Transient effects, fluorescence quenching

In this chapter we describe more advanced topics in quenching. Quenching in membranes is desoibed in some detail because of the numerous applications. These include localization of membrane-bound probes, estimation of diffusion coefficients in membranes, and the effect of quencher partitioning into membranes. We also describe transient effects in quenching, which result in nonexponential decays whenever diffusive quenching occurs. These effects can complicate the interpretation of the time-resolved data, but they also provide additional information about the diffusion coefficient of die quencher, the interaction radius, and the medianism of quendiing. For those interested in an introduction to fluorescence, the reading of this chi ter can be postponed. 

Table 5. Effect of ground-state CT complexation on fluorescence quenching and the transient yield of MV+- for APh-x (8), QPh-x (12), and their monomer models AM (15) and QM (16) in aqueous solution [76]...

It has been shown in Chapter 5, the fluorescence quenching of the DPA moiety by MV2 + is very efficient in an alkaline solution [60]. On the other hand, Delaire et al. [124] showed that the quenching in an acidic solution (pH 1.5-3.0) was less effective (kq = 2.5 x 109 M 1 s 1) i.e., it was slower than the diffusion-controlled limit. They interpreted this finding as due to the reduced accessibility of the quencher to the DPA group located in the hydrophobic domain of protonated PMA at acidic pH. An important observation is that, in a basic medium, laser excitation of the PMAvDPA-MV2 + system yielded no transient absorption. This implies that a rapid back ET occurs after very efficient fluorescence quenching. 

Dynamic quenching of fluorescence is described in Section 4.2.2. This translational diffusion process is viscosity-dependent and is thus expected to provide information on the fluidity of a microenvironment, but it must occur in a time-scale comparable to the excited-state lifetime of the fluorophore (experimental time window). When transient effects are negligible, the rate constant kq for quenching can be easily determined by measuring the fluorescence intensity or lifetime as a function of the quencher concentration the results can be analyzed using the Stern-Volmer relation ... 

R. W. Wijnaendts van Resandt, Picosecond transient effect in the fluorescence quenching of tryptophan, Chem. Rhys. Lett. 95, 205-208 (1983). 

A mathematical model of PSII reaction centre containing 6 different states of the complex is represented. A possibility for cycling of electrons around PSII is included. The model describes dark-light-dark transients in fluorescence, including the tip effect. The mechanism of fluorescence quenching and regulation of the PSII activity based on the futile cycle or pheophytin redox potential shifts were studied. In the first case the tip effect is present in the fluorescence induction but it is absent in the second case. Fluorescence induction curves are sensitive to the rate of electron donation from the water-splitting system which can be controlled either by the redox state of the donor or by the rate constant of electron donation. 

The effect is significant for reactions with short half-times. In an ordinary diffusion-limited reaction under conditions where the species B is in great excess, the half-time is l/(/ diff[B]). If / diff 10 ° sec-, a steady-state treatment of the kinetics will suffice as long as [B] < 0.1 If [B] is larger, the effect of transient behavior cannot be ignored. Another instance where transients are important is in the kinetics of fluorescence quenching. Here, the intrinsic lifetimes of the photoexcited species are typically 10 sec. The photostationary state is not established and effects attributable to the time dependence of fcapp can be observed. 

In some real systems, it is possible to encounter a combination of both types of quenching. The Stem-Volmer plots are frequently not linear because various transient effects may cause the upward curvature of the plot. On the other hand, downward curvature and leveling-off of the plots can be a result of uneven (hindered) accessibility of a fraction of fluorophores in micro-heterogeneous media. A number of specific models for analyzing fluorescence decays affected by quenching have been proposed in the literature [8, 15]. 

Holub, O., Seufferheld, M. J., Gohlke, C., Govindjee, G. J., Heiss, G. J. and Clegg, R. M. (2007). Fluorescence lifetime imaging microscopy of Chlamydomonas reinhardtii Non-photochemical quenching mutants and the effect of photosynthetic inhibitors on the slow chlorophyll fluorescence transients. J. Microsc. 226, 90-120. 

Another recent study makes use of the participation of the T2 state in the S - TISC process in anthracene [31]. 1,3-Octadiene was used to intercept some of the T2 states before they relaxed to Tx and the decrease in 7, yield was used to estimate the T2 lifetime. Further, this study compensated for the effects of static and time-dependent quenching that comes into play at the relatively large quencher concentrations that are required when quenching sub-nanosecond-lifetime transients. The lifetimes obtained (given in Table 5) were significantly less than previously estimated from other quenching studies and are in line with the lifetimes implied from the T-T fluorescence quantum yields discussed above. 

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