Single molecule tracking in three dimensions (3D) in a live cell environment promises to reveal important new insights into cell biological mechanisms. However, classical microscopy techniques suffer from poor depth discrimination which severely limits single molecule tracking in 3D with high temporal and spatial resolution. We introduced a novel imaging modality, multifocal plane microscopy (MUM) for the study of subcellular dynamics in 3D. We have shown that MUM provides a powerful approach with which single molecules can be tracked in 3D in live cells. MUM allows for the simultaneous imaging at different focal planes, thereby ensuring that trajectories can be imaged continuously at high temporal resolution. A critical requirement for 3D single molecule tracking as well as localization based 3D super-resolution imaging is high 3D localization accuracy. MUM overcomes the depth discrimination problem of classical microscopy based approaches and supports high accuracy 3D localization of singe molecule/particles. In this way, MUM opens the way for high precision 3D single molecule tracking and 3D super-resolution imaging within a live cell environment. We have used MUM to reveal complex intracellular pathways that could not be imaged with classical approaches. In particular we have tracked quantum dot labeled antibody molecules in the exo/endocytic pathway from the cell interior to the plasma membrane at the single molecule level. Here, we present a brief review of these results.© (2010) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.