Simultaneous measurements of the flow velocities in a microchannel by wide/evanescent field illuminations with particle/single molecules.

A laser-induced fluorescence imaging method was developed to simultaneously measure flow velocities in the middle and near wall of a channel with particles or single molecules, by selectively switching from the wide field excitation mode to the evanescent wave excitation mode. Fluorescent microbeads with a diameter of 175 nm were used to calibrate the system, and the collisions of microbeads with channel walls were directly observed. The 175 nm microbeads velocities in the main flow and at 275 nm from the bottom of the channel were measured. The measured velocities of particles or single molecules in two positions in a microchannel were consistent with the calculated value based on Poiseuille flow theory when the diameter of a microbead was considered. The errors caused by Brownian diffusion in our measurement were negligible compared to the flow velocity. Single lambda DNA molecules were then used as a flowing tracer to measure the velocities. The velocity can be obtained at a distance of 309.0 +/- 82.6 nm away from bottom surface of the channel. The technique may be potentially useful for studying molecular transportation both in the center and at the bottom of the channel, and interactions between molecules and microchannel surfaces. It is especially important that the technique can be permitted to measure both velocities in the same experiment to eliminate possible experimental inconsistencies.