Single-molecule detection of Rhodamine 6G in ethanolic solutions using continuous wave laser excitation

The ultimate in analytlcal sensltlvity is the ability to detect analytes on a single-molecule level. Laser-Induced fluores- cence (LIF) detection of single molecules in solution is ham- pered by specular, Rayleigh, and Raman scattering that contribute slgnlflcantly to the background. In order to ob- serve indlvidual 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 me- thod to increase the number of photons per event Is to in- crease 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 cy- cled 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 pho- tons per molecule than may be obtained in H,O. Observation of single molecular events of R-6G in EtOH is substantiated by autocorrelation analysis and from shlfts In the histograms of the frequency of photoelectron counts. Results from Monte Carlo simulatlons also support our experimental results.