Tracking objects in 3d is a common challenge in microscopy. It is often required when studying fluid mechanics at small scales, as well as the dynamics of soft matter and biological systems.  

I will present a method based on out-of-focus fluorescence microscopy imaging, where the 3d positions of fluorescent labelled micron sized spheres are determined from diffraction rings, imaged on a single camera.

The image analysis algorithm is based on the Hough transform and addresses the need to efficiently trace the trajectories of several particles concurrently, when their number in not necessarily fixed.  

It is currently implemented in a micro-fluidics flow experiment to allow real time multi-particle tracking, while achieving a high detection rate (>95%) and a low false-detection rate (<2%).

In addition, the current implementation is robust to overlaps and inclusions of diffraction rings, which result from particles being near to each other.