Fluorescence recovery after photobleaching: direct measurement of diffusion anisotropy

Fluorescence recovery after photobleaching (FRAP) is a widely used technique for studying difusion in biological tissues. Most of the existing approaches for the analysis of FRAP experiments assume isotropic difusion, while only a few account for anisotropic difusion. In fbrous tissues, such as articular cartilage, tendons and ligaments, difusion, the main mechanism for molecular transport, is anisotropic and depends on the fbre alignment. In this work, we solve the general difusion equation governing a FRAP test, assuming an anisotropic difusivity tensor and using a general initial condition for the case of an elliptical (thereby including the case of a circular) bleaching profle. We introduce a closed-form solution in the spatial coordinates, which can be applied directly to FRAP tests to extract the difusivity tensor. We validate the approach by measuring the difusivity tensor of 3 kDa FITC-Dextran in porcine medial collateral ligaments. The measured difusion anisotropy was 1.42 ± 0.015 (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