Single-molecule detection by two-photon excitation of fluorescence

Using a mode-locked titanium: sapphire laser at 700 nm for two-photon excitation we studied fluorescence bursts from individual coumarin 120 molecules in water and triacetin. Fluorescence lifetimes and multichannel scaler traces have been measured simultaneously. Due to the fact that scattered excitation light as well as Raman scattered photons can be suppressed by a short-pass filter a very low background level was achieved. To identify the fluorophore by its characteristic fluorescence lifetime the time-resolved fluorescence signals were analyzed by a maximum likelihood estimator. The obtained average fluorescence lifetimes (tau) av equals 4.8 +/- 1.2 ns for coumarin 120 in water and (tau) av equals 3.3 +/- 0.6 for coumarin 120 in triacetin are in good agreement with results obtained from separate measurements at higher concentrations.