Fluorescence Recovery after Photobleaching in Ultrathin Polymer Films

Abstract Fluorescence recovery after photobleaching (FRAP) is a widely used technique to study the transport of molecules in biological systems. Recently, FRAP has been used to study molecular transport in polyelectrolyte multilayers (PEMs). Through numerical simulations verified by experiments, it is shown that the FRAP behavior of PEM films in an aqueous medium differs significantly from that in previously explored systems such as single cells. This is because fluorescence recovery can take place through the aqueous medium surrounding the PEM film. The simulations show the critical role of the time scale of the different processes, namely, diffusion through PEM, diffusion through surrounding medium, and the unbinding rate of fluorophore‐labeled species in the interpretation of FRAP data. An important conclusion from the numerical and experimental study is that, for ultrathin PEM films with ≈100 nm thicknesses, recovery is dominated through the solution medium and hence, classical FRAP analysis is not sufficient to probe diffusion in PEM. The numerical study reveals several aspects of the FRAP phenomena in thin polymer films that are critical for the proper interpretation of experimental data.