In frequency-domain lifetime spectroscopy, the apparent fluorescence lifetimes obtained from phase-shift measurements are independent of modulation frequency only in the special case of a single exponential fluorescence decay. For heterogeneous fluorescence decay, the apparent fluorescence lifetimes measured by the phase-shift methods are functions of the modulation frequency. This modulation-frequency dependent property of apparent fluorescence lifetimes may be used to identify heterogeneous fluorescence decays by measuring Lifetimes at multiple frequencies. in this article we explore the requirements and experimental design considerations for making such measurements in flow, We report a phase-sensitive now cytometric method that allows one to probe the excited state-lifetimes of labeled cells by using multiple simultaneous modulation frequencies, Application of this method is demonstrated by measuring fluorescence Lifetimes of labeled cells at two frequencies simultaneously, using a continuous-wave, dual-frequency modulated excitation in now, The dual-frequency method presented herein can be used to rapidly identify heterogeneity in the fluorescence decay on a cell-by-cell basis in real time, Information on the nature of the fluorescence decay is important in biological measurements because it can provide insight into intermolecular interactions at the subcellular level. (C) 1995 Wiley-Liss, Inc.
