Super-Resolution Mapping and Quantification of Molecular Diffusion via Single-Molecule Displacement/Diffusivity...

Area of Science:

• Biophysics
• Cell Biology
• Nanotechnology

Background:

• Mapping molecular diffusion at subcellular scales is challenging.
• Existing single-particle tracking methods are limited for wide-field mapping of fast diffusion.

Purpose of the Study:

• To develop and validate a novel method for super-resolution mapping of molecular diffusion.
• To investigate nanoscale diffusion heterogeneities and their origins in various cellular compartments.
• To quantify molecular diffusion with high precision in biological systems.

Main Methods:

• Single-molecule displacement/diffusivity mapping (SMdM) utilizing a tandem excitation scheme for rapid, wide-field imaging.
• Correlated single-molecule localization microscopy (SMLM) and spectrally resolved SMLM (SR-SMLM) for enhanced spatial and spectral analysis.
• Analysis of single-molecule displacements to determine local diffusion coefficients and identify interactions.

Main Results:

• SMdM successfully achieved super-resolution diffusivity mapping in diverse cellular and in vitro systems.
• Revealed nanoscale diffusion heterogeneities in the cytoplasm and nucleus, linked to macromolecular crowding.
• Uncovered a charge effect impeding the diffusion of positively charged proteins and identified ubiquitous charge-driven protein interactions.

Conclusions:

• SMdM provides a powerful tool for super-resolution mapping and high-precision quantification of molecular diffusion.
• The study elucidates spatiotemporal heterogeneities in living cells, driven by crowding and charge effects.
• This technique opens new avenues for understanding molecular dynamics and interactions in complex biological environments.