Fluorescence recovery after photobleaching in material and life sciences: putting theory into practice

Abstract

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.

F-Actin Mediated Focusing of Vesicles at the Cell Tip Is Essential for Polarized Growth

“…Instead, P. patens only exhibits two modes of vesicle transport, namely diffusion and active transport along the cytoskeleton. We visualized these modes of transport and performed fluorescence recovery after photobleaching (FRAP;McNally, 2008;Lorén et al, 2015) during polarized growth. To probe the utility of actin-mediated active transport, we used the small molecule inhibitor latrunculin B to depolymerize actin and uncouple vesicle diffusion from active transport.…”

“…To probe the utility of actin-mediated active transport, we used the small molecule inhibitor latrunculin B to depolymerize actin and uncouple vesicle diffusion from active transport. With FRAP as well as number and brightness analyses Lorén et al, 2015;Kingsley et al, 2017), we measured vesicle diffusion rates and concentrations, respectively. We then developed a diffusion-limited analytical model and numerically solved it using these parameters as well as previously measured cellular growth rates (Furt et al, 2013).…”

Lattice Boltzmann simulations of diffusion through native and steam-exploded softwood bordered pits

“…An indication of this can clearly be seen from comparing the 40 kDa dextran with the 40 kDa protein. The smallest probe was in the higher range of what can be measured reliably with the FRAP technique (Lorén et al 2015), which was reflected in the standard deviation. Previous studies by Arrio-Dupont et al (1996) on dextran probes agree well with the results here.…”

“…Fluorescence recovery after photobleaching (FRAP) is an established technique (Lorén et al 2015) used to obtain information on the diffusion dynamics of fluorescent molecules commonly used with confocal microscopes. Labeled fluorescent probes in solution are irreversibly bleached in a volume by high intensity light, which causes a concentration gradient within the volume.…”

“…By using confocal laser scanning microscopy (CLSM) with the method of fluorescent recovery after photobleaching (FRAP) (Lorén et al 2015), the diffusion coefficients were determined for larger molecules in solution. Combined with simulations in a generated geometry based on SEM images of pit membrane structure, the effective diffusion coefficient of the entire structure was computed.…”