Single particle tracking (SPT) in biological systems is a quickly growing field. Many new technologies are being developed providing new tracking capabilities, which also lead to higher demands and expectations for SPT. Following a single biomolecule as it performs its function provides quantitative mechanistic information that cannot be obtained in classical ensemble methods. From the 3D trajectory, information is available over the diffusional behavior of the particle and precise position information can also be used to elucidate interactions of the tracked particle with its surroundings. Thus, three-dimensional (3D) SPT is a very valuable tool for investigating cellular processes. This review presents recent progress in 3D SPT, from image-based techniques toward more sophisticated feedback approaches. We focus mainly on the feedback technique known as orbital tracking. We present here a modified version of the original orbital tracking in which the intensities from two z-planes are simultaneously measured allowing a concomitant wide-field imaging. The system can track single particles with a precision down to 5 nm in the x–y plane and 7 nm in the axial direction. The capabilities of the system are demonstrated using single virus tracing to follow the infection pathway of Prototype Foamy Virus in living cells.