High efficiency molecular counting in solution: Single-molecule detection in electrodynamically focused microdroplet streams

We report fluorescence detection of individual rhodamine 6G molecules using a linear quadrupole to focus streams of microdroplets through the waist of a counterpropagating cw Ar+ laser. Since the terminal velocity scales as the square of the droplet diameter, the droplet-laser interaction time was ''tunable'' between 5 and 200 ms by using water samples spiked with a small, variable (2-5% v/v) amount of glycerol. Fluorescence bursts from droplets containing single molecules were clearly distinguished from the blanks in real time with an average signal-to-noise ratio of about 10, limited primarily by photobleaching and droplet size fluctuations (<1%). The volume throughput rates associated with this approach (similar to 10 pL/s) are roughly 10(3) higher than those associated with particle levitation techniques, with minimal sacrifice in sensitivity, Total molecular detection efficiencies of about 80% (at >99% confidence) were obtained for 100 and 15 fM rhodamine 6G solutions, in good agreement with detailed theoretical calculations and statistical limitations.