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 la ser 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 so lution. Multichannel scaler traces and fluorescence decay times were m easured simultaneously. The fluorescence decays were determined by the time-correlated single-photon counting technique. The time-resolved f luorescence signals of the dyes were analyzed and identified by a maxi mum likelihood estimator. It turned out that 40 photons per dye molecu le are sufficient to distinguish two rhodamine derivatives with a misc lassification of less than 1% via their characteristic fluorescence li fetimes of 3.61 +/- 0.45 ns (JA167) and 1.41 +/- 0.3 ns (cyanorhodamin e B).