Nanoscale organization of mitochondrial microcompartments revealed by combining tracking and localization microscopy.

While detailed information on the nanoscale-organization of proteins within intracellular membranes has emerged from electron microcopy, information on their spatiotemporal dynamics is scarce. By use of a photostable rhodamine attached specifically to Halo-tagged proteins in mitochondrial membranes, we were able to track and localize single protein complexes such as Tom20 and ATP synthase in suborganellar structures in live cells. Individual membrane proteins in the inner and outer membrane of mitochondria were imaged over long time periods with localization precisions below 15 nm. Projection of single molecule trajectories revealed diffusion-restricting microcompartments such as individual cristae in mitochondria. At the same time, protein-specific diffusion characteristics within different mitochondrial membranes could be extracted.