Single particle tracking as a method to resolve differences in highly colocalized proteins.

Single particle tracking fluorescence microscopy was used to study two late endosomal proteins, Rab7 and LAMP1, that appear to be highly colocalized in static fluorescence microscopy images. Imaging these proteins simultaneously reveals that Rab7 and LAMP1 undergo periods of separation within the cell. Single particle tracking carried out during these periods of separation shows that Rab7-vesicles have greater velocities, but undergo less efficient transport than LAMP1-vesicles. This research demonstrates the use of single particle tracking as a tool to resolve functional differences in highly colocalized proteins in intact live cells.