Spherical fluorescent particle are micrometer-scale spherical beads used in various areas of physics, chemistry or biology as markers associated with local physical media. They are useful for example in fluid dynamics to characterize flows, diffusion coefficients, viscosity or temperature ; they are used in cells dynamics to estimate mechanical strain and stress at the micrometer scale. In order to estimate these physical measurements , tracking these particles is necessary. Numerous approaches and existing packages, both open-source and proprietary are available to achieve tracking with a high degree of precision in 2D. However, little such software is available to achieve tracking in 3D. One major difficulty is that 3D confocal microscopy acquisition is not typically fast enough to assume that the beads are stationary during the whole 3D scan. As a result, beads may move between planar scans. Classical approaches to 3D segmentation may yield objects are not spherical. In this article, we propose a 3D bead segmentation that deals with this situation.