Due to the crucial role of physical cues in regulating cell behaviour, the mechanical properties of hydrogels are a key design parameter in tissue engineering applications. The shear elastic properties of viscoelastic materials are commonly measured by mechanical rheometers. Storage and loss moduli of a material can be measured by application of strain while measuring stress or vice versa. In contrast, recently developed optical micro-rheology techniques use nanometer- or micrometer-sized particles embedded in the material to obtain the viscoelastic response parameters. Thermal or passive micro-rheology for viscoelastic materials is based on an extension of the concepts of Brownian motion of particles in simple liquids. The movement of the embedded particles can be monitored using particle tracking. Initially developed to investigate the rheological properties of uniform complex fluids, particle tracking micro-rheology (PTM) is becoming a popular technique to analyze polymer blends and gels, as well as the deformability and elasticity within cells. However, if the beads locally modify the structure of the gel or are contained in a pore in an inhomogeneous matrix, the bulk rheological properties will not be retrieved. A solution is to use the cross-correlated thermal fluctuation of pairs of tracer particles, ‘two-point micro-rheology’. This method provides a better agreement between micro and macro-rheology, even in complex micro-structured fluids. However, technical constrains limit the wide application of this technique. One of the major limitations of two-point micro-rheology is the reduced number of trajectories that can be used for analysis. During particle tracking micro-rheology, the length of the calculated trajectories is limited by the time spent by the tracers in the field of view (x,y) and depth of focus (z). Consequently, mechanical characterization of complex polymer matrixes at the micrometer scale would benefit greatly of a new method for (fast) tracking in 3D. We are developing a new method for fast tracking of (fluorescent) beads in 3D using a multi-plane wide field microscope. This will allow a better mechanical characterization of soft materials, at the microscale.