Fluorescence emission anisotropy

The fluorescence depolarization technique excites a fluorescent dye by linearly polarized light and measures the polarization anisotropy of the fluorescence emission. The fluorescence anisotropy, r, is defined as... 

Dependencies of luminescence bands (both fluorescence and phosphorescence), anisotropy of emission, and its lifetime on a frequency of excitation, when fluorescence is excited at the red edge of absorption spectrum. Panel a of Fig. 5 shows the fluorescence spectra at different excitations for the solutes with the 0-0 transitions close to vI vn, and vra frequencies. Spectral location of all shown fluorescence bands is different and stable in time of experiment and during lifetime of fluorescence (panel b)... 

Figure 4.9 illustrates time-gated imaging of rotational correlation time. Briefly, excitation by linearly polarized radiation will excite fluorophores with dipole components parallel to the excitation polarization axis and so the fluorescence emission will be anisotropically polarized immediately after excitation, with more emission polarized parallel than perpendicular to the polarization axis (r0). Subsequently, however, collisions with solvent molecules will tend to randomize the fluorophore orientations and the emission anistropy will decrease with time (r(t)). The characteristic timescale over which the fluorescence anisotropy decreases can be described (in the simplest case of a spherical molecule) by an exponential decay with a time constant, 6, which is the rotational correlation time and is approximately proportional to the local solvent viscosity and to the size of the fluorophore. Provided that... 

Fluorescence anisotropy decay measurements, which are based on the excitation of probes with polarized light and subsequent polarized fluorescence emission, can... 

With the probe position moving toward the center of the aqueous channel, we detected more ultrafast and less slow solvation components. Note the negligible change of the quasi-bound water contributions, which indicates the complete detection of the two layers of quasi-bound water by all four Trp-probes. For TME, the fluorescence emission peak shifts to 338 nm, and its location moves to the lipid interface (Fig. 18). We did observe a smaller fraction of slow solvation dynamics decreasing from 53% in TBE to 43% in TME and an increase of the ultrafast component from 17% to 26%. The corresponding anisotropy dynamics drops from 726 to 440 ps with a less hindered local motion at the lipid interface. 

Wahl, Ph., Paoletti, J. and Le Pecq, J.-B. (1970). Decay of fluorescence emission anisotropy of the ethidium bromide-DNA complex evidence for an internal motion in DNA. Proceedings of the National Academy of Sciences, USA, 65, 417-421. 

Davenport, L., and Targowski, P. (1996), Submicrosecond phospholipid dynamics using a long-lived fluorescence emission anisotropy probe, Biophys. 1,71,1837-1852. 

The simplest steady-state measurements of fluorescence properties such as the fluorescence emission spectrum or the steady-state anisotropy can be carried out in a standard fluo-rometer with excitation from a lamp source and a monochromator. Common lamp sources in commercial fluorometers include xenon lamps for excitation from the UV to the near-IR (250 nm to llOOnm). 

As a final example of the influence of solvent on dynamical processes in biopolymers, we examine the decay of the fluorescence emission anisotropy for Trp-62 and Trp-63 in lysozyme the model used is described in Chapt. XI.C).324 In this model the fluorescence anisotropy, r(t), as a function of time for a given residue is given by (see Eq.110)... 

The fluorescence emission anisotropy, r(f), at time t after excitation of the chromophore is defined by the expression... 

Direct evidence of energy migration in the different films was found in polarized fluorescence emission studies. The fluorescence anisotropy indicated that intramolecular energy migration (with a Dexter hopping mechanism) existed in both the solution and spin-cast films. Further energy migration and transfer studies were... 

Similar aggregation to form nanotubes, see flgure 5, was observed with / - and y-CD linked by strings of all trans l,6-diphenyl-l,3,5-hexatriene (DPH). The formation of such aggregates, containing 20 -CD or 20-35 y-CD, is characterized by red shift in absorption and fluorescence emission spectra of DPH and by an anisotropy value indicating scarce reorientation of DPH during the life of the excited state [135]. 

Lakowicz, J. R., Gratton, E., Cherek, H., Maliwal, B. P. and Laczko, G, 1984, Determination of time-resolved fluorescence emission spectra and anisotropies at a fluorophore-protein complex using frequency-domain phase-modulation fluorometry. Journal of Biological Chemistry 259, 10967-10972. 

The timescale of fluorescence emission is comparable to that of rotational diffusion of proteins and the timescale of segmental motions of protein domains or individual amino acid residues. The polarization or anisotropy of the emission provides a measure of these processes. Suppose a sample is excited with vertically polarized light (Fig. 11), and that the sample is viscous so that the fluorophores do not rotate during the lifetime of the excited state. Then the emission is polarized, usually also in the vertical direction. This polarization occurs because the polarized excitation selectively excites those fluorophores in the isotropic solution whose absorption... 

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