Surfaces fluorescence recovery after photobleaching

To increase the speed of the TIRF-based kinetic techniques, the perturbation can be optical rather than chemical. If the evanescent wave intensity is briefly flashed brightly, then some of the fluorophores associated with the surface will be photobleached. Subsequent exchange with unbleached dissolved fluorophores in equilibrium with the surface will lead to a recovery of fluorescence, excited by a continuous but much attenuated evanescent wave. The time course of this recovery is a measure of the desorption kinetic rate k2. This technique1-115) is called TIR/FRAP (or TIR/FPR) in reference to fluorescence recovery after photobleaching (or fluorescence photobleaching recovery). 

A very suitable method for measurement of the lateral diffusion of molecules adsorbed at the foam film surfaces is Fluorescence Recovery after Photobleaching (FRAP) ([491-496], see also Chapter 2). Measurements of the lateral diffusion in phospholipid microscopic foam films, including black foam films, are of particular interest as they provide an alternative model system for the study of molecular mobility in biological membranes in addition to phospholipid monolayers at the air/water interface, BLMs, single unilamellar vesicles, and multilamellar vesicles. 

Consider surfaces that are inert and may be made (molecularly) smooth, so that, optically speaking, they may be treated as Fresnel surfaces. Mica, certain polished glasses, quartz and silicon wafer surfaces may belong to this category. For such well-defined systems the optical techniques introduced in sec. 1.7.10 come to mind reflectometry, ellipsometry, and (to study the dynamics) fluorescence recovery after photobleaching (FRAP). The principles of these techniques have been outlined in that section. 

Figure 12.29. Fluorescence Recovery After Photobleaching (FRAP) Technique. (A) The cell-surface fluoresces because of a labeled surface component. (B) The fluorescent molecules of a small part of the surface are bleached by an intense light pulse. (C) The fluorescence intensity recovers as bleached molecules diffuse out of the region and unbleached molecules diffuse into it. (D) The rate of recovery depends on the diffusion coefficient.

Jacobson K, Dersko Z, Wu ES, Hou Y, Poste G (1976) Measurement of the lateral mobility of cell surface components in single living cells by fluorescence recovery after photobleaching. J Supramol Struct 5 565-576... 

A EXPERIMENTAL FIGURE 5-6 Fluorescence recovery after photobleaching (FRAP) experiments can quantify the lateral movement of proteins and lipids within the plasma membrane, (a) Experimental protocol. Step H Cells are first labeled with a fluorescent reagent that binds uniformly to a specific membrane lipid or protein. Step B A laser light is then focused on a small area of the surface, irreversibly bleaching the bound reagent and thus reducing the fluorescence in the illuminated area. Step B In time, the fluorescence of the bleached patch increases as unbleached fluorescent surface molecules diffuse into it and bleached ones diffuse outward. The extent of recovery of fluorescence in the bleached patch is... 

One of the recent techniques most commonly used to measure diffusion in 2-dimensions is fluorescence recovery after photobleaching (FRAP). This method uses a laser beam focused through a fluorescence microscope to follow the diffusion of fluorescent molecules in a plane perpendicular to the laser beam. The fluorescence intensity from a laser spot of known diameter, typically a few microns, is measured. The laser intensity is then increased by approximately 1000 times. This irreversibly photobleaches any fluorophore in the spot. The intensity is then decreased again and the recovery in fluorescence intensity measured as unbleached molecules diffuse into the spot. The time function of fluorescence intensity is then analysed to give surface self-diffusion coefficient (Clark et al. 1990a, b, Wilde Clark 1993, Ladhaetal. 1994). 

Noiiy Ring methods, pendant and spinning drop methods, for surface and interfacial dilational elasticity, thin-film techniques, and surface lateral diffusion when using fluorescence recovery after photobleaching (FRAP) methods. 

Figure 6 a) Fluorescence recovery after photobleaching for hexadecane flowing aver a sapphire surface (filled symbols) and over a dense OTS monolayer (open symbols), b) Evolution of the FRAP curves with incubation time t for a 1% solution of stearic acid in hexadecane. t = 0 means less than one hour. 

The FRAP (fluorescence recovery after photobleaching) technique uses photobleaching to measure molecular diffusion and can be used if the material in question is confined to a specific plane (e.g., a membrane or cytoskeletal filaments adsorbed on a surface). The fluorophores... 

Pit and co-workers used a fluorescence recovery after photobleaching (FRAP) technique to assess the velocity of liquid close to a solid surface under simple Couette shear [5][6]. A 90 pm thick film of... 

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