Abstract

The detection of individual fluorescent molecules in liquids has been of great interest in recent years. Various fluorescence-based techniques shown to provide single molecule sensitivities include confocal microscopy [1], flow cell techniques [2], and levitated microdroplets [3]. The application of the microdroplet technique to single molecule detection offers many advantages. First, fluoresence decay rates and total fluoresence yield have been shown to be enhanced in glycerol microdroplets [4]. Additionally, the droplet confines the single fluorophore to a small volume thereby removing difficulties arising from diffusion of the fluorophore. Furthermore, the discrete detection unit of the droplet is ideally suited to the application of digital molecular detection for the analysis of ultradilute solutions [5]. Previous liquid microdroplet work has exhibited single molecule detection with signal-to-noise ratios in the range of 10-40 [3]. In our previous work, an electrodynamic trap was employed to trap glycerol microdroplets for a period much longer than the average photochemical lifetime, thus obtaining the maximum possible signal from the analyte. However, the application of digital molecular analysis to real systems requires tens of thousands of droplet measurements [5]; the time required to trap (and to size) the droplet in a levitated system prohibits its application in a high-speed molecular counting technique. In addition, many biological applications of single molecule fluorescence detection require aqueous samples. The present work discusses the development of an instrument designed to permit single molecule detection in water microdroplets at count rates in the range of 10 - 1000 Hz.

© 1996 Optical Society of America