Fluorescence Recovery after Photobleaching in Ultrathin Polymer Films

• Published in: Macromolecular chemistry and physics 2018-04, Vol.219 (7), p.n/a
• Main Authors: Prakash, Praneet, Pahal, Suman, Varma, Manoj
• Format: Article
• Language: English
• Subjects: Computer simulation; Diffusion rate; Fluorescence; fluorescence recovery; Multilayers; photobleaching; polyelectrolyte multilayer (PEM); Polyelectrolytes; Polymer films; Polymers; Recovery; Thin films; Transport; ultrathin polymer films
• ISSN: 1022-1352; 1521-3935
• DOI: 10.1002/macp.201700543

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. The fluorescence recovery after photobleaching in a multispecies system such as polyelectrolyte multilayer (PEM) is initiated after it is introduced to an aqueous medium. Often, researchers neglect the recovery through aqueous medium, citing low diffusion coefficient. Surprisingly, the routinely used analysis can have significant error in the case of ultrathin (≈100 nm) PEM.