Time-resolved identification of single molecules in solution with a pulsed semiconductor diode laser

We used a confocal microscope to study bursts of fluorescence photons from single dye molecules excited at 638 nm by a short-pulsed diode laser with a repetition rate of 17 MHz. Four newly synthesized dyes (JA 167, DR 333, cyanorhodamine B and MR 121) as well as two commercially available dyes (Cy5 and rhodamine 700) were used in ethylene glycol solution. Multichannel scaler traces and fluorescence decay times were measured simultaneously. The fluorescence decays were determined by the time-correlated single-photon counting technique. The time-resolved fluorescence signals of the dyes were analyzed and identified by a maximum likelihood estimator. It turned out that 40 photons per dye molecule are sufficient to distinguish two rhodamine derivatives with a misclassification of less than 1% via their characteristic fluorescence lifetimes of 3.61 ± 0.45 ns (JA167) and 1.41 ± 0.3 ns (cyanorhodamine B).