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 (∼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.

We report observation of fluorescence from individual rhodamine 6G molecules in streams of charged 1-μm-diameter water droplets. With this approach, illumination volumes comparable to diffraction-limited fluorescence microscopy techniques (≤500 aL) are achieved, resulting in similarly high contrast between single-molecule fluorescence signals and nonfluorescent background. However, since the fluorescent molecules are confined to electrically charged droplets, in situ electrodynamic manipulation (e.g., focusing, switching, or merging) can be accomplished in a straightforward manner, allowing experimental control over both the delivery of molecules of interest to the observation region and the laser-molecule interaction time. As illustrated by photocount statistics that are independent of molecular diffusion and spatial characteristics of the excitation field, individual rhodamine 6G molecules in 1-μm droplets are reproducibly delivered to a target a few micrometers in diameter at a rate of between 10 and 100 Hz, with laser beam transit times more than 1 order of magnitude longer than diffusion-limited laser-molecule interaction times in equivalent volumes of free solution.

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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.

We show that homogeneous polymer blend microparticles can be prepared in situ from droplets of dilute solution of codissolved polymers. Provided that the droplet of solution is small enough (<10 mum), solvent evaporation is rapid enough to inhibit phase separation. Thus the polymers that are being mixed need not be miscible, which greatly enhances the applicability of the technique. From analysis of two-dimensional Fraunhofer diffraction (angular scattering) patterns, we show that both the real and the imaginary parts of the refractive index can be tuned by adjustment of the relative weight fractions of polymers in solution.