Improving parameter estimation for cell surface FRAP data

Fluorescence Recovery After Photobleaching (FRAP) using the confocal laser scanning microscope has become a standard method used to determine the diffusion coefficient and mobile fraction of cell surface proteins. A common experimental approach is to bleach a stripe on the cell surface and fit the ensuing FRAP curve to a 1D diffusion model. This model is derived from the time course of recovery to an infinitely long stripe bleached on an infinite flat plane. This choice of model dictates the use of a long bleach stripe. We demonstrate that, in the case of a long bleach stripe, the finite extent of the cell leads to significant errors in parameter estimation. We further show that these errors are reduced when a relatively small stripe is bleached. Unfortunately, diffusion to such a region is fundamentally two dimensional and therefore applying the 1D model of diffusion leads to significant errors. We derive an equation suitable for fitting to FRAP data acquired from small bleach regions and analyze its accuracy using simulated data. We propose that the use of a small bleach region along with a two dimensional diffusion model is the ideal protocol for cell surface FRAP.