Nature Commun. 5, 4494 (2014)

Intracellular structures can be mapped with a resolution of tens of nanometres by using a few imaging techniques, such as super-resolution light microscopy, cryo-electron microscopy or fluorescence correlation spectroscopy. However, most microscopy techniques lack sufficient temporal resolution to track molecules that diffuse quickly, or they need fixed samples. Living cells can be tracked with fluorescence spectroscopy, but following hundreds of inert fluorescence trackers at high spatial and temporal resolution is not straightforward. Building on previous work on intracellular fluorescence imaging, Michael Baum et al. now show that this can be done by using multiscale fluorescence cross-correlation spectroscopy. With this technique, the researchers found that both the cell's nucleus and cytosol impose similar constraints on the mobility of most proteins, that chromatin is the dominant obstacle that prevents protein translocation into the nucleus, and that the cytoskeleton has only a small effect on protein transport, which suggests that cytosolic organelles play a significant role in the topology of the cell's porous interior. The researchers' approach should be readily applicable to the study of the effects of drug-induced perturbations on protein mobility.