SINGLE-MOLECULE COUNTING AND IDENTIFICATION IN A MICROCAPILLARY

Using a confocal microscope we studied photon bursts from individual m olecules (dye-labeled mononucleotides) flowing in a cone-shaped microc apillary with an inner diameter of 0.5 mu m at the small end of the co ne. The flow of the conjugates was established by electrokinetic force s. Excitation of the fluorophore was provided by a pulsed diode laser (lambda = 640 nm, average power-800 mu W, repetition rate 56 MHz). The characteristic diffusion and flow time through the laser focus and hu rst size statistics were determined in the microcapillary as well as i n an open volume. Applying time-correlated single-photon counting, two different conjugate species (Cy5-dCTP, JA53-dUTP) can be distinguishe d due to their characteristic fluorescence decay time with a probabili ty of correct classification of 80%. (C) 1998 Elsevier Science B.V.