Molecular shot noise, burst size distribution, and single-molecule detection in fluid flow: Effects of multiple occupancy

We present a new mathematical approach for calculating burst size distributions for the detection of fluorescent molecules introduced into a fluid flow at different rates. The burst size distributions reflect the passage of more than one molecule through the detection volume in close succession. The calculations are based upon a physical model appropriate for the fluorophore phycoerythrin under the conditions of no saturation of excitation, negligible triplet-state dynamics, and negligible detector dead time. The model includes photophysical properties of the fluorophore phycoerythrin (absorption cross section, fluorescence quantum yield, and photostability); diffusion; sample stream hydrodynamics; spatially dependent optical detection efficiency; and excitation laser beam characteristics. Good agreement is found between the mathematical model and experimental results with phycoerythrin.