Single-molecule identification in flowing sample streams by fluorescence burst size and intraburst fluorescence decay rate.

We report a multiplex technique for identification of single fluorescent molecules in a flowing sample stream by correlated measurement of single-molecule fluorescence burst size and intraburst fluorescence decay rate. These quantities were measured simultaneously for single fluorescent molecules in a flowing sample stream containing a dilute mixture of fluorescent species:  Rhodamine 6G and tetramethylrhodamine isothiocyanate. Using a detailed Monte Carlo simulation of our experiment, we calculate single-molecule detection efficiencies and confidence levels for identification of these species and identify major sources of error for single-molecule identification. The technique reported here is applicable to distinguishing between fluorophores with similar spectroscopic properties and requires only a single excitation wavelength and single fluorescence emission detection channel.