Fluorescence recovery after photobleaching (FRAP) is a versatile tool for determining diffusion and interaction/binding properties in biological and material sciences. An understanding of the mechanisms controlling the diffusion requires a deep understanding of structure-interaction-diffusion relationships. In cell biology, for instance, this applies to the movement of proteins and lipids in the plasma membrane, cytoplasm and nucleus. In industrial applications related to pharmaceutics, foods, textiles, hygiene products and cosmetics, the diffusion of solutes and solvent molecules contributes strongly to the properties and functionality of the final product. All these systems are heterogeneous, and accurate quantification of the mass transport processes at the local level is therefore essential to the understanding of the properties of soft (bio)materials. FRAP is a commonly used fluorescence microscopy-based technique to determine local molecular transport at the micrometer scale. A brief high-intensity laser pulse is locally applied to the sample, causing substantial photobleaching of the fluorescent molecules within the illuminated area. This causes a local concentration gradient of fluorescent molecules, leading to diffusional influx of intact fluorophores from the local surroundings into the bleached area. Quantitative information on the molecular transport can be extracted from the time evolution of the fluorescence recovery in the bleached area using a suitable model. A multitude of FRAP models has been developed over the years, each based on specific assumptions. This makes it challenging for the non-specialist to decide which model is best suited for a particular application. Furthermore, there are many subtleties in performing accurate FRAP experiments. For these reasons, this review aims to provide an extensive tutorial covering the essential theoretical and practical aspects so as to enable accurate quantitative FRAP experiments for molecular transport measurements in soft (bio)materials.

Confocal or multi-photon laser scanning microscopes are convenient tools to perform FRAP diffusion measurements. Despite its popularity, accurate FRAP remains often challenging since current methods are either limited to relatively large bleach regions or can be complicated for non-specialists. In order to bring reliable quantitative FRAP measurements to the broad community of laser scanning microscopy users, here we have revised FRAP theory and present a new pixelbased FRAP method relying on the photobleaching of rectangular regions of any size and aspect ratio. The method allows for fast and straightforward quantitative diffusion measurements due to a closed-form expression for the recovery process utilising all available spatial and temporal data. After a detailed validation, its versatility is demonstrated by diffusion studies in heterogeneous biopolymer mixtures. 2010 Optical Society of America

The time-dependent diffusion and mechanical properties of gelatin in solution, in the gel state, and during the sol/gel transition were determined using fluorescence recovery after photobleaching (FRAP) and rheology. The parameters in the experimental design were 2% w/w and 5% w/w gelatin concentration; 15, 20, and 25 °C end quench temperatures; and Na(2)-fluorescein, 10 kDa FITC-dextran, and 500 kDa FITC-dextran as diffusion probes. The samples were monitored in solution at 60 °C, during quenching, for 75 min at end quench temperatures and after 1, 7, and 14 days of storage at the end quench temperature. The effect of temperature on the probe diffusion was normalized by determining the free diffusion of the probes in pure water for the different temperatures. The results gained by comparing FRAP and rheology showed that FRAP is able to capture structural changes in the gelatin before gelation occurs, which was interpreted as a formation of transient networks. This was clearly seen for 2% w/w gelatin and 20 and 25 °C end quench temperatures. The structural changes during sol/gel transition are detected only by the larger probes, giving information about the typical length scales in the gelatin structure. The normalized diffusion rate increased after 7 and 14 days of storage. This increase was most pronounced for fluorescein but was also seen for the larger probes.

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