Spatially mapping the diffusivity of proteins in live cells based on cumulative area analysis.

Molecular motion provides a way for biomolecules to mix and interact in living systems. Quantifying their motion is critical to the understanding of how biomolecules perform its function. However, it has been a challenged task to spatially map the fast diffusion of unbound proteins in the heterogenous intracellular environment. Here we reported a new imaging technique named cumulative area based on single-molecule diffusivity mapping (CA-SMdM). The strategy is based on the comparison of single-molecule images between a shorter and longer exposure time. With longer exposure time, molecules will travel further, thus giving more blurred single-molecule images, hence implying its local diffusion rates. We validated our technique through measuring the fast diffusion rates (10–40 µm²/s) of fluorescent dye in glycerol-water mixture, and found the values fit well with Stokes-Einstein equation. We further showed that the spatially mapping of diffusivity in live cells is plausible through CA-SMdM, and it faithfully reported the local diffusivity heterogeneity in cytosol and nucleus. CA-SMdM provides an efficient way to mapping the local molecular motion, and therefore will have profound applications in probing the biomolecular interactions for living systems. [ABSTRACT FROM AUTHOR]