Wavefronts matching: a novel paradigm for three-dimensional holographic particle tracking

Digital Holography (DH) in microscopy allows to retrieve in an accurate way the spatial coordinates of multiple moving particles, performing 3D tracking of the sample in the entire field of view. In particular, a posteriori quantitative multi-focus phase-contrast imaging, suitable for 3D tracking of micro-objects, is one of the main features of the holographic approach. However, classical methods need to decouple amplitude and phase contributions of the reconstructed complex wavefronts to calculate target positions in 3D, due to the fact that the lateral displacements can be calculated only after refocusing step. In order to overcome this limitation, recently, a novel method of the simultaneous calculation of both axial and lateral coordinates of moving particles has been proposed. This is based on the novel concept of wavefronts matching, i.e. the 3D positions of micro-object, moving in 3D volume, are obtained by aligning wo subsequent holographic complex reconstructions, calculated at the same distance. We test this approach in different experimental conditions in order to highlight its effectiveness in bio-microfluidic applications.