Fluorescence redistribution after

The measurement of fluorescence redistribution after photobleaching (FRAP) is a unique, noninvasive method for direedy analyzing dynamic processes in living cells (13, 18-23, 69-73). FRAP experiments have provided important insights into membrane structure (18, 20), mechanisms of hormone action (82, 83), nucleocytoplasmic communication (13, 21), cytoplasmic organization and structure (84), actin and tubulin assembly (85, 22), cell-cell communication (13, 65, 67-69), cell differentiation and proliferation (86), parasite membrane structure (87), and bacterial membrane biosynthesis (88-89). 

Salmon, E.D., et al. Diffusion coefficient of fluorescein-labeled tubulin in the cytoplasm of embryonic cells of a sea urchin video image analysis of fluorescence redistribution after photobleaching. Journal of Cell Biology, 1984, 99(6), 2157-2164. 

Houtsmuller, A.B., Vermeulen, W. Macromolecular dynamics in living cell nuclei revealed by fluorescence redistribution after photobleaching. Histochem. Cell Biol. 115, 13-21 (2001)... 

ABSTRACT. Over the past 15 years, a number of transient optical grating techniques have been developed for measurements of the transport properties of materials. Such methods have been used to measure the tracer diffusion coefficients of polymer molecules which have been labeled with photochromic or fluorescent dyes. The present paper describes the common features of these techniques, and gives an example of how Fluorescence Redistribution After Pattern Photobleaching has been used to study the diffusion of polymer molecules in the melt. 

In Fluorescence Redistribution After Pattern Photobleaching (FRAPP), fluorescence emission is used to detect the concentration of the molecules of interest as a function of position and time. The light bleaches, or irreversibly photochemically destroys, the fluorescence of dye molecules in the illuminated regions of the sample. The pattern decays by the diffusion of the remaining, unbleached molecules. 

In 1978, Salcedo, et al at Stanford University reported the measurement of singlet electronic excitation transport in a molecular crystal. The time scale of their measurement was 1 ns. Also in 1978, the first reports were published of the measurement of mass transport by transient grating techniques. Hervet, et al of the College de France reported the translational diffusion coefficient of the photochromic dye methyl red in an aligned liquid crystal, as a function of direction. The time scale was 0.1 s. Smith and McConnell at Stanford University used Fluorescence Redistribution After Pattern Photobleaching to measure the diffusion coefficient of dye-labeled phospholipid in oriented multibilayer films. The time scale was 100 s. 

FLUORESCENCE REDISTRIBUTION AFTER PATTERN PHOTOBLEACHING 3.1. Principles... 

Such layer structure does not allow ns to say a priori that hybridization of DNA will be possible, for it is protected by the octadecylamine layer. In order to control for this possibility, fluorescence measurements were performed. The first indication that hybridization was successful is that after the process, the sample surface became wettable, while before it and after cold hybridization it was not wettable at all. The results of the fluorescence measurements are summarized in Table 10. The results of the specific hybridization are three times more with respect to unspecific hybridization and one order of magnitude more with respect to cold hybridization. Thus, it appears that during a normal hybridization (100% homology) some structural changes and redistribution of the layer takes place. As a result, DNA becomes available for the specific reaction. Such a model also explains why the fluorescence level after unspecific hybridization (10% homology) is higher with respect to cold hybridization. Because the molecules have some mobility when the film is warmed, some DNA from the film could be hybridized on itself, while during cold hybridization this is impossible. 

Recently, an agonist-induced redistribution of FPR in human neutrophils consistent with the lateral segregation model was demonstrated by direct measurement of receptor lateral mobility with fluorescence recovery after photobleaching [43]. 

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. 

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. 

Measurement of transient concentration profiles of deuterated polymers in polymer blends using forward recoil spectroscopy Measurement of transient concentration profiles of fluorescently labeled molecules in capillary tubes using epifluorescence microscopy Measurement of transient concentration profiles of fluorescently labeled molecules in biological tissues using epifluorescence microscopy Fluorescence recovery (or redistribution) after photobleaching (FPR)... 

Figure 5.2 Schematic showing the redistribution of labeled molecules after fusion and after photobleaching. (a) Redistribution of membrane proteins that had different fluorescent labels (shown as black or white circles) after cell fusion, (b) Fluorescent recovery after photobleaching, in which the fluorescent molecules (black circles) in an isolated section of the membrane are bleached (white circles). The recovery of fluorescence within the isolated region is an indicator of mobility in the membrane.

In Fig. 2, the rate constants of S2 fluorescence decay (A =1/t ) and the inverses of S, fluorescence rise by S, state formation Xp) of ZP-I systems in Tol and THF are plotted against-AGCS. In Tol solutions, Xp at the top regions are little bit delayed relative to X, which shows that the charge recombination (CR) to the S[ state after charge separation from the S2 state is the main process for the S, formation in these systems. The results in MCH basically showed the same features with those in Tol (data not shown). On the other hand, X, and Xp values are rather close in THF which seems to suggest the ultrafast S, state formation by CR from the vibrationally unrelaxed CS state in the course of the vibrational redistribution relaxation. 

Additive and impurity rejection at the growing crystal front leads to uneven distribution in a crystalline polymer. This redistribution process has been studied by UV and fluorescence microscopy and by an electron microscope with energy dispersive x-ray analysis. In polymer samples which are quenched after rapid crystallization, the additive distribution is kinetically determined and may be modeled in a computer as a three-dimensional zone-refining process. In annealed polymer samples, low molecular weight additives are uniformly concentrated in the amorphous phase. The additive distribution reflects that of crystalline material within the polymer. Antioxidant and uv stabilzer redistribution probably does not have a major effect on polymer stability, but the redistribution of partially oxidized, impure polymer may be important... 

I.r. emission has been seen from the vibrationally excited CO2 product of MPD of vinylacetic and pyruvic acids, from hydrogen halides formed by chemical reaction of CI2 with H atoms produced in the MPD of various hydrocarbons, and of HBr with F and Cl atoms formed in the MPD of CFjCl. Infrared fluorescence from CjFjCl following MPA shows emission from both discrete levels and the quasicontinuum, with efficient intramolecular vibrational redistribution out of the pumped mode evident after absorption of only 2—3 photons. Emission in the i.r. has been seen following MPA in N2p4, and has been used to study the interconversion of perfluorocyclobutene to perfluoro-butadiene isomers following MPA. Further isomerization reactions induced by CO2 lasers have been reported. 

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