Analysis of simulated and experimental fluorescence recovery after photobleaching. Data for two diffusing components.

Fluorescence recovery after photobleaching has been a popular technique to quantify the lateral mobility of membrane components. A variety of analysis methods have been used to determine the lateral diffusional mobility, D. However, many of these methods suffer from the drawbacks that they are not able to discern two-component diffusion (i.e., three-point fit), cannot solve for two components (linearization procedures), and do not perform well at low signal-to-noise. To overcome these limitations, we have adopted the approach of fitting fluorescence recovery after photobleaching curves by the full series solution using a Marquardt algorithm. Using simulated data of one or two diffusing components, determinations of the accuracy and reliability of the method with regard to extraction of diffusion parameters and the differentiation of one- versus two-component recovery curves were made under a variety of conditions comparable with those found in actual experimental situations. The performance of the method was also examined in experiments on artificial liposomes and fibroblast membranes labeled with fluorescent lipid and/or protein components. Our results indicate that: 1) the method was capable of extracting one- and two-component D values over a large range of conditions; 2) the D of a one-component recovery can be measured to within 10% with a small signal (100 prebleach photon counts per channel); 3) a two-component recovery requires more than 100-fold greater signal level than a one-component recovery for the same error; and 4) for two-component fits, multiple recovery curves may be needed to provide adequate signal to achieve the desired level of confidence in the fitted parameters and in the differentiation of one- and two-component diffusion.