FLOW CYTOMETER FOR QUANTIFYING FLUORESCENCE LIFETIME

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The technology for measuring fluorescence lifetime by flow cytometry (FCM) in single cells labeled with fluorochromes is under development. The long-term goals are to develop the FCM technology for resolving 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 for studying fluorochrome interactions with cellular constituents and each other. The specific aims of this project are: (1) to evaluate the first generation instrument in terms of sensitivity, precision, dynamic range, and accuracy of lifetime measurements; (2) to determine the instrument's capability to resolve the emission signals from spectrally-overlapping fluorochromes, including the effectiveness in suppressing background interference caused by cellular autofluorescence, nonspecific staining, free and unbound dye, and Raman scatter; (3) to apply the technology as a spectroscopic tool for probing the interactions of fluorochromes with cells and chromosomes; and (4) to improve on the technology as user needs arise from the biomedical community. Stained cells are analyzed as they flow through a chamber and intersect an intensity-modulated laser beam. The pulse-- modulated fluorescence signals, which are shifted in phase from a reference, are processed by phase-sensitive detection (PSD) electronics to suppress one component in a multicomponent mixture based on lifetime differences or to determine the phase shift and extent of demodulation which allows calculation of fluorescence lifetimes. The flow system, including laser modulator, improved flow chamber, fluorescence detector, signal amplifiers, and phase-sensitive detectors, is operational and a model of the signal detection theory has been developed. Preliminary experiments have demonstrated (1) a fluorescence detection threshold (sensitivity) of 300 to 800 fluorescein isothiocyanate (FITC) molecules equivalence by phase sensitive measurement for excitation frequencies ranging from 10 to 30 MHz, (2) coefficients of variation less than 1.5% on alignment fluorospheres and less than 4% on propidium iodide (PI)-stained (DNA content) line CHO cells excited at 10 MHz, and (3) the capability to quantify nanosecond lifetimes by fluorescence phase and amplitude measurements on ethidium bromide-, PI-, and FITC-stained cells. Fluorescence signals from cells stained with Pi and FITC in combination also have been resolved and the individual histograms displayed based on differences in lifetimes (phase shift). We will continue to evaluate the first generation instrument in terms of instrumental capabilities and its usefulness as a tool for probing the interactions of fluorochromes with cells and chromosomes. Studies of chromatin structure, DNA synthesis in relationship to cell cycle, heterogeneity of antibody labeling, and membrane receptor structure are planned. Improvements are proposed to develop a more advanced and versatile instrument as needs arise from the biological community.