Quenching anisotropy

The theory of high order corrections to the Lamb shift described above for H and D may also be applied to other light hydrogenlike ions. The simplest such ion is He+. Originally the classic Lamb shift in 17e+ was measured in [50] by the quenching-anisotropy method with the result L 2Si — 2Pi,17e+) = 14 042.52 (16) MHz. Later the authors of [50] discovered a previously unsuspected source of systematic error in their experiment. Their new measurement of the classic Lamb shift in 17e+ by the anisotropy method resulted in the value L 2Si — 2Pi,He ) = 14 041.13 (17) MHz [51]. Besides the experimental data this result depends also on the theoretical value of the hne structure interval. In [51] the value AE 2Pz —2Pi) = 175 593.50 (2) MHz was used. We recalculated this interval using tire latest theoretical results discussed above and obtained AE 2P3 — 2Pi) = 175 593.33 (1) MHz. Then the value of the... 

It appears likely that the statistical uncertainty will eventually be reduced to around 100 kHz, so we consider sources of systematic error which may be expected to enter at this level. The uncertainty in the second-order Doppler shift (450kHz/eV) will be reduced to 100 kHz by a 5% measurement of the beam energy. The AC Stark shift of the 2S-3S transition will be around 70 kHz for the present laser intensity, and can be extrapolated to zero intensity by varying the UV power. Finally, as mentioned above, the systematic uncertainties will be quite different from those in the microwave and quench anisotropy measurements. 

When anisotropy increases due to fluorescence lifetime decrease being coupled to any effect of dynamic quenching. 

This means that if the intensity of one of the forms is zero (static quenching), such anisotropy sensor is useless since it will show anisotropy of only one of the forms. The account of fractional intensity factor R Fb // , (the ratio of intensities of bound and free forms) leads to a more complicated function for the fraction of bound target,/ ... 

Exploration of collective effects in multiple transfers that appear when the donor and acceptor are the same molecules and display the so-called homotransfer. In this case, the presence of only one molecular quencher can quench fluorescence of the whole ensemble of emitters coupled by homotransfer [32]. The other possibility of using homo-FRET is the detection of intermolecular interactions by the decrease of anisotropy [33]. 

Homotransfer does not cause additional de-excitation of the donor molecules, i.e. does not result in fluorescence quenching. In fact, the probability of de-excitation of a donor molecule does not depend on the fact that this molecule was initially excited by absorption of a photon or by transfer of excitation from another donor molecule. Therefore, the fluorescence decay of a population of donor molecules is not perturbed by possible excitation transport among donors. Because the transition dipole moments of the molecules are not parallel (except in very rare cases), the polarization of the emitted fluorescence is affected by homotransfer and information on the kinetics of excitation transport is provided by the decay of emission anisotropy. 

The mobility of tyrosine in Leu3 enkephalin was examined by Lakowicz and Maliwal/17 ) who used oxygen quenching to measure lifetime-resolved steady-state anisotropies of a series of tyrosine-containing peptides. They measured a phase lifetime of 1.4 ns (30-MHz modulation frequency) without quenching, and they obtained apparent rotational correlation times of 0.18 ns and 0.33 ns, for Tyr1 and the peptide. Their data analysis assumed a simple model in which the decays of the anisotropy due to the overall motion of the peptide and the independent motion of the aromatic residue are single exponentials and these motions are independent of each other. 

There should exist a correlation between the two time-resolved functions the decay of the fluorescence intensity and the decay of the emission anisotropy. If the fluorophore undergoes intramolecular rotation with some potential energy and the quenching of its emission has an angular dependence, then the intensity decay function is predicted to be strongly dependent on the rotational diffusion coefficient of the fluorophore.(112) It is expected to be single-exponential only in the case when the internal rotation is fast as compared with an averaged decay rate. As the internal rotation becomes slower, the intensity decay function should exhibit nonexponential behavior. 

M. Eftink, Quenching-resolved emission anisotropy studies with single and multitryptophan-containing proteins, Biophys. J. 43, 323-334 (1983). 

The long lifetime of phosphorescence allows it to be used for processes which are slow—on the millisecond to microsecond time scale. Among these processes are the turnover time of enzymes and diffusion of large aggregates or smaller proteins in a restricted environment, such as, for example, proteins in membranes. Phosphorescence anisotropy is one method to study these processes, giving information on rotational diffusion. Quenching by external molecules is another potentially powerful method in this case it can lead to information on tryptophan location and the structural dynamics of the protein. 

A, Intensity and lifetime B, anisotropy C, quenching D, energy transfer E solvent effects. 

The measurement of fluorescence lifetimes is an integral part of the anisotropy, energy transfer, and quenching experiment. Also, the fluorescence lifetime provides potentially useful information on the fluorophore environment and therefore provides useful information on membrane properties. An example is the investigation of lateral phase separations. Recently, interest in the fluorescence lifetime itself has increased due to the introduction of the lifetime distribution model as an alternative to the discrete multiexponential approach which has been prevalent in the past. 

If a collisional quencher of the fluorophore is also incorporated into the membrane, the lifetime will be shortened. The time resolution of the fluorescence anisotropy decay is then increased,(63) providing the collisional quenching itself does not alter the anisotropy decay. If the latter condition does not hold, this will be indicated by an inability to simultaneously fit the data measured at several different quencher concentrations to a single anisotropy decay process. This method has so far been applied to the case of tryptophans in proteins(63) but could potentially be extended to lipid-bound fluorophores in membranes. If the quencher distribution in the membrane differed from that of the fluorophore, it would also be possible to extract information on selected populations of fluorophores possibly locating in different membrane environments. 

Figure 5.4. EfTect of the phase transition on the partition coefficient of 5-DOXYL-decane calculated on the basis of its ability to quench the fluorescence of DPH in dimyristoyl-phosphatidylcholine vesicles. The fluorescence anisotropy of the DPH is also shown. (From Ref. 104, with permission.)...

J. R. Lakowicz, H. Cherek, I. Gryczynski, N. Joshi, and M. L. Johnson, Enhanced resolution of fluorescence anisotropy decays by simultaneous analysis of progressively quenched samples, Biophys. J. 51, 755-768 (1987). 

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