High-Sensitivity Single-Molecule Fluorescence Detection in Theory and Practice

Abstract

The number of emitted photons that can be obtained from a fluorophore 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.1 The fluorescence lifetime and the photodestruction quantum yield are the key parameters that determine the optimum light intensity and exposure time. To test this theory we have performed single molecule detection of phycoerythrin (PE). The laser power was selected to give a mean time between absorptions approximately equal to the fluorescence decay time. The transit time was selected to be nearly equal to the photodestruction time of ~600 μs. Under these conditions the photon count distribution function, the photon count autocorrelation function, and the concentration dependence clearly show that we are detecting bursts of fluorescence from individual fluorophores as they pass through the laser beam. A hard-wired version of this single-molecule detection system was used to measure the concentration of PE down to 10-15 M.2 This single-molecule counter is three orders-of-magnitude more sensitive than conventional fluorescence detection systems. The approach presented here should be useful in the optimization of fluorescence-detected DNA sequencing gels and in HPLC and capillary electrophoresis detectors.