SINGLE-MOLECULE IDENTIFICATION OF COUMARIN-120 BY TIME-RESOLVED FLUORESCENCE DETECTION - COMPARISON OF ONE-PHOTON AND 2-PHOTON EXCITATION IN SOLUTION

Using two-photon excitation (TPE) at 700 nm as well as one-photon exci tation (OPE) at 350 nm, we applied confocal fluorescence microscopy to detect single Coumarin-120 molecules in the solvents water and triace tin. To study the behavior of Coumarin-120 under different excitation conditions, fluorescence Lifetimes, multichannel scaler traces, and au tocorrelation curves have been measured simultaneously. A signal-to-ba ckground ratio of 1300 was achieved for TPE due to a very low backgrou nd level. The detection efficiency of TPE is limited by other competin g nonlinear processes, in particular continuum generation in the solve nt. The applicable laser intensity for OPE is limited by two-step phot olysis of the dye as shown by fluorescence correlation spectroscopy (F CS). The time-resolved fluorescence signals were analyzed by a maximum likelihood estimator to identify the fluorophore through its characte ristic fluorescence lifetime. The average fluorescence lifetimes 4.8 /- 1.2 ns in water and 3.3 +/- 0.6 ns in triacetin are in good agreeme nt with results obtained from separate measurements at higher concentr ations.