Single-Molecule Detection in Biology with Multiplex Dyes and Pulsed Semiconductor Lasers

Abstract

In recent times, researchers have made considerable efforts to achieve the detection of individual atoms and molecules in solids [1–6], on surfaces [7–10], and in liquids [11–20] using laser-induced fluorescence techniques. In particular, single-molecule detection in condensed phases has many important biological applications, including rapid DNA sequencing, medical diagnosis, and forensic analysis [21–23]. For the detection of single dye molecules it is essential to minimize the background from scattering and luminescent impurities in the solvent, i.e. the ability to detect fluorescence bursts emitted by individual dye molecules is not as much an issue of sensitive detection as it is of background reduction. The predominant source of background is Rayleigh scattering, as well as reflected light, which can be efficiently suppressed by suited optical filters. Since Raman scattering is directly proportional to the detection volume, this background signal can be greatly reduced if a confocal setup in combination with a detection volume of only a few femto liters or even less is employed [24–27]. The detection volume is usually defined by the intersection of the focused excitation laser and the image of a spatial filter. The reduction of the detection volume improves the signal-to-background ratio by many orders of magnitude without measurable photodestruction of the dye molecules under study. Poisson statistics predicts, for a concentration of less than 10⁻¹⁰ M, that the number of molecules fluctuates predominantly between 0 and 1 in an applied detection volume in the femto liter region. Hence, the probability of two or more molecules being present in the detection volume simultaneously is negligible at concentrations below 10⁻¹⁰ M.