SIMULTANEOUS DUAL-FREQUENCY PHASE-SENSITIVE FLOW CYTOMETRIC MEASUREMENTS FOR RAPID IDENTIFICATION OF HETEROGENEOUS FLUORESCENCE DECAYS IN FLUOROCHROME-LABELED CELLS AND PARTICLES

In frequency-domain lifetime spectroscopy, the apparent fluorescence l ifetimes obtained from phase-shift measurements are independent of mod ulation frequency only in the special case of a single exponential flu orescence decay. For heterogeneous fluorescence decay, the apparent fl uorescence lifetimes measured by the phase-shift methods are functions of the modulation frequency. This modulation-frequency dependent prop erty of apparent fluorescence lifetimes may be used to identify hetero geneous fluorescence decays by measuring Lifetimes at multiple frequen cies. in this article we explore the requirements and experimental des ign considerations for making such measurements in flow, We report a p hase-sensitive now cytometric method that allows one to probe the exci ted state-lifetimes of labeled cells by using multiple simultaneous mo dulation frequencies, Application of this method is demonstrated by me asuring fluorescence Lifetimes of labeled cells at two frequencies sim ultaneously, using a continuous-wave, dual-frequency modulated excitat ion 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.