Recent advances in microscopy and cytolabeling methods enable the real time imaging of cells as they move and interact in their real physiological environment. Scenarios in which multiple cells move autonomously in all directions are not uncommon in biology. A remarkable example is the swimming of marine spermatozoa in search of the conspecific oocyte. Imaging cells in these scenarios, particularly when they move fast and are poorly labeled or even unlabeled requires very fast three-dimensional time-lapse imaging (3D+t). This 3D+t imaging poses challenges not only to the acquisition systems but also to the image analysis algorithms. It is in this context that this work describes an original automated multi-particle segmentation method to analyze motile translucent cells in 3D microscopical volumes. The proposed segmentation technique takes advantage of the way the cell appearance changes with the distance to the focal plane position. The cells translucent properties and their interaction with light produce a specific pattern: when the cell is within or close to the focal plane, its two-dimensional appearance matches a bright spot surrounded by a dark ring, while when it is farther from the focal plane the cell contrast is inverted looking like a dark spot surrounded by a bright ring. The proposed method analyses the acquired video sequence frame-by-frame taking advantage of 2D image segmentation algorithms to identify and select candidate cellular sections. The crux of the method is in the sequential filtering of the candidate sections, first by template matching of the in-focus and out-of-focus templates and second by considering adjacent candidates sections in 3D. These sequential filters effectively narrow down the number of segmented candidate sections making the automatic tracking of cells in three dimensions a straightforward operation.