Single-molecule counting and identification in a microcapillary

Using a confocal microscope we studied photon bursts from individual molecules (dye-labeled mononucleotides) flowing in a cone-shaped microcapillary with an inner diameter of 0.5 μm at the small end of the cone. The flow of the conjugates was established by electrokinetic forces. Excitation of the fluorophore was provided by a pulsed diode laser (λ=640 nm, average power 800 μW, repetition rate 56 MHz). The characteristic diffusion and flow time through the laser focus and burst size statistics were determined in the microcapillary as well as in an open volume. Applying time-correlated single-photon counting, two different conjugate species (Cy5-dCTP, JA53-dUTP) can be distinguished due to their characteristic fluorescence decay time with a probability of correct classification of 80%.