamber and intersect a high-frequency, intensity-modulated laser beam. The modulated fluorescence signals, which are shifted in phase from a reference, are processed electronically to suppress one signal component and pass the other in a multicolored mixture based on fluorescence lifetime/phase differences. Experiments have demonstrated (1) phase shift in flow on propidium iodide (PI)- and ethidium bromide (EB)-stained cells, (2) a fluorescence detection threshold (sensitivity) of 300 to 500 fluorescein molecules equivalence for excitation frequencies ranging from 10 to 30 MHz, (3) measurement precision less than 1.5% on fluorospheres and 4% on PI- stained (DNA content) cells, and (4) lifetime measurement (nanosecond range) by phase shift using DNA check fluorospheres and fluorescein isothiocyanate (FITC)- and PI- stained cells. In addition, we have demonstrated the resolution of signals from simultaneous fluorescence emissions in cells stained with PI and FITC and with PE and Red613 conjugated Thy 1.2 antibody and PI. We continue to evaluate the first generation phase sensitive-flow cytometer instrumental capabilities, the ability to resolve fluorescence emissions based on lifetime differences using a variety of fluorochromes, and the ability to quantify lifetime directly by the amplitude demodulation.
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