Single-molecule displacement mapping unveils nanoscale heterogeneities in intracellular diffusivity

Intracellular diffusion underlies vital cellular processes. However, it remains difficult to elucidate how an unbound protein diffuses inside the cell with good spatial resolution and sensitivity. Here we introduce single-molecule displacement/diffusivity mapping (SMdM), a super-resolution strategy that enables the nanoscale mapping of intracellular diffusivity through local statistics of the instantaneous displacements of freely diffusing single molecules. We thus show that the diffusion of an average-sized protein in the mammalian cytoplasm and nucleus to both be spatially heterogeneous at the nanoscale, and such variations in local diffusivity correlate with the ultrastructure of the actin cytoskeleton and the chromosome, respectively. SMdM of differently charged proteins further unveils that the possession of positive, but not negative, net charges drastically impedes diffusion, and that the exact degree of slowdown is determined by the specific subcellular environments. We thus open a new door to probing intracellular properties and functions at the nanoscale.