Complex systems of fluorophores undergoing energy transfer can exhibit a variety of anomalous lifetime behavior when probed with frequency domain methods. When presented in traditional apparent lifetime format the data from such systems can exhibit "nodal"behavior in which the computed lifetime approaches ±∞. The location of the nodes is system and frequency dependent. In addition, simpler systems, not undergoing energy transfer, show ill behavior in the region of zero lifetime (τ_m) and long lifetime (τ_π) due to noise in typical measurements. Here, we systematically investigate systems of multiple fluorophores with and without energy transfer to provide insight into frequency domain investigations of complex systems of fluorophores. The results of simulations are compared to data collected from a multi-fluorophore system designed to exhibit fluorescence resonance energy transfer (FRET) using imaging spectroscopic fluorescence lifetime imaging microscopy (ISFLIM). The results are applicable to both cuvette and imaging arrangements.

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