An improved strip FRAP method for estimating diffusion coefficients: Correcting for the degree of photobleaching

Summary Fluorescence recovery after photobleaching is a widely established method for the estimation of diffusion coefficients, strip bleaching with an associated recovery curve analysis being one of the simplest techniques. However, its implementation requires near 100% bleaching in the region of interest with negligible fluorescence loss outside, both constraints being hard to achieve concomitantly for fast diffusing molecules. We demonstrate that when these requirements are not met there is an error in the estimation of the diffusion coefficient D, either an under-or overestimation depending on which assumption is violated the most. We propose a simple modification to the recovery curve analysis incorporating the concept of the relative bleached mass m giving a revised recovery time parametrization τ = m2w 2/4πD for a strip of width w. This modified model removes the requirement of 100% bleaching in the region of interest and allows for limited diffusion of the fluorophore during bleaching. We validate our method by estimating the (volume) diffusion coefficient of FITC-labelled IgG in 60% glycerol solution, D = 4.09 ± 0.21 μm2 s-1, and the (surface) diffusion coefficient of a green-fluorescent protein-tagged class I MHC protein expressed at the surface of a human B cell line, D = 0.32 ± 0.03 μm2 s-1 for a population of cells. © 2009 The Royal Microscopical Society.