A noninvasive fluorescence imaging-based platform measures 3D anisotropic extracellular diffusion

Nat Commun. 2021 Mar 26;12(1):1913. doi: 10.1038/s41467-021-22221-0.


Diffusion is a major molecular transport mechanism in biological systems. Quantifying direction-dependent (i.e., anisotropic) diffusion is vitally important to depicting how the three-dimensional (3D) tissue structure and composition affect the biochemical environment, and thus define tissue functions. However, a tool for noninvasively measuring the 3D anisotropic extracellular diffusion of biorelevant molecules is not yet available. Here, we present light-sheet imaging-based Fourier transform fluorescence recovery after photobleaching (LiFT-FRAP), which noninvasively determines 3D diffusion tensors of various biomolecules with diffusivities up to 51 µm2 s-1, reaching the physiological diffusivity range in most biological systems. Using cornea as an example, LiFT-FRAP reveals fundamental limitations of current invasive two-dimensional diffusion measurements, which have drawn controversial conclusions on extracellular diffusion in healthy and clinically treated tissues. Moreover, LiFT-FRAP demonstrates that tissue structural or compositional changes caused by diseases or scaffold fabrication yield direction-dependent diffusion changes. These results demonstrate LiFT-FRAP as a powerful platform technology for studying disease mechanisms, advancing clinical outcomes, and improving tissue engineering.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Anisotropy
  • Collagen / chemistry
  • Collagen / metabolism
  • Cornea / metabolism*
  • Diffusion
  • Extracellular Space / metabolism*
  • Fluorescence Recovery After Photobleaching / methods*
  • Fourier Analysis
  • Microscopy, Confocal / methods
  • Microscopy, Electron, Scanning / methods
  • Microscopy, Fluorescence, Multiphoton / methods*
  • Rats, Sprague-Dawley
  • Reproducibility of Results
  • Swine
  • Tendons / metabolism*
  • Tissue Engineering / methods
  • Tissue Scaffolds / chemistry


  • Collagen