Quantitative FRAP by Means of Diffusion through 3D Polyelectrolyte Shells Using Confocal and Two-photon Excitation Approaches. (2005)

In a typical FRAP (fluorescence recovery after photobleaching) experiment, the fluorophores in a region of interest (ROI) are irreversibly "turned off" by photobleaching Then fluorescence recovery within the ROI, due to the mobility of the marked molecules, is monitored as function of time and fluorescence intensity. The analysis of the fluorescence recovery behavior with a suitable theoretical model allows the evaluation of the diffusion coefficient of the species bound to fluorescent molecules. Unfortunately, the theoretical models reported in literature require precise conditions that often cannot be satisfied by the experimental set-up First, many of the algorithms neglect diffusion processes during the bleach time. Consequently, a sort of "corona" effect arises, resulting in a understimated diffusion coefficient Unfortunately, this approach works only for homogeneous media (same viscosity in a large area surrounding the chosen region), a condition that often cannot be satisfied when performing FRAP experiments on biological samples. Moreover, due to the spot size of the laser beam, a mismatch between the region chosen and the one effectively bleached is always induced. This phenomenon can cause a significant error when evaluating diffusion processes in structures of a size comparable or smaller than the laser beam spot (~200nm). We are investigating the influence of the "corona effect" as well as the mismatch between the bleaching area and the ROI on the evaluated diffusion. As a heterogeneous model system, a micrometer sized cubic polyelectrolyte capsule with unknown pore width was used It was through this multilayer system that the diffusion coefficients of different molecular weight dextran molecules were determined. The error induced by the "corona effect" can be eliminated by using the approach described by Weiss [2] for each sample, different FRAP experiments have been performed by varying the bleaching time t bleach . The curves of the estimated diffusion coefficients in dependence of t bleach can be fitted with the empirical function: D = (a/(1+(t bleach /t limit ) b ) + c, where t limit represents the shortest bleach time used. Then, the value of D at t bleach = D /15, for which the corona effect can be neglected, can be extrapolated. Due to the fact that FITC-dex molecules at room temperature diffuse quite fast through capsule walls ( D /15 5ms), the value of D can only be extrapolated to t bleach = 0. Then, in order to estimate the error induced by the mismatch of the bleaching spot with the ROI, two different FRAP experiments on cubic polylectrolyte shells have been developed. In the first case, a bleaching region slightly larger than the shell has been chosen, while in the second a region that exactly matches the inner part of the shell was chosen. The results of the measurements performed for the diffusion of 40kDa FITC-dex through the polyelectrolyte multilayer are shown in figure The determined values for the diffusion from extrapolation with the Weiss function [2] are D 40kDa, out = (0.17 0.07) m 2 /s and D 40kDa, in = (0.028 0.008) m 2 /s. The diffusion coefficients measured by bleaching the capsule inside should give a better estimation of the real diffusion through the multi-layered wall than that which relies partly on the bleaching of outside regions. In the second case, also free diffusion plays a role. In case of the linear 10kDa-dextran, the diffusion in presence and in absence of NaCl was