A method improving the accuracy of fluorescence recovery after photobleaching analysis.

Fluorescence recovery after photobleaching, FRAP, has been an established technique of quantifying the mobility of molecular species in cells and cell membranes for more than 30 years. However, under non-ideal experimental conditions the current methods of analysis still suffer from occasional problems, for example, when the signal to noise ratio is low, when there are temporal fluctuations in the illumination or when there is bleaching during the recovery process. We here present a method of analysis that overcomes these problems, yielding accurate results even under non-ideal experimental conditions. The method is based on circular averaging of each image, followed by spatial frequency analysis of the averaged radial data, and requires no prior knowledge of the shape of the bleached area. The method was validated using both simulated and experimental FRAP data, illustrating that the diffusion coefficient of a single diffusing component can be determined to within ~1 %, even for small signal levels (100 photon counts), and that at typical signal levels (5000 photon counts) a system with two diffusion coefficients can be analyzed with less than 10 % error