The effects of laser pulsewidth and molecular lifetime on the experimental determination of one-photon and two-photon excitation spectra

We demonstrate that it is important to know both the observed fluorescence lifetime (τ) and the excitation pulsewidth ( ) in order to determine the effect of changes in on the measurement of one- and two-photon excitation spectra. If is greater than 3, it is sufficient to measure or for -photon excitation spectra ( and 2), where IFis the peak fluorescence intensity, ILis the peak intensity of the excitation pulse, and NLis the total number of photons per pulse. When is less than 3, however, the pulsewidth dependence of or should be treated by using the pulse-width correction functions defined in this paper. A simple method to estimate observed fluorescence lifetimes is also described where one needs only to measure and the time interval between the peak intensity of an excitation pulse with a Gaussian or near Gaussian temporal profile and the peak fluorescence intensity. Tables of correction factors are provided which can be used to generate accurate values of these functions for arbitrary ratios of using interpolation techniques. The treatment of the effect of non-Gaussian excitation pulses on the measurement of one- and two-photon excitation spectra is discussed.