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Application of fluorescence recovery after photobleaching to diffusion of a polyelectrolyte in a multilayer film

The diffusion coefficient, D, and the proportion of mobile molecules, p, is measured for a fluorescently labeled polyelectrolyte in a multilayer film using fluorescence recovery after photobleaching (FRAP). The film was composed of poly(L‐lysine) (PLL) and hyaluronan (HA). The labeled polyelectrolyte (PLLFITC) was either deposited on top of the film or embedded within it. A circular area of diameter ≈ 60 μm was bleached using a confocal laser scanning microscope. Because molecules do already diffuse during the bleaching step, the initial light intensity profile is not characteristic of a uniformly bleached circular area. A formalism is developed in which a simple mathematical representation of a measured profile serves as starting profile. This radial distribution is introduced in the equation describing the time evolution of the labeled molecule concentration under the hypothesis that the recovery results from pure, two‐dimensional Brownian diffusion of the mobile molecules according to Fick's law. The analysis of a series of images taken at successive times after bleaching (up to approximately one hour) leads to estimates of D of the order of 0.2 μm2 s−1 for labeled molecules deposited on top of the film and a 5‐fold smaller value for the molecules embedded in the film. However, p is remarkably insensitive to the position in the multilayer architecture (p ≈ 0.40). Microsc. Res. Tech. 66:43–57, 2005. © 2005 Wiley‐Liss, Inc.