RESOLUTION OF FLUORESCENCE SIGNALS FROM CELLS LABELED WITH FLUOROCHROMES HAVING DIFFERENT LIFETIMES BY PHASE-SENSITIVE FLOW-CYTOMETRY

A flow cytometric method has been developed that uses phase-sensitive detection to separate signals from simultaneous fluorescence emissions in cells labeled with fluorochromes having different fluorescence decay lifetimes. By CHO cells were stained with propidium iodide (PI) and fluorescein isothiocyanate (FITC). These dyes bind to DNA and protein and the fluorescence lifetimes of the bound dyes are 15.0 and 3.6 ns, respectively. Cells were analyzed as they passed through a modulated (sinusoidal) laser excitation beam. Fluorescence was measured using only a long-pass filter to block scattered laser excitation light and a single photomultiplier tube detector. The fluorescence detector output signals were processed by dual-channel phase-sensitive detection electronics and the phase-resolved PIand FITC signals were displayed as frequency distribution histograms and bivariate plots. By shifting the phase of one detector channel reference signal by pi/2 + phi1 degrees and the phase of the other detector channel reference signal by -pi/2 + phi2 degrees, where phi1 and phi2 are the phase shifts associated with the PI and FITC lifetimes, the PI and FITC signals were separately resolved at their respective phase-sensitive detector outputs. This technology is also applicable to suppressing background interferences caused by cellular autofluorescence, unbound/free dye, nonspecific dye binding, and Raman and Rayleigh scattering.