This article presents a novel method for determining the three-dimensional location of fluorescent particles that is suitable for three-dimensional particle tracking velocimetry measurements in microfluidic flows. This method determines the depth of a particle by inserting a convex lens and axicon into the optical path between a microscope and camera. For particles close to the focal plane, this converts the wavefront from a particle into a Bessel beam, the frequency, and center of which can be directly related to the three-dimensional position of the particle. A robust image analysis method is presented that can determine the properties of the Bessel beam necessary to calculate the particle position. The theory and data analysis method are verified by comparing the calculated position of 1-lm particles to the known position of the particles which scanned through a depth of 100 μm. The average error in the calculated position was 4 μm. Finally, the method is applied to 3D3C particle tracking velocimetry of Poiseuille flow in a 200-μm-deep channel. Uniquely, this method requires no calibration procedure and is insensitive to variations in particle size and brightness.
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