Fluorescence recovery after photobleaching , probe diffusion technique

A third technique for studying foam films is the fluorescence recovery after photobleaching (FRAP). This techniques was applied by Clarke et al. [36] for lateral diffusion in foam films, and involves irreversible photobleaching by intense laser light of fluorophore molecules in the sample. The time of redistribution of probe molecules (which are assumed to be randomly distributed within the constitutive membrane lipids in the film) is monitored. The lateral diffusion coefficient, D, is calculated from the rate of recovery of fluorescence in the bleaching region due to the entry of unbleaching fluoroprobes of adjacent parts of the membranes. 

A number of macromolecular diffusion and conformational properties can be studied using fluorescence anisotropy, fluorescence correlation spectroscopy (ECS), and fluorescence recovery after photobleaching (FRAP). These techniques most commonly are applied to proteins labelled with highly fluorescent probes, but can exploit intrinsic fluorescence in some instances. In fluorescence anisotropy studies, polarized light is used to selectively excite molecules whose transition dipole moments are aligned with the electric field vector. Steady-state... 

Fluorescence recovery after photobleaching (FRAP) is a technique that allows the diffusion coefficients of a fluorescent probe to be measured. With a highly intense laser pulse all fluorophores in a selected spot are destroyed irreversibly, and the subsequent diffusion of fresh probe molecules into the area is followed by an increase in the fluorescence intensity. To give an example, gelatine-based organogels in microemulsions were investigated with this method [80]. 

In micro- and nanoscale fluid mechanics, measurements of mass transport and fluid velocity are used to probe fundamental physical phenomena and evaluate the performance of microfluidic devices. Evanescent wave illumination has been combined with several other diagnostic techniques to make such measurements within a few hundred nanometers of fluid—solid interfaces with a resolution as small as several nanometers. Laser Doppler velocimetry has been applied to measure single-point tracer particle velocities in the boundary layer of a fluid within 1 pm of a wall. By seeding fluid with fluorescent dye, total internal reflection fluorescence recovery after photobleaching (FRAP) has been used to measure near-wall diffusion coefficients and velocity (for a summary of early applications, see Zettner and Yoda [2]). 

The nature of polymer motion in semidilute and concentrated solutions remains a major question of macromolecular science. Extant models describe polymer dynamics very differently 3-11). Many experimental methods have been used to study polymer dynamics (12). One meAod is probe diffusion, in which inferences about polymer dynamics are made by observing the motions of dilute mesoscopic probe particles diffusing in the polymer solution of interest. Probe diffusion can be observed by several experimental techniques, for example, quasi-elastic light scattering spectroscopy (QELSS), fluorescence recovery after photobleaching (FRAP), and forced Rayleigh scattering (FRS). 

A variety of physical techniques has been used to measure probe diffusion in polymer solutions. In the fluorescence recovery after photobleaching (FRAP) technique, a small fluorescent label is attached to the probes. An intense pulse of light (the pump laser beam) then destroys ( bleaches ) the fluorescent labels in some volume of the solution. Much weaker illumination (the probe beam) is used to monitor recovery of the fluorescence intensity as unbleached fluorescent molecules diffuse back into the regions in which the bleaching occurred. 

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