Fluorescence FRAP

Key words Laser, Microbeam, Antibody, Fluorescence, FRAP, FRET, Ablation, Photobleaching... 

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)... 

FRAP (fluorescence recovery after photo bleaching) analysis was originally developed in the mid-1970s to study the diffusion of biomolecules in living cells (Edidin et al, 1976). Flowever, due to the increased availability of GFP tags and advances in the bleaching capabilities of confocal microscopes, this... 

These values can be normalized to allow comparison with other FRAP experiments. This involves the generation of a relative fluorescence value expressed as a percentage of the total pre-bleached value. 

The membrane-associated small G proteins H-Ras and K-Ras have been studied with respect to their association with cytoplasmic leaflets. These two proteins have nearly identical structures and functions but different membrane anchors, membrane distributions and effector responses. Application of the FRAP method to fluorescent constructs of H-Ras and K-Ras revealed that only H-Ras in its guanosine 5 diphosphate (GDP)-bound form associates with cholesterol-dependent rafts [26]. 

CPK creatine phosphokinase FRAP fluorescence recovery after photolysis... 

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

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). 

Another TIR/FRAP study on biological cell membranes has examined the reversible but specific binding kinetics of fluorescence-labeled epidermal growth factor to the surface of cells.(125) The background problem here was solved simply by choosing cells with a very large concentration of epidermal... 

Although TIR/FCS and TIR/FRAP both give similar information about kinetic rates and surface diffusion, and the mathematics of the two is similar,(U5) there is an interesting and perhaps useful difference. Thompson(128) has shown theoretically that with TIR/FCS, but not with TIR/FRAP, one can infer kinetic rates of a nonfluorescent species as it competes with fluorescent species for the same nearly saturated surface sites. 

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. 3. Schematic diagram of the spot photobleaching method of FRAP. (A) Darkened circles represent fluorescently labeled molecules evenly distributed over a two-dimensional surface (assumed to be an infinite plane). (B) White and light gray circles represent the initial postbleach distribution of photobleached molecules within a 1-pm diameter spot. (C) Redistribution of photobleached and unbleached molecules as a consequence of random diffusion over time. (D) Curve representing the fluorescence intensity within the l-pm diameter spot monitored over time arrows a, b, and c indicate the time-points that correspond to their respective panels. The rate of recovery from point b to point c is used to determine the diffusion constant. The magnitude of the recovery is determined by comparing the fluorescence intensity at point c with the initial intensity at point a, and is used to determine the mobile fraction.

Another variation of the FRAP technique scans the laser in one dimension across the cell producing a line-scan profile of fluorescence intensity (14). For a round cell whose surface is evenly labeled with a fluorescently conjugated antibody, a line scan typically produces a profile with two peaks of fluorescence, since the laser beam is illuminating more fluorophores at the edges... 

Duplicating the GFP-H2B experiments with both GFP-H3 and GFP-H4 vectors, very little H3 and H4 exchange outside of S phase was observed. Fluorescence of GFP-H3 and GFP H4 in HeLa cells in G1 recovered rapidly. The extremely rapid recovery rate is similar to that of a diffuse soluble protein, indicating that at this stage of the cell cycle, GFP-tagged H3 and -H4 are not incorporated into chromatin. FRAP experiments of transfected cells in S or G2 show that there is very little recovery of GFP-H3 or -H4 fluorescence. The fluorescence imaging indicates that once the H3 and H4 proteins are incorporated into the chromatin, they are essentially immobile for the remainder of the cell cycle. Unlike histones H2A and H2B, which associate as dimers in the nucleosome histone octamer, there is very little exchange of the components of the H3/H4 tetramer. 

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.

The observation was that bleached GFP-GR is rapidly replaced with unbleached GFP-GR (FRAP) (Fig. 7), and if GFP-GR is bleached elsewhere in the nucleus, GFP-GR is replaced with non-fluorescent GR molecules (either bleached GFP-GR or endogenous unlabelled GR) on the array (FLIP). The conclusion from these observations is that GFP-GR exchanges at a high rate between the array promoters and the nucleoplasm. Observations made in live cells has led to a completely... 

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.

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