<title>High-sensitivity single-molecule fluorescence detection</title>

Abstract

The number of photons that can be obtained from a fluorescent chromophore increases with the incident light intensity and the duration of illumination. However, saturation of the absorption transition and photodestruction place natural limits on the ultimate signal-to-noise ratio that can be obtained. Equations have been derived to describe the fluorescence-to-background-noise ratio in the presence of saturating light intensities and photodestruction. The fluorescence lifetime, the extinction coefficient, and the photodestruction quantum yield are the key parameters that determine the optimum light intensity and exposure time. This theory indicates that the laser power should be selected to give a mean time between absorptions approximately equal to the fluorescence decay rate, and the transit time should be selected to be nearly equal to the photodestruction time. Using these optimum conditions we have performed experiments to detect individual molecules of phycoerythrin (PE). The photocount distribution function, the photocount autocorrelation function, and the concentration dependence from phycoerythrin clearly show that we are detecting bursts of fluorescence from individual fluorophores. A hard-wired version of this single-molecule detection system was used to measure the concentration of PE down to 1015 M. This single-molecule counter is three orders-of-magnitude more sensitive than conventional fluorescence detection systems. The approach presented here has also been used to optimize fluorescence-detected DNA sequencing gels. Using a confocal microscope configuration we have detected DNA sequencing gels at concentrations as low as i0 fluorescent DNA fragments per band.