Parallax: high accuracy three-dimensional single molecule tracking using split images

Three-dimensional (3D) tracking can provide valuable biological insights that are missing in conventional microscopy. Here we developed a single molecule 3D tracking microscopy technique, named Parallax, with high localization precision and temporal resolution. We demonstrated its capabilities by studying the 3D trafficking of glucose-transporter-4 containing vesicles in living adipocytes as well as the walking path of single myosin VI molecules along actin filaments.