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The technique of multifrequency phase and modulation fluorometry may be applied to the measurement and analysis of heterogeneous fluorescence lifetimes. This paper addresses two approaches to the data analysis: (1) the exact analytical solution (Weber's algorithm) for determining the lifetimes of N components and their relative contributions using N modulation frequencies and (2) a least-squares method. The mathematical rationale for each approach is described and the effects of random or systematic errors on the results obtained with each method are discussed. The relative merits of each approach are considered in light of results on both real and simulated data.
heterogeneous fluoresence lifetimes, phase/modulation fluorometry, Weber's algorithm, least-squares analysis, molecular luminescence
Assistant professor, University of Texas, Health Science Center at Dallas, Dallas, TX
Assistant professor, University of Illinois at Urbana-Champaign, Urbana, IL