Abstract

Ultrasensitve detection has recently become possible with the development of the single­molecule detection (SMD) technique, a laser-based method that allows the detection of individual dye molecules in solution. For the detection of single dye molecules in solution it is essential to minimize the background due to scattering and luminescent impurities in the solvent. Therefore a confocal setup with a detection volume of only a few femtoliters seems to be ideally suited for SMD techniques. Although most of the background luminescence can be removed by the use of small detection volumes and appropriate optical filter systems, bursts from impurities which are excitable especially in the green wavelength region can prevent the defenite detection and identification of single analyte molecules. The limited number of compounds which show intrinsic absorption and emission above 620 nm and the availability of low-cost diode lasers in this spectral region has led us to develop so called multiplex dyes. These dyes show similar absorption and emission characteristics but exhibit well distinct fluorescence lifetimes. The multiplex dyes absorb in the wavelength range 630-670 nm and are most suitable for ultrasensitive detection techniques in combination with pulsed diode laser excitation.

© 1998 IEEE