Single-molecule identification by spectrally and time resolved fluorescence detection

A method to identify single molecules rapidly and with high efficiency base d on simple probability considerations is proposed. In principle, any prope rty of a detected photon in a single-molecule fluorescence experiment, e.g. , emission wavelength, arrival time after pulsed excitation, and polarizati on, can be analyzed within the framework of the outlined methodology. Monte Carlo simulations show that less than 500 photons are needed to assign an observed single molecule to one out of four species with a confidence level higher than 99.9%. We show that single dye molecules of four different dye s embedded in a polymer film can be identified with time-correlated single- photon counting spectrally resolved in two channels.