Anal. Chem. 1998, 70, 658-661
Correspondence
Confinement and Manipulation of Individual Molecules in Attoliter Volumes C.-Y. Kung, M. D. Barnes,* N. Lermer, W. B. Whitten, and J. M. Ramsey
Chemical and Analytical Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, Tennessee 37831-6142
We report observation of fluorescence from individual rhodamine 6G molecules in streams of charged 1-µmdiameter water droplets. With this approach, illumination volumes comparable to diffraction-limited fluorescence microscopy techniques (e500 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 diffusionlimited laser-molecule interaction times in equivalent volumes of free solution. The exciting potential of single-molecule probes as bioanalytical tools for detecting, sorting, and manipulating individual molecules in solution has significantly expanded interest in ultrasensitive fluorescence methods in recent years. In addition to its utility as an analytical tool, spectroscopic studies of individual molecules1,2 offer insight into fundamental photophysical phenomena which are often obscured by ensemble averaging. To date, several elegant experimental approaches to single-molecule fluorescence detection3-6 and imaging7-9 in liquids have been developed. In addition, far-field fluorescence microscopy10,11 has recently re(1) Moerner, W. E. Science 1994, 265, 46-52, and references cited therein. (2) Lu, H. P.; Xie, X. S. Nature 1997, 143-146. (3) For reviews, see: Barnes, M. D.; Whitten, W. B.; Ramsey, J. M. Anal. Chem. 1995, 67, A418-A423. Keller, R. A; Ambrose, W. P.; Goodwin, P. M.; Jett, J. H.; Martin, J. C.; Wu, M. Appl. Spectrosc. 1996, 50, A12-A32, and references cited therein. (4) Shera, E. B.; Seitzinger, N. K.; Davis, L. M.; Keller, R. A.; Soper, S. A. Chem. Phys. Lett. 1990, 174, 553-557. (5) Wilkerson, C. W.; Goodwin, P. M.; Ambrose, W. P.; Martin, J. C.; Keller, R. A. Appl. Phys. Lett. 1993, 62, 2030-2032. (6) Li, L. Q. ; Davis, L. M. Appl. Opt. 1995, 34, 3208-3217.
658 Analytical Chemistry, Vol. 70, No. 3, February 1, 1998
ceived a great deal of attention as a probe of individual solvated molecules in free solution. The central feature of this technique is the illumination of an extremely small volume (limited by diffraction in the radial dimension and by spherical aberration along the optical axis), typically ∼0.5 fL, which produces high contrast between single-molecule fluorescence signals from the background. However, fluorophore diffusion often limits both the amount of time that the molecule of interest can be forced to interact with the excitation field and the rate at which molecules enter the observation region. In the limit of zero throughput, significantly extended residence times for molecules in the pulled section (∼0.5-µm i.d.) of a capillary have recently been observed. This effect is believed to be derived from strong electrostatic interactions between the ionic molecule and the silica surface.12 In this paper, we show that individual fluorescent molecules can be confined and efficiently probed in volumes of solution (∼500 aL) roughly equal to illumination volumes defined by diffraction-limited high-numerical-aperture optics. As a result of molecular confinement, laser-molecule interaction times can be extended well beyond the diffusion-limited interaction times in comparable volumes of free solution. In addition, since the droplets are produced with a small electrical charge, in situ electrodynamic manipulation is achieved in a straightforward manner. While optical trapping methods13,14 are well established as a means of isolating macromolecules such as large peptides or DNA fragments, the trapping potential associated with such an approach is insufficient to confine relatively small molecules such as rhodamine 6G. We show that droplets of solution containing individual molecules can be directed to a desired target with a precision of a few microns via application of a suitable (7) Schmidt, Th.; Schultz, G.; Baumgartner, J. W.; Gruber, H. T.; Schindler, H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 2926-2929. (8) Dickson, R. M.; Norris, D. J.; Tzeng, Y.-L.; Moerner, W. E. Science 1996, 274, 966-969. (9) Xu, X.; Yeung, E. S. Science 1997, 275, 1106-1109. (10) Eigen, M.; Rigler, R. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5740-5747; Rigler, R. J. Biotechnology 1995, 41, 177-186. (11) Nie, S.; Chiu, D. T.; Zare, R. N. Science 1994, 266, 1018-1021. Nie, S.; Chiu, D. T.; Zare, R. N. Anal. Chem, 1995, 67, 2849-2857. (12) Lyon, W. A; Nie, S. Anal. Chem. 1997, 69, 3400-3405. (13) Chiu, D. T.; Zare, R. N. J. Am. Chem. Soc. 1996, 118, 67512-6513. (14) Chiu, D. T.; Hsiao, A.; Gaggar, A.; Garza-Lopez, R. A.; Orwar, O.; Zare, R. N. Anal. Chem. 1997, 69, 1801-1807. S0003-2700(97)01107-4 CCC: $15.00
© 1998 American Chemical Society Published on Web 02/01/1998
Figure 1. Comparison of experimental and calculated Fraunhofer diffraction patterns from water droplets ejected from a 1-µm-diameter orifice. The 0.5-µm uncertainty figure represents the error in accuracy due to the lack of well-defined calibration of the scattering angle (see ref 19). Shot-to-shot size fluctuations are estimated to be of the order of 1-2%.
electric potential. Thus, molecules of interest are actively delivered to the interrogation region at a rate limited only by the concentration and droplet production rate. Moreover, the fluorescent molecules (ionic dyes) being probed cannot diffuse out of the droplet volume, allowing nearly arbitrary extension of lasermolecule interaction over a range of 1-20 ms in the present configurationsat least a factor of 50 larger than the average diffusion-limited residence time in a 0.5-fL volume.15 While our previous work on single-molecule fluorescence detection involved using droplets made of low vapor pressure solvents such as glycerol levitated in an electrodynamic trap,16,17 or focused in a linear quadrupole,18 many applications of singlemolecule probes relevant to the biotechnology community demand the ability to detect fluorescent molecules directly in aqueous solution. However, the nearly 2 order of magnitude reduction in photostability of rhodamine dyes in water relative to ethanolic solvents places much more stringent demands on droplet production; the average number of signal counts expected from an individual R6G molecule under photobleaching conditions in our experimental configuration is only ∼10, which implies a maximum tolerable average droplet diameter of 2 µm with e2% shot-to-shot size fluctuations. In the experiments described here, streams of 1-µm-diameter water droplets were produced at a rate of ∼10 Hz with a piezoelectric droplet generator specifically designed and built in our laboratory for on-demand production of small (
