Confidence in fluorescence lifetime determinations: a ratio correction for the photomultiplier time response with wavelength

Abstract

The photomultipliers used in single photon counting fluorescence lifetime Systems have a time response function which varies with wavelength. This variation results in a reduced confidence in the kinetic parameters obtained after deconvolution of the lamp intensity decay profile measured at the excitation wavelength from the sample emission decay profile measured at the emission wavelength. Moreover in the case of multi-component decays, inaccurate kinetic parameters are calculated because of this PMT variation. Using weighted residual plots, we demonstrate how one may make a satisfactory subjective appraisal of the adequacy of the deconvolution and the physical model of the decay processes. We have developed a ratio correction technique which accounts for the PMT time response variation with wavelength. This technique requires no apriori assumption of the nature of the PMT effect. Only when the data are corrected using the ratio correction technique can sufficient confidence in the accuracy of the singlet excited state parameters and the validity of the physical model be obtained.