Theory of Fluorescence Quenching

The processes of excitation of a solute molecule A by absorption of a photon of ultraviolet light, and of fluorescence by emission of a photon of visible light, may be represented as 

Emission from individual excited molecules is a random process, occurring over a range of times the result is that the intensity of emission after the cessation of illumination decays exponentially. The corresponding relaxation time, which may be shown to be equal to the average interval between excitation and emission [3], is known as the fluorescence lifetime (tq). This is typically around 10 s (10 ns). Within this lifetime, some of the excited molecules are deactivated, either by thermal equilibration within the molecule ( internal conversion ), or rarely by interaction with the solvent, before they can emit  

Under constant-intensity illumination, in the absence of any reactive partner, a steady state is set up, in which the production of excited molecules A by absorption of photons at a rate /abs (process (6.1)) is balanced by fluorescence (6.2) and deactivation by internal conversion (6.3) and/or solvent interaction (6.4). The situation may be summarised as follows  

As the concentration of Q increases, the intensity of fluorescence from A decreases. If the product is an excited molecule or complex, the intensity of its fluorescence increases. The two intensities can be separately measured if the two fluorescence spectral frequency distributions are different enough. 

In the next section we show that, in the simplest cases, this scheme accounts for the observed relations between the concentration of the quenching solute and the intensity and lifetime of fluorescence. In subsequent sections we introduce additional processes, such as complex-formation or proton-transfer, in which the product may itself be a fluorophore, so that there are two excited states to be considered. This two-state model covers a variety of fluorescence phenomena. We discuss also the physical mechanisms of the various processes and the ways in which excited molecules lose their excitation energy. 

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B3.6 Determining the Fluorescence Spectrum of a Protein B3.6.1 Strategic Planning B3.6.1 Basic Protocol l Recording a Fluorescence Emission Spectrum B3.6.5 Basic Protocol 2 Determination of Fluorescence Quenching B3.6.9 Support Protocol Basic Theory and Interpretation of Fluorescence Spectra B3.6.12 Commentary B3.6.19... 

Fluorescence spectroscopy, of proteins basic theory and interpretation, 257-263 determination of fluorescence quenching, 253-255... 

Since the transfer of protons is truly contact, this reaction is best suited for comparison with the contact and spinless theory given above. However, the authors who first monitored it in the time domain tried to fit the fluorescence signal as a biexponential one [59]. The similar reaction but of a more reactive photoacid (2-naphthol-6-sulfonate) with an acetate anion has been studied, and its kinetics, which is neither exponential nor biexponential, was fitted to the tme theory of contact quenching [60]. It is especially important that the fluorescence... 

The energy dependence of charge separation is most readily scrutinized and it was investigated first in considerable detail. Rehm and Weller studied rate constants of fluorescence quenching for a series of more than 60 organic donor-acceptor pairs and found a maximum rate of electron transfer, essentially diffusion limited, without any indication for an inverted region (Fig. 6) [136]. These results were at variance with the existing theories, but they could be rationalized on the basis... 

Re-examination of what are generally considered to be well established theories are needed for extension of photophysics into new areas. For example, the theory of concentration quenching of fluorescence in 1, 2 and 3 dimensional media has been examined by Sienicki and the time dependence of decay and fluorescence quenching in a one dimensional lattice analysed by Dudkiewicz and Twardowski. ... 

Kusba, J., Bogdanov, V., Qryczynski, I., Lakowicz, J.R. Theory of light quenching. Effects on fluorescence polarization, intensity, and anisotropy decays. Biophys. J. 67, 2024-2040 (1994)... 

According to the Marcus theory of electron transfer discussed in Sections 8.9-8.12, the rate of electron transfer from ground or excited states, and therefore in this context the rate of fluorescence quenching, depends on... 

Fast concentration and sample injection are considered with the use of a theory of vibrational relaxation. A possibility to reduce a detection limit for trinitrotoluene to 10 g/cnf in less than 1 min is shown. Such a detection limit can by obtained using selective ionization combined with ion drift spectrometry. The time of detection in this case is 1- 3 s. A detection technique based on fluorescent reinforcing polymers, when the target molecules strongly quench fluorescence, holds much promise for developing fast detectors. 

The remainder of this section considers several experimental studies of reactions to which the Smoluchowski theory of diffusion-controlled chemical reaction rates may be applied. These are fluorescence quenching of aromatic molecules by the heavy atom effect or electron transfer, reactions of the solvated electron with oxidants (where no longe-range transfer is implicated), the recombination of photolytically generated radicals and the reaction of carbon monoxide with microperoxidase. 

The first reasonably successful theory of diffusion-limited chemical reactions which specifically endeavoured to develop a model that could described, in principle, the competitive effect was introduced by Wilemski and Fixman [51], They considered the fluorescence of a species A which can be quenched by natural decay (lifetime t) and by a quencher, Q, of concentration [Q]... 

A simplified theory of FRET is sufficient to describe affinity sensors used in fluorescence transduction of glucose concentrations. A key quantity that describes the potential FRET interaction between a donor-acceptor pair is the Forster distance, Ro, the distance at which half the donor molecules are quenched by the acceptor molecules. Ro is proportional to several parameters of the fluorophores, in accordance with Ro = K6 Jx2n 4 cf>DJ l], where K is a constant. The variable k2 refers to the relative spatial orientation of the dipoles of D and A, taking on values from 0 to 4 for completely orthogonal dipoles and collinear and parallel transitional dipoles k2 = 4,... 

Popovic et al. (1987) studied photogeneration of N,N-bis(methyl)perylene-3,4,9,10-tetracarboxyldiimide by field modulation of the time-resolved fluorescence. The results show that the field increases the rate of decay of fluorescence but leaves the initial intensity unchanged. The results were described by a process which occurs by the field-assisted dissociation of the first-excited singlet state. The absence of quenching of the initial fluorescence (amplitude quenching) was interpreted as evidence that the photogeneration process cannot be described by Onsager theories. 

Quenching Excimers and Exciplexes.—By measurements of decay times and fluorescence anisotropy of pyrene and the excimer in cellulose acetate films it has been found that the medium consists of spaces where small pyrene molecules have considerable freedom, Dissado and Walmsley have developed a complete theory of excimer formation and exciton-induced lattice distortion in crystals. Reference is made to data on 9-cyanoanthracene. The spectroscopy of chemically linked dimers of l,3-(l,l -dinaphthyl)propane in a... 

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