Emission decay kinetics

Decay and Quenching of Fluorescence 2.3.1. Emission Decay Kinetics 

In this section, the information on structure and dynamics of proteins which may be obtained from direct observations of fluorescence decay will be considered. This type of information is afforded by methods which permit fluorescence decay kinetics to be followed with picosecond and nanosecond resolution. 

The single-exponential decay kinetics, described by the equation 

Nonexponential fluorescence decay would be expected to be observed in the following cases  

There are several conformational arrangements with different interactions of the fluorophores with surrounding groups of atoms. Such interactions may affect differently nonradiative deexcitation processes in the excited state, and the decay times for these conformational states will differ. If each of these states is characterized by singleexponential decay kinetics, then the number of constants f, will correspond to the number of aromatic groups in the protein that are in conformationally different states. 

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In addition, the quenching of the fluorescence of fluorophore groups in protein molecules by neighboring groups(35) and its temperature dependence, t36) energy transfer of electronic excitation and its dependence on excitation wavelength,(1) the type of emission decay kinetics,(1,2) and changes... 

The electron- and hole-trapping dynamics in the case of WS2 are elucidated by electron-quenching studies, specifically by the comparison of polarized emission kinetics in the presence and absence of an adsorbed electron acceptor, 2,2 -bipyridine [68]. In the absence of an electron acceptor, WS exhibits emission decay kinetics similar to those observed in the M0S2 case. The polarized emission decays with 28-ps, 330-ps, and about 3-ns components. For carrier-quenching studies to resolve the dynamics of electron trapping, it is necessary that the electron acceptor quenches only conduction-band (not trapped) electrons. It is therefore first necessary to determine that electron transfer occurs only from the conduction band. The decay of the unpolarized emission (when both the electron and the hole are trapped) is unaffected by the presence of the 2,2 -bipyridine, indicating that electron transfer docs not take place from trap states in the WS2 case. Comparison of the polarized emission kinetics in the presence and absence of the electron acceptor indicates that electron transfer does occur from the conduction band. Specifically, this comparison reveals that the presence of 2,2 -bipyridine significantly shortens the slower decay component of the polarized... 

Figure 2. Emission decay kinetics measured using time-correlated single-photon counting under normal conditions for AR25 in DMF (1 X 10 M) and adsorbed onto a 4 pm thick transparent Ti02 film. Dashed lines correspond to the adjusted fit decay. The excitation wavelength was = 405 nm, and the emission was mon-...

Fig. 3. Emission decay kinetics of 9-bromoanfhnicene dissolved in hexanes. The ssonpk was excited with a 3SS ran pulse. 310 < fn< ran, tempeiatuK, 20°C. (a) l-btomonaphthalaie, (b) 1-bromo-2-inethylnapblhalene, (c) 2-bn>monaphthalene in hexane.

Below is the function Data Em iss ion. m to generate the data. Note that experimental emission decays are exponentials and that the lifetime x is used instead of the more customary rate constant in kinetics. Also, we use the notation C representing the concentration of the exited states, not the normal concentration. 

Proteins having one chromophore per molecule are the simplest and most convenient in studies of fluorescence decay kinetics as well as in other spectroscopic studies of proteins. These were historically the first proteins for which the tryptophan fluorescence decay was analyzed. It was natural to expect that, for these proteins at least, the decay curves would be singleexponential. However, a more complex time dependence of the emission was observed. To describe the experimental data for almost all of the proteins studied, it was necessary to use a set of two or more exponents.(2) The decay is single-exponential only in the case of apoazurin.(41) Several authors(41,42) explained the biexponentiality of the decay by the existence of two protein conformers in equilibrium. Such an explanation is difficult to accept without additional analysis, since there are many other mechanisms leading to nonexponential decay and in view of the fact that deconvolution into exponential components is no more than a formal procedure for treatment of nonexponential curves. 

In the majority of cases, fluorescent labels and probes, when studied in different liquid solvents, display single-exponential fluorescence decay kinetics. However, when they are bound to proteins, their emission exhibits more complicated, nonexponential character. Thus, two decay components were observed for the complex of 8-anilinonaphthalene-l-sulfonate (1,8-ANS) with phosphorylase(49) as well as for 5-diethylamino-l-naphthalenesulfonic acid (DNS)-labeled dehydrogenases.(50) Three decay components were determined for complexes of 1,8-ANS with low-density lipoproteins.1 51 1 On the basis of only the data on the kinetics of the fluorescence decay, the origin of these multiple decay components (whether they are associated with structural heterogeneity in the ground state or arise due to dynamic processes in the excited state) is difficult to ascertain. 

In the time-resolved quenching method, the decay kinetics of the monomer and the excimer emission are monitored in the presence of a micelHzed medium. If the micellar system is viewed as a group of individual micelles with probe occupancies 0, 1, 2, 3, etc., the probabihty of micelles with n probes, Pn, may be related to n, the average number of probes per micelle by Poissonian statistics through relation. 

Long-lived emissions of very low intensities can be observed against very high intensities of short-lived emissions. It is also possible to observe the decay kinetics of long-lived emissions directly on an oscilloscope, by varying the speed of rotation of the phosphoroscope. 

Electron transfer kinetics from the triplet excited state of TMPD to PA in polystyrene has been monitored by phosphorescence emission decay in ref. 85. The rate constant has been found to be invariant over the temperature interval 77-143 K. Parameters ae and ve calculated from the phosphorescence decay using eqn. (12) were found to be ae = 3.46 A and vc = 104 s 1. 

Edmond Becquerel (1820-1891) was the nineteenth-century scientist who studied the phosphorescence phenomenon most intensely. Continuing Stokes s research, he determined the excitation and emission spectra of diverse phosphors, determined the influence of temperature and other parameters, and measured the time between excitation and emission of phosphorescence and the duration time of this same phenomenon. For this purpose he constructed in 1858 the first phosphoroscope, with which he was capable of measuring lifetimes as short as 10-4 s. It was known that lifetimes considerably varied from one compound to the other, and he demonstrated in this sense that the phosphorescence of Iceland spar stayed visible for some seconds after irradiation, while that of the potassium platinum cyanide ended after 3.10 4 s. In 1861 Becquerel established an exponential law for the decay of phosphorescence, and postulated two different types of decay kinetics, i.e., exponential and hyperbolic, attributing them to monomolecular or bimolecular decay mechanisms. Becquerel criticized the use of the term fluorescence, a term introduced by Stokes, instead of employing the term phosphorescence, already assigned for this use [17, 19, 20], His son, Henri Becquerel (1852-1908), is assigned a special position in history because of his accidental discovery of radioactivity in 1896, when studying the luminescence of some uranium salts [17]. 

The time-resolved emission spectra were reconstructed from the fluorescence decay kinetics at a series of emission wavelengths, and the steady-state emission spectrum as described in the Theory section (37). Figure 4 shows a typical set of time-resolved emission spectra for PRODAN in a binary supercritical fluid composed of CO2 and 1.57 mol% CH3OH (T = 45 °C P = 81.4 bar). Clearly, the emission spectrum red shifts following excitation indicating that the local solvent environment is becoming more polar during the excited-state lifetime. We attribute this red shift to the reorientation of cosolvent molecules about excited-state PRODAN. 

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