High-Efficiency Molecular Counting in Solution: Single-Molecule Detection in Electrodynamically Focused Microdroplet Streams

Abstract

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 (99% confidence) were obtained for 100 and 15 fM rhodamine 6G solutions, in good agreement with detailed theoretical calculations and statistical limitations.