Measuring Diffusion in Non-Sectioned Articular Cartilage: A FRAP Sensitivity Study

Measuring the diffusion of molecules within articular cartilage is essential in characterizing its behavior. Information about this important mechanism may be useful to understanding changes in cartilage during degeneration or osteoarthritis. One method used in quantifying diffusion is fluorescence recovery after photobleaching (FRAP). The FRAP technique has been used in previous studies for cartilage [1] and various tissues [2]. In FRAP, a small region of interest (ROI) is selected within the tissue and fluorescent molecules are bleached using a higher laser power than would be used for imaging. Immediately following the ROI bleaching, bleached molecules diffuse out of the ROI as unbleached molecules diffuse into it. The average intensity data within the ROI is collected as a function of time. This data is then fit to a diffusion model usually resulting in a calculation of the diffusion constant, D (μm²/second) [3].