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

• Published in: Science China. Chemistry 2023-11, Vol.66 (11), p.3307-3313
• Main Authors: Gao, Huihui, Han, Chu, Xiang, Limin
• Format: Article
• Language: English
• Subjects: Biomolecules; Chemistry; Chemistry and Materials Science; Chemistry/Food Science; Diffusion rate; Diffusivity; Einstein equations; Electrons; Fluorescent dyes; Glycerol; Glycerol-Water; Heterogeneity; Imaging techniques; Mapping; Molecular motion; Proteins
• ISSN: 1674-7291; 1869-1870
• DOI: 10.1007/s11426-023-1764-x

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 µm 2 /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.