  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.
