Confinement and Manipulation of Individual Molecules in Attoliter Volumes

Abstract

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.