FRAP技术及数学模型的应用.ppt

Using FRAP and mathematical modeling to determine thein vivo kinetics of nuclear proteins Methods 29 (2003) 14–28 Estimating diffusion within a membrane-bounded domain * Example of photobleaching. An Indian muntjac fibroblast nucleus expressing ASF/SF2-GFP is shown before (left) and after (right) photobleaching of a 2um spot within the nucleus. Bar=10 um. Example of a FRAP recovery curve. Images collected at different points in the recovery time course are shown. Analyzing the data The panel shows the normalized plot of intensity-versus-time for the cell shown. Green lines represent the half-time of recovery. Relative Intensity= Ii I0 Hi H0 u u u u u u The most commonly used approach to describe the mobility of nuclear proteins during FRAP experiments is to assume the diffusion coefficient. u(x,t): the concentration of fluorescent species x : the position of fluorescent species Deff : the effective diffusion coeffcient, f (x) : the initial condition of ?uorescent species right after photobleaching. A simple 2D model for diffusion The initial condition depends on the intensity profile I(x) of the laser beam that is used to photobleach a certain region. Then the theoretical recovery curve is given where q is the product of all the quantum effciencies of light absorption, emission, and detection and A is the attenuation factor of the laser beam during observation of recovery Axelrod et al. obtained explicit theoretical fluorescence recovery curve, which can be used to fit the normalized data F (tj), w and K are parameters that characterize the bleaching profile of the laser beam, w is the half-width of the intensity at e-2 height, and K is a parameter describing the amount of bleaching a fixed specimen, with an initial uniform fluorophore concentration Ci, is photobleached under the same conditions