Single-molecule identification of Coumarin-120 by time-resolved fluorescence detection: Comparison of one- and two-photon excitation in solution

Using two-photon excitation (TPE) at 700 nm as well as one-photon excitation (OPE) at 350 nm, we applied confocal fluorescence microscopy to detect single Coumarin-120 molecules in the solvents water and triacetin. To study the behavior of Coumarin-120 under different excitation conditions, fluorescence Lifetimes, multichannel scaler traces, and autocorrelation curves have been measured simultaneously. A signal-to-background ratio of 1300 was achieved for TPE due to a very low background level. The detection efficiency of TPE is limited by other competing nonlinear processes, in particular continuum generation in the solvent. The applicable laser intensity for OPE is limited by two-step photolysis of the dye as shown by fluorescence correlation spectroscopy (FCS). The time-resolved fluorescence signals were analyzed by a maximum likelihood estimator to identify the fluorophore through its characteristic fluorescence lifetime. The average fluorescence lifetimes 4.8 +/- 1.2 ns in water and 3.3 +/- 0.6 ns in triacetin are in good agreement with results obtained from separate measurements at higher concentrations.