Single-molecule analysis of ultradilute solutions with guided streams of 1--microm water droplets

We describe instrumentation for real-time detection of single-molecule fluorescence in guided streams of 1-microm (nominal) water droplets. In this technique, target molecules were confined to droplets whose volumes were comparable with illumination volumes in diffraction-limited fluorescence microscopy and guided to the waist of a cw probe laser with an electrostatic potential. Concentration detection limits for Rhodamine 6G in water were determined to be approximately 1 fM, roughly 3 orders of magnitude lower than corresponding limits determined recently with diffraction-limited microscopy techniques for a chemical separation of similar dyes. In addition to its utility as a vehicle for probing single molecules, instrumentation for producing and focusing stable streams of 1-2-microm-diameter droplets may have other important analytical applications as well.