Excitation pulse-shape mimic technique for improving picosecond-laser-excited time-correlated single-photon counting deconvolutions

Abstract

The determination of accurate subnanosecond fluorescence lifetimes by the time-correlated single-photon counting technique is often limited by difficulties in obtaining the correct instrumental response function f(λ,t) to the excitation pulse. These difficulties are increased when a grating monochromator is used to disperse the emission due to the introduction of an additional temporal broadening of f(λ,t). A technique for determining the correct f(λ,t) at the wavelength of sample emission, λem, is described. The technique consists of using a very short-lived fluorophore to ‘‘mimic’’ the excitation pulse shape at λem, yielding the convoluted mimic decay function Cm(λem,t), and then to computationally extract f (λem,t) from Cm(λem,t). The technique is experimentally and computationally simple and yields the desired instrumental response function at λem which eliminates problems due to the sensitivity of f(λ,t) to λ (color shift artifact). The photomultiplier tube receives spatially equal illumination when both the sample decay and Cm(λem,t) are determined because both the mimic and sample emission are spectrally broad, eliminating the problem of the variation of f(λ,t) with position of incident light on the photomultiplier tube photocathode (targeting artifact). Artifacts due to monochromator temporal broadening are eliminated since both the mimic and sample emission are monitored at constant wavelength.