Three-dimensional tracking of fluorescent particles applied to micro-fluidic measurements

The diffraction patterns of submicron fluorescent particles were obtained using an air immersion objective ( M = 40, NA = 0.6) under non-design optical conditions through a thick cover glass with a mismatched refractive index. The particle diffraction patterns changed with the defocus distance out of the front focal plane in the optical axis direction. A scalar diffraction model was applied to analyze the diffraction patterns for various optical conditions and to determine the defocus distance of the particle according to the measured intensity distribution of the diffraction pattern image. The results showed that the defocus distances have a submicron accuracy (0.2–0.3 µm), which can be used to determine the three-dimensional flow velocity distribution in a micro-channel and to track the three-dimensional Brownian motion of the particles for the specified defocus conditions.