Three-dimensional particle tracking in microfluidic channel flow using in and out of focus diffraction

Three-dimensional particle tracking is important to accurately understand the motion of particles within complex flow fields. We show that three-dimensional trajectories of particles within microfluidic flow can be extracted from two-dimensional bright field video microscopy. The method utilizes the defocusing that occurs as particles move out of the objective focal plane when viewed through a high numerical aperture objective lens. A fast and simple algorithm based on cross correlation to a set of reference images taken at prescribed amounts of defocus is used to extract out-of-plane particle position. In-plane particle position is determined through center point detection and therefore the particle position in all three dimensions can be constructed at each time point. Particle trajectories at high flow velocity of greater than 2 mm/s can be tracked by utilizing a high speed camera to obtain unblurred images. Three dimensional computational fluid simulations are used to validate the particle tracking methods.