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Diffusion Tensor Analysis by Two-Dimensional Pair Correlation of Fluorescence Fluctuations in Cells.

Carmine Di Rienzo1, Francesco Cardarelli2, Mariagrazia Di Luca3

  • 1NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, Piazza San Silvestro, Pisa, Italy; Center for Nanotechnology Innovation @NEST, Istituto Italiano di Technologia, Piazza San Silvestro, Pisa, Italy.

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Summary

We developed a new method using 2D pair correlation function (pCF) analysis to map molecular diffusion and barriers within live cells. This technique reveals preferential diffusion routes and distinguishes between isotropic and anisotropic molecular motion.

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Area of Science:

  • Cellular and Molecular Biophysics
  • Biomolecular Dynamics and Transport

Background:

  • Cellular organization into subcompartments regulates biomolecule movement, creating diffusion barriers and directing molecules to targets.
  • Existing experimental tools lack the spatiotemporal resolution to fully probe intracellular connectivity and accessibility.
  • Understanding nanoscale molecular diffusion is crucial for deciphering cellular organization and function.

Purpose of the Study:

  • To develop and validate a novel experimental approach for mapping molecular dynamics and diffusion barriers within live cells.
  • To overcome limitations of current methods in characterizing complex intracellular environments.
  • To provide insights into the regulation of molecular diffusion by the intracellular milieu.

Main Methods:

  • Utilized a two-dimensional (2D) extension of the pair correlation function (pCF) analysis on time-series image data.
  • Calculated 2D pCF near each point for each time delay to analyze spatial distribution and diffusion routes.
  • Combined 2D pCF with image-derived mean-square displacement (iMSD) to determine directional molecular displacements.

Main Results:

  • The 2D pCF analysis accurately describes preferential diffusive routes within the cell.
  • The combined iMSD approach provides directional information on nanoscopic molecular displacements.
  • Developed a fluorescence-fluctuation-based diffusion tensor capable of distinguishing between isotropic and anisotropic local diffusion.

Conclusions:

  • The novel 2D pCF and iMSD tensor approach offers a powerful tool for characterizing heterogeneous molecular dynamics in live cells.
  • This method can map diffusion barriers and reveal directional molecular motion, overcoming limitations of traditional techniques.
  • The diffusion tensor measurement advances the understanding of how the intracellular environment regulates nanoscale molecular diffusion.