We describe the development of a tracking device, mounted on an epi-fluorescent inverted microscope, suited to obtain time resolved 3D Lagrangian tracks of fluorescent passive or active micro-objects in micro-fluidic devices. The system is based on real-time image processing, determining the displacement of a x,y mechanical stage to keep the chosen object at a fixed position in the observation frame. The z displacement is based on the refocusing of the fluorescent object determining the displacement of a piezo mover keeping the moving object in focus. Track coordinates of the object with respect to the micro-fluidic device, as well as images of the object are obtained at a frequency of several tenths of Hertz. This device is particularly well adapted to obtain trajectories of motile micro-organisms in micro-fluidic devices with or without flow.
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