Determination of single particle flow velocities in microchannels using a maximum likelihood estimator method

A statistical method based on the maximum likelihood estimator is used in order to discriminate between single particle flow velocities using confocal spectroscopy. Particles or molecules travelling at specific flow rates through a confocal volume are expected to have a definable burst widths, heights, and areas. Hence, single particles travelling with different velocities should be distinguishable based on such parameters. With this approach we show that for particles travelling with a flow rate difference of 20x, there is virtually a 0% error rate in assigning a particle to a given flow velocity, based on the analysis of a single fluorescence burst. When a flow rate difference of 4x is used, 90% of the particles travelling through the confocal probe volume are still assigned a correct flow velocity. Use of this method technique allows a statistically meaningful flow velocity to be assigned from the analysis of a photon burst from a single particle.