Fluorescence recovery after photobleaching: direct measurement of diffusion anisotropy.

ABSTRACT

Fluorescence recovery after photobleaching (FRAP) is a widely used technique for studying diffusion in biological tissues. Most of the existing approaches for the analysis of FRAP experiments assume isotropic diffusion, while only a few account for anisotropic diffusion. In fibrous tissues, such as articular cartilage, tendons and ligaments, diffusion, the main mechanism for molecular transport, is anisotropic and depends on the fibre alignment. In this work, we solve the general diffusion equation governing a FRAP test, assuming an anisotropic diffusivity tensor and using a general initial condition for the case of an elliptical (thereby including the case of a circular) bleaching profile. We introduce a closed-form solution in the spatial coordinates, which can be applied directly to FRAP tests to extract the diffusivity tensor. We validate the approach by measuring the diffusivity tensor of [Formula see text] FITC-Dextran in porcine medial collateral ligaments. The measured diffusion anisotropy was [Formula see text] (SE), which is in agreement with that reported in the literature. The limitations of the approach, such as the size of the bleached region and the intensity of the bleaching, are studied using COMSOL simulations.