The Correct Use of "Average" Fluorescence Parameters

When more than one fluorophore is present or when one fluorophore displays a multiple exponential decay "average" fluorescence parameters are derived, which can be combined with "average" lifetimes for further interpretation. However, two kinds of average lifetimes are used in this context: the intensity and the amplitude average lifetime. In this paper the different average parameters are carefully analyzed and their "best" combinations are derived. These average parameters are analyzed in the context of external and internal dynamic and static quenching, Föster energy transfer and the calculation of the radiative rate constant. The use of the amplitude average lifetime for the analysis of multiple fluorophore-containing systems and the detection of interactions is discussed.