Fluorescent recovery after photobleaching FRAP

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)... 

Total internal reflection fluorescence (TIRF) microscopy, fluorescence in situ hybridization (FISH), fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging microscopy (FLIM). 

Lateral diffusion of phospholipids in model membranes at ambient pressure has been studied over the years by a variety of techniques including fluorescence recovery after photobleaching (FRAP), spin-label ESR, pulse field gradient NMR (PFG-NMR), quasielastic neutron scattering (QENS), excimer fluorescence and others.In general, the values reported for the lateral diffusion coefficient (D) range from 10 to 10 cm /s in the... 

Fig. 2. Exchange of histones Hl.l and H2B from chromatin in interphase cells by analysis with fluorescence recovery after photobleaching (FRAP). Half of a nucleus of an SK-N cell expressing GFP-Hl.l was bleached (upper panel), and the recovery monitored over the times shown. Similarly, a region of a nucleus of an SK-N cell stably expressing H2B-CFP was bleached (lower panel), and the recovery monitored over the times shown. Whereas unbleached HI molecules move into the bleached region after a few minutes, the H2B histones are much less mobile, since the bleached region shows no recovery (from Ref [23]). Scale bar 5 pm.

Fig. 7. GFP-GR bound to the MMTV array was analyzed by fluorescence recovery after photobleaching (FRAP). The bleached region is indicated in the image of the pre-bleached nucleus (A). The pre-bleach array is shown in (B), the post-bleach image (C), and at 4.1 s (D) and 11.6 s (E) post-bleach. This analysis, along with Fluorescence Loss in Photobleaching (FLIP) experiments, show that GFP-GR undergoes rapid exchange with the array (from Ref. [58]). Scale bar 5 pm.

The velocity of interstitial fluid in solid tumors is often lower than the resolution of experimental techniques, which is 0. lpm/sec, except in some special tumor models. For example, Chary and Jain (1989) have examined interstitial fluid velocity in granulation tissues and VX2 mammary carcinoma grown in rabbit ear chambers, using the fluorescence recovery after photobleaching (FRAP) technique. The average velocities in both tissues are about 0.6 pm/sec. 

The direct measurement of the various important parameters of foam films (thickness, capillary pressure, contact angles, etc.) makes it possible to derive information about the thermodynamic and kinetic properties of films (disjoining pressure isotherms, potential of the diffuse electric layer, molecular characteristics of foam bilayer, such as binding energy of molecules, linear tension, etc.). Along with it certain techniques employed to reveal foam film structure, being of particular importance for black foam films, are also considered here. These are FT-IR Spectroscopy, Fluorescence Recovery after Photobleaching (FRAP), X-ray reflectivity, measurement of the lateral electrical conductivity, measurement of foam film permeability, etc. 

Fluorescence recovery after photobleaching (FRAP) of foam films... 

The fluorescent spectroscopy is one of the optical techniques that are widely used in the study of structure and dynamic properties of lipids in lamellar phases [76]. The Fluorescence Recovery after Photobleaching (FRAP) is successfully applied in the lateral diffusion studies of BLM (e.g. 77]. FRAP has been employed to study similar phenomena at the air/water interface of Langmuir trough [78]. 

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. 

Ras trafficking to cellular membranes can be measured by fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) (54). Both techniques rely on the expression of fluorescent-labeled Ras proteins to monitor different parameters of Ras movement across and between cellular membranes. FRAP involves photobleaching a membrane subdomain and measuring the kinetics of fluorescence recovery—and hence Ras trafflcking—into the bleached area. With FLIP, a cellular membrane is photobleached repeatedly and the subsequent intercellular movement of the photobleached area is monitored. 

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.

Various new techniques suitable for estimating D are now available fluorescence correlation spectroscopy (FCS) [5], fluorescence recovery after photobleaching (FRAP) [6], pulsed field gradient nuclear magnetic resonance (PFG-NMR) [7], diffusion ordered NMR spectroscopy (DOSY) [8], and others. Among these, FCS and FRAP are popular in biological studies because they are often installed on a commercial LSM system and conveniently coupled with it. 

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. 

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