We describe instrumentation for real-time detection of single-molecule fluorescence in guided streams of 1-μm (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 ~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-μm-diameter droplets may have other important analytical applications as well.

© 1999 Optical Society of America

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