Fluorescence recovery after photobleaching determination

One of the most popular applications of molecular rotors is the quantitative determination of solvent viscosity (for some examples, see references [18, 23-27] and Sect. 5). Viscosity refers to a bulk property, but molecular rotors change their behavior under the influence of the solvent on the molecular scale. Most commonly, the diffusivity of a fluorophore is related to bulk viscosity through the Debye-Stokes-Einstein relationship where the diffusion constant D is inversely proportional to bulk viscosity rj. Established techniques such as fluorescent recovery after photobleaching (FRAP) and fluorescence anisotropy build on the diffusivity of a fluorophore. However, the relationship between diffusivity on a molecular scale and bulk viscosity is always an approximation, because it does not consider molecular-scale effects such as size differences between fluorophore and solvent, electrostatic interactions, hydrogen bond formation, or a possible anisotropy of the environment. Nonetheless, approaches exist to resolve this conflict between bulk viscosity and apparent microviscosity at the molecular scale. Forster and Hoffmann examined some triphenylamine dyes with TICT characteristics. These dyes are characterized by radiationless relaxation from the TICT state. Forster and Hoffmann found a power-law relationship between quantum yield and solvent viscosity both analytically and experimentally [28]. For a quantitative derivation of the power-law relationship, Forster and Hoffmann define the solvent s microfriction k by applying the Debye-Stokes-Einstein diffusion model (2)... 

Determination of translational diffusion rates of proteins requires measurements at longer timescales, one-tenth of a second to several minutes. Eosin derivatives are also commonly used to measure translational diffusion coefficients using the Fluorescence Recovery After Photobleaching technique [138-141],... 

Unambiguous determination of the conditions under which slippage occurs requires a technique able to measure the velocity of the fluid in the immediate vicinity of the solid wall over a thickness comparable to the size of a polymer chain, i.e. a few tens of nanometers. Classical laser Doppler velocimetry does not meet this requirement even if it allows for the determination of velocity profiles which clearly reveal a non-zero velocity within typically a few 10 pm from the wall. We have developed a new optical technique. Near Field Velocimetry (N.F.V.) [14], which combines Evanescent Wave Induced Fluorescence (E.WF.) [27] and Fringe Pattern Fluorescence Recovery After Photobleaching (F.P.F.R.A.P.) [28]. The former technique gives the spatial resolution normal to the solid wall, while the latter one enables the determination of the local velocity of the fluid. A major constraint of the technique is that it needs polymer molecules labelled with an easily photobleachable fluorescent probe. 

In another approach, the interfacial diffusion of the nanoparticles was determined using two photobleaching methods fluorescence loss induced by photobleaching (FLIP) and fluorescence recovery after photobleaching (FRAP). It was found that the lateral diffusion of the nanoparticles at the interface as well as the diffusion normal to and from the interface deviated by about four orders of magnitude from the values obtained in free solution [46],... 

The lateral mobility of proteins and lipids in natural and artificial lipid bilayer membranes was determined by different methods. For long-range mobility, fluorescence recovery after photobleaching (13-15) and electrophoresis of membrane components (16) were employed. We employed the electrophoresis method for determination of the eletrophoretic and diffusional mobilities of PSI in the plane of hypotonically inflated, spherical thylakoid vesicles. To monitor the redistribution of PSI particles, we made use of the spatial characteristics of the contribution of PSI particles to electrophotoluminescence (EPL) (17, 18). The contribution of PSII to EPL was eliminated by heat treatment of the chloroplasts (19). The EPL originates from the PSI particles at the hemisphere of the vesicles at which the induced electrical field destabilizes the photoinduced charge separation (18). The electrophoretic and diffusional mobilities were measured in vesicular suspensions to avoid immobilization for microscopic visualization (20). The photosynthetic membranes are devoid of cytoskeletal elements that might interfere with the lateral mobility. 

Fluorescence recovery after photobleaching has been used to determine lateral diffusion in block copol5uner bilayers (Fig. 21). The experiments 5deld a time constant x from which the lateral diffusion coefficient D = P/2x is calculated. In the fluid La phase, lipid molecules have lateral diffusion coefficients of... 

Fluorescence recovery after photobleaching (FRAP) and the measurement of the fluorescence resonance energy (FRET), which allow the diffu-sional mobility of cellular components and their interaction at the molecular level to be monitored are other varieties of time-resolved methods [30]. A novel method is fluorescence correlation spectroscopy (FCS), which measures the statistical fluctuations of fluorescence intensity within a con-focally illuminated volume. Correlation analysis allows the concentration of particles and their diffusion to be determined [31], [32], FCS has proved... 

A most powerful method for an unambiguous determination of selfdiffusion coefficients in polymer melts, involving fluorescence recovery after photobleaching, has been developed in Sillescu s laboratory (45). Data were obtained for polystyrene up to M = 150,000 at 177°C, at which D = 4 X 10 cm -s" was obtained. The high precision of the data permitted the authors to determine the effect of the labeling on the diffusion rate Increasing the number of fluorescein labels from one to two on a polystyrene with M = 43,700 reduced D by about a third. It is obvious that our method is crude in comparison, but it recommends itself by its great experimental simplicity and the ability to study even slower processes. 

II.1. Experimental Procedure. The micelles self-diffusion coefficient has been determined by fluorescence recovery after fringe pattern photobleaching (FRAP). A fluorescent probe was incorporated in the micelles it was checked that its finite residence time in the micelle did not affect the diffusion process. 

Such an approach has been used to determine the average a-relaxation time based on second harmonic generation measurements [60, 61] and fluorescence recovery after pattern photobleaching measurements [62, 63]. 

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