Fluorescence Recovery After Photobleaching in Ultrathin Polymer Films

Fluorescence recovery after photobleaching (FRAP) is a widely used technique to study 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, we show that the FRAP behaviour 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. Our 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 labelled species in the interpretation of FRAP data. An important conclusion from our numerical and experimental study is that, for ultrathin PEM films ~ 100 nm thicknesses, recovery is dominated through solution medium and hence, classical FRAP analysis is not sufficient to probe diffusion in PEM. Our numerical study reveals several aspect of FRAP phenomena in thin polymer films which are critical for the proper interpretation of experimental data.