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The ultimate in analytical sensitivity is the ability to detect analytes on a single-molecule level. Laser-induced fluorescence (LIF) detection of single molecules in solution is hampered by specular, Rayleigh, and Raman scattering that contribute significantly to the background. In order to observe individual fluorescent molecules as they transit the laser beam in the presence of large backgrounds, it is necessary to detect a large number of photons per molecule. One method to increase the number of photons per event is to increase the residence time of the molecule in the laser beam. However, with long residence times, photostability sets an upper limit on the number of times the molecule can be cycled between the ground and first excited singlet state. We have observed the passage of individual rhodamine 6G (R-6G) molecules in ethanol (EtOH). The use of EtOH as a solvent allows one to obtain nearly 2 orders of magnitude more photons per molecule than may be obtained in H2O. Observation of single molecular events of R-6G in EtOH is substantiated by autocorrelation analysis and from shifts in the histograms of the frequency of photoelectron counts. Results from Monte Carlo simulations also support our experimental results.
Soper et al. (Fri,) studied this question.