This book describes general and robust algorithms that are devoted to automate the analysis process in the 3D space and in time of dynamic objects present in a volume studied by a specific microscope that permits to record images containing their 3D information. This imaging system using a coherent light records not only the intensity information of the light, but also the direction of propagation for each pixel in the image. Together with a numerical model for coherent light diffraction, it is possible to generate numerically other images parallel to the recorded plane and as a consequence to refocus numerically objects. The acquisition time of 3D data is as a consequence only limited by the acquisition rate of the CCD camera of in the imaging system. The extraction of 3D information is performed by algorithm applied on the recorded data. The studied objects are either of biological nature or latex particles. A focus criterion to determine the best focus plane of an object is proposed. From the knowledge of the 3D position of objects, several analyses are proposed like shape or object trajectories analysis in time and in 3D.