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.
Highlights
Diffusion is a major molecular transport mechanism in biological systems
Noninvasive 3D diffusion tensor measurement of fastdiffusing molecules was achieved by the development of a LiFTFRAP data acquisition system and an accompanying 3D fluorescence recovery after photobleaching (FRAP) data analysis method
Our results demonstrate that LiFT-FRAP is a robust technique for accurately and noninvasively measuring 3D diffusion tensors of biorelevant molecules with various sizes in a diverse range of biological systems, including fibrous tissues and tissue-engineered scaffolds, resolving the technical hurdle that prevents our understanding of tissue pathophysiology, the development of clinical treatment for translational applications, and standardization of biofabrication for the biotech industry
Summary
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 define tissue functions. As evidenced by computational modeling, 3D anisotropic diffusion generates significantly different molecular concentration distributions compared to direction-independent (i.e., isotropic) diffusion[12,13,14] (Fig. 1c and Supplementary Note 1) These altered concentration profiles and diffusion rates yield significant downstream effects on cellular responses and tissue function[12,13,14]. Biotransport research field in studying molecular diffusion is suffering from a lack of analytical tools capable of quantitative 3D diffusion measurement of various biomolecules in biological tissues, as well as biomaterials[15]. Increasing efforts have been made to determine the 3D diffusion properties in various biological tissues (e.g., intervertebral discs[17,18,21], temporomandibular joint discs[23], and corneas24) based on the existing 1D or 2D approaches through physical tissue sectioning Such a strategy suffers from invasiveness and laborintensiveness. In situ magnetic resonance imaging (MRI)-based 3D diffusion tensor imaging in intact ovine corneas reported anisotropic extracellular diffusion[26]
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