Single-Molecule Displacement Mapping Indicates Unhindered Intracellular Diffusion of Small (≲1 kDa) Solutes

While fundamentally important, the intracellular diffusion of small (≲1 kDa) solutes has been difficult to elucidate due to challenges in both labeling and measurement. Here we quantify and spatially map the translational diffusion patterns of small solutes in mammalian cells by integrating several recent advances. In particular, by executing tandem stroboscopic illumination pulses down to 400 μs separation, we extend single-molecule displacement/diffusivity mapping (SMdM), a super-resolution diffusion quantification tool, to small solutes with high diffusion coefficients D of >300 μm²/s. We thus show that for multiple water-soluble dyes and dye-tagged nucleotides, intracellular diffusion is dominated by vast regions of high diffusivity ∼60-70% of that in vitro, up to ∼250 μm²/s in the fastest cases. Meanwhile, we also visualize sub-micrometer foci of substantial slowdowns in diffusion, thus underscoring the importance of spatially resolving the local diffusion behavior. Together, these results suggest that the intracellular diffusion of small solutes is only modestly scaled down by the slightly higher viscosity of the cytosol over water but otherwise not further hindered by macromolecular crowding. We thus lift a paradoxically low speed limit for intracellular diffusion suggested by previous experiments.