Relaxation kinetics by fluorescence correlation spectroscopy: Determination of kinetic parameters in the presence of fluorescent impurities

The use of fluorescence correlation spectroscopy (FCS) in combination with Förster resonance energy transfer (FRET) is gaining popularity as a tool to investigate kinetics in equilibrium conditions. The technique is based on the study of fluorescence fluctuations in small numbers of molecules and is particularly well-suited to investigate conformational dynamics in biopolymers. In practice, its applicability is often hindered by the presence of certain impurities such as partially labeled biomolecules, excess of free fluorophore, or partially dissociated multisubunit complexes. Here, we show that the simultaneous measurement of the fluctuations in the donor and acceptor intensities allows the determination of the kinetic relaxation time of the reaction in the presence of donor-only particles when cross-talk is negligible or in cases where all species have the same diffusion coefficient. Theoretical predictions are supported with the results of Monte Carlo simulations and demonstrate that the applicability of the technique is more general than previously thought.