Three-dimensional tracking of submicron particles in flows in a micro-channel was carried out using in-line holographic microscopy.A fixed single 0.5 μm fluorescent particle was identified and isolated from dust particles or overlapped particle pair using the laser induced fluorescent (LIF) method.Then in-line microscopic holograms of the fixed single particle were obtained at different positions on the optical axis

i.e.

the defocus distances.The holograms of the single particle were used as the model templates with the known defocus distances.The particles in the in-line microscopic holograms of flow in the microchannel were then identified and located to obtain their two-dimensional positions.The defocus distances of those particles were determined by matching each hologram pattern to one of the model templates obtained in the single particle test.Finally the three-dimensional position and velocity of each particle were obtained.