A systematic study of global analysis of diffusion-mediated fluorescence quenching data

The recovery of the Smoluchowski-Collins-Kimball (SCK) model parameters from simulated fluorescence quenching decays and stationary Stern-Volmer (SV) quenching data is studied for the direct method where the instrument response function (IRF) is measured together with a sample, and the reference method where the monoexponential fluorescence decay of a reference compound provides information on the shape of the IRF. The zero-time shift was assumed to be an adjustable parameter in the direct method, and a fixed parameter in the reference method. We found that the quality of parameter estimates from the direct method is similar to that from the reference method. Single-curve analysis, where individual decay traces are fitted to the SCK model, leads to rather poor parameter estimates. Better results can be obtained when quenching decays are analyzed globally, i.e., simultaneously. In particular, global analysis of quenching decays with different time resolutions (=channel widths) improves the recovery compared to single curve analysis. For decays with high counts at a peak channel the inclusion of stationary SV data into the global analysis does not improve significantly parameter recovery.