Perturbation quenching

Perturbation quenching occurs when orbital overlap between the probe and quencher allows some property of the quencher to be shared with the probe excited-state. Neither probe nor quencher is chemically altered in the process. For example, the quencher may act as a heavy atom, causing non-radiative relaxation of the excited singlet state. Si, to the Ti state via intersystem crossing and/or enhanced radiative relaxation from Ti. Detection may then be based on quenching of Si, i.e. fluorescence quenching, or phosphorescence from the Ti state. 

Several colloidal systems, that are of practical importance, contain spherically symmetric particles the size of which changes continuously. Polydisperse fluid mixtures can be described by a continuous probability density of one or more particle attributes, such as particle size. Thus, they may be viewed as containing an infinite number of components. It has been several decades since the introduction of polydispersity as a model for molecular mixtures [73], but only recently has it received widespread attention [74-82]. Initially, work was concentrated on nearly monodisperse mixtures and the polydispersity was accounted for by the construction of perturbation expansions with a pure, monodispersive, component as the reference fluid [77,80]. Subsequently, Kofke and Glandt [79] have obtained the equation of state using a theory based on the distinction of particular species in a polydispersive mixture, not by their intermolecular potentials but by a specific form of the distribution of their chemical potentials. Quite recently, Lado [81,82] has generalized the usual OZ equation to the case of a polydispersive mixture. Recently, the latter theory has been also extended to the case of polydisperse quenched-annealed mixtures [83,84]. As this approach has not been reviewed previously, we shall consider it in some detail. 

The experimental principle is illustrated in Fig. 3. The interaction of the polymer with the liposomal membranes causes the perturbation of the bilayer. This perturbation follows the leakage of calcein from the liposome. Calcein in high concentration in the liposome is self-quenched, but has strong fluorescence intensity by the leak from the liposome. Therefore, the extent of the membrane interaction can be estimated quantitatively from the fluorescence spectroscopy. 

To test this sensitivity of photoinduced quenching to perturbations in the intervening base pair stack, an assembly was prepared containing an intervening CA base mismatch which disrupts locally the /r-stack between the donor and acceptor. We observed that the yield of CT was reduced in the DNA... 

Hence the steady-state population of triplets should increase under heavy-atom perturbation. However, this conclusion is valid only if unimolecular decay is the main route leading to triplet state depopulation. If bimolecular triplet quenching as shown below is more important than unimolecular decay by several orders of magnitude, kd could be increased as much or more than klte without decreasing the steady state triplet population<136) ... 

Following an external perturbation, the fluorescence quantum yield can remain proportional to the lifetime of the excited state (e.g. in the case of dynamic quenching (see Chapter 4), variation in temperature, etc.). However, such a proportionality may not be valid if de-excitation pathways - different from those described above - result from interactions with other molecules. A typical case where the fluorescence quantum yield is affected without any change in excited-state lifetime is the formation of a ground-state complex that is non-fluorescent (static quenching see Chapter 4). 

The serious drawback of the methods of evaluation of fluidity based on intermolecular quenching or excimer formation is that the translational diffusion can be perturbed in constrained media. It should be emphasized that, in the case of biological membranes, problems in the estimation of fluidity arise from the presence of proteins and possible additives (e.g. cholesterol). Nevertheless, excimer formation with pyrene or pyrene-labeled phospholipids can provide interesting in-... 

The choice of method depends on the system to be investigated. The methods of intermolecular quenching and intermolecular excimer formation are not recommended for probing fluidity of microheterogeneous media because of possible perturbation of the translational diffusion process. The methods of intramolecular excimer formation and molecular rotors are convenient and rapid, but the time-resolved fluorescence polarization technique provides much more detailed information, including the order of an anisotropic medium. 

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. 

S. S. Lehrer, Solute perturbation of protein fluorescence. The quenching of other tryptophan fluorescence of model compounds and of lysosome by iodide ion, Biochemistry 10, 3254-3263 (1971). 

In an investigation of the physical basis of the interaction of histones with DNA, De Petrocellis et al.< 95 > have examined the effect of phosphate ions on histone HI. Binding results have shown that there are high-affinity sites for phosphate ions. In addition, phosphate ions were found to perturb the absorption spectra of HI and quench tyrosine fluorescence. Binding of the phosphate group resulted in positive difference absorption bands near 275 and 293 nm, which are similar to those produced at acid and alkaline pH, respectively. 

Figure 3 The collapse of the peptide Ace-Nle30-Nme under deeply quenched poor solvent conditions monitored by both radius of gyration (Panel A) and energy relaxation (Panel B). MC simulations were performed in dihedral space 81% of moves attempted to change angles, 9% sampled the w angles, and 10% the side chains. For the randomized case (solid line), all angles were uniformly sampled from the interval —180° to 180° each time. For the stepwise case (dashed line), dihedral angles were perturbed uniformly by a maximum of 10° for 4>/ / moves, 2° for w moves, and 30° for side-chain moves. In the mixed case (dash-dotted line), the stepwise protocol was modified to include nonlocal moves with fractions of 20% for 4>/ J/ moves, 10% for to moves, and 30% for side-chain moves. For each of the three cases, data from 20 independent runs were combined to yield the traces shown. CPU times are approximate, since stochastic variations in runtime were observed for the independent runs. Each run comprised of 3 x 107 steps. Error estimates are not shown in the interest of clarity, but indicated the results to be robust.

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