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

Abstract

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.

Fluorescence Recovery after Photobleaching in Colloidal Science: Introduction and Application

“…Diffusion characteristics of diverse materials are critical for nonbiological as well as biological applications, such as drug delivery, cell culture, and tissue engineering. − The behavior of material components determines material stability and mechanical characteristics; similarly, diffusion of active chemicals in the bulk material determines their presentation or release. In 1827, Robert Brown observed the free migration of plant organelles expelled from pollen grains into a surrounding aqueous solution.…”

Versatile Types of Inorganic/Organic NIR-IIa/IIb Fluorophores: From Strategic Design toward Molecular Imaging and Theranostics

“…Molecular imaging is an emerging technique that can noninvasively detect, and monitor the physiological or pathological processes in vivo at the cellular and molecular level, which is essential for the uncovering of molecular and cellular mechanisms in pathophysiologic processes. − Different from conventional imaging techniques which mainly reveal the anatomical changes caused by accumulated molecular changes, molecular imaging aims at detecting the abnormalities at the cellular and molecular levels in the super early stages, providing valuable information for the occurrence, development, and outcome of diseases, evaluating the efficacy of drugs, thus bridging the molecular biology and the clinical medicine. Among the existing imaging modalities, NIR-IIa/IIb fluorescence imaging with high sensitivity and resolution can monitor the rapid biological processes in detail.…”

Tracking Molecular Diffusion across Biomaterials’ Interfaces Using Stimulated Raman Scattering

“…Current methods to quantify the rates of diffusion often involve labeling, include the use of fluorescent markers with confocal imaging in a z-stack, nuclear magnetic resonance (NMR), dynamic light scattering (DLS), , fluorescence recovery after photobleaching (FRAP), and neutron transmission . However, tagging with such labels is not always practical and may influence the molecule’s interaction with tissues or cells.…”

“…Effective characterization of mass transfer, through either convective or non-convective mechanisms, provides a key predictor of the transport process kinetics and may be important in informing new biomaterials' formulations and formats. 2 Current methods to quantify the rates of diffusion often involve labeling, include the use of fluorescent markers with confocal imaging in a z-stack, 3 nuclear magnetic resonance (NMR), 4 dynamic light scattering (DLS), 5,6 fluorescence recovery after photobleaching (FRAP), 7 and neutron trans-mission. 8 However, tagging with such labels is not always practical and may influence the molecule's interaction with tissues or cells.…”