Fluorescence lifetime studies decay kinetics

The scope of fluorescence lifetime studies The kinetics of fluorescence decay... 

A number of studies on the fluorescence decay of tyrosine, tyrosine derivatives, and small tyrosyl peptides have been carried out. 36-38 Whereas the tyrosine zwitterion and tyrosine derivatives with an ionized a-carboxy group exhibited monoexponential fluorescence decay (x = 3.26-3.76 ns), double- or triple-exponential decay was observed in most other cases. As in the case of the tryptophan model compounds, the complex decay kinetics were again interpreted in terms of rotamer populations resulting from rotation around the C —Cp bond. There is evidence to indicate that the shorter fluorescence lifetimes may arise from rotamers in which the phenol ring is in close contact with a hydrated carbonyl group 36 37 and that a charge-transfer mechanism may be implicated in this quenching process. 39 ... 

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]. 

In other media like micelles, cyclodextrin, binary solvent mixtures, and proteins (47-55), lifetime distributions are routinely used to model the decay kinetics. In all of these cases the distribution is a result of the (intrinsic or extrinsic) fluorescent probe distributing simultaneously in an ensemble of different local environments. For example, in the case of the cyclodextrin work from our laboratory (53-55), the observed lifetime distribution is a result of an ensemble of 1 1 inclusion complexes forming and coexisting. These complexes are such that the fluorescent probe is located simultaneously in an array of environments (polarities, etc.) in, near, and within the cyclodextrin cavity, which manifest themselves in a distribution of excited-state lifetimes (53-55). In the present study our experimental results argue for a unimodal lifetime distribution for PRODAN in pure CF3H. The question then becomes, how can a lifetime distribution be manifest in a pure solvent ... 

When a fluorophore is encapsulated in heterogeneous media or immobilized on a surface, single exponential emission decays are rarely observed. Multi-exponential kinetics are attributed to the slow reorientation of the molecular environment after photoexcitation, and the heterogeneity of the microenvironment. Different species in the excited ensemble are oriented differently or exist in different microenvironments on the timescale of the emission which influences the excited-state lifetimes of the immobilized species. Studying the number and distribution of decays can provide information on the microenvironment of the immobilized fluorophore. When combined with fluorescence depolarization studies, detailed information on the motion of these species and their interaction with their environment can be obtained. 

Earlier studies on dye-sensitized Ti02 reported nanosecond time constants for the injection kinetics [16, 40-42]. These results were obtained indirectly from the measurement of the injection quantum yield and implicitly assumed that the interfacial electron transfer reaction was competing only with the decay of the dye excited state. Other studies were based on the same assumption but used measurements of the dye fluorescence lifetime, which provided picosecond-femtosecond time resolution [43-45]. Direct time-resolved observation of the buildup of the optical absorption due to the oxidized dye species S+ has been employed in more recent studies [46-51]. This appears to be a more reliable way of monitoring the charge injection process as it does not require any initial assumption on the sensitizing mechanism. 

The effect of oxygen on cyclic 1,3-diradicals shows that conformation can affect the triplet state lifetime ST Time resolved resonance Raman spectroscopy has been used to examine triplet states produced from different isomers of p-carotene. A theoretical study has also been reported on the a-cleavage of the triplet states of symmetric and non-symmetric ketones S mechanism for triplet state relaxation of aromatic molecules has been used to explain experimental data for substituted benzenes. The decay kinetics of triplet-triplet fluorescence in the mesitylene biradical (two sub-levels) have been measured between 10 and 77K in Shpolski matrices triplet state of dimesityl... 

A more discriminating picture of the role of traps in triplet exciton mobility can be drawn from the work by Webber (64, 65) who studied the kinetics of triplet quenching using a quencher molecule with a short lived triplet state (biacetyl) as a probe of the triplet exciton lifetime. In a study on P2VN (64), PACN (64) and PVCA (65) it has been shown that trapping is more efficient in PVCA compared to P2VN where triplet excitons exist long after the decay of delayed fluorescence (next section). 

Pyrene excimer formation still continues to be of interest and importance as a model compound for various types of study. Recent re-examinations of the kinetics have been referred to in the previous section. A non a priori analysis of experimentally determined fluorescence decay surfaces has been applied to the examination of intermolecular pyrene excimer formation O. The Kramers equation has been successfully applied to the formation of intermolecular excimer states of 1,3-di(l-pyrenyl) propane . Measured fluorescence lifetimes fit the predictions of the Kramer equation very well. The concentration dependence of transient effects in monomer-excimer kinetics of pyrene and methyl 4-(l-pyrenebutyrate) in toluene and cyclohexane have also been studied . Pyrene excimer formation in polypeptides carrying 2-pyrenyl groups in a-helices has been observed by means of circular polarized fluorescence" . Another probe study of pyrene excimer has been employed in the investigation of multicomponent recombination of germinate pairs and the effect on the form of Stern-Volmer plots ". 

Figure 4.6B shows typical fluorescence decays of the rhodopsin chromophore, protonated Schiff base of 11-cis-retinal, in methanol solution at 605 and 695 nm. The kinetic features are very similar to those of rhodopsin in terms of ultrafast and nonexponential components (Fig. 4.6A), but the kinetics are considerably slower. The fluorescence lifetimes for five wavelengths obtained in the study [53] were classified by two features the fast femtosecond (90-600 fs) and the slow picosecond (2-3 ps) components. The populations of fast and slow components were 25 and 75%, respectively. Figure 4.6C shows typical fluorescence decays of protonated Schiff base of 11-cis-locked 5-membered retinal in methanol solution... 

The determination of the rates of decay (lifetime) of the excited states of fluorophores, initiated by a light flash, is itself an exercise in kinetics. Some of the methods for deconvoluting exponentials which were developed for this purpose, have influenced the approach to the analysis of other rate processes (see Brand Johnson, 1992). In the present section those aspects of lifetime studies will be discussed which can contribute to our knowledge of the dynamics of proteins. While it was emphasized in section 2.1 that exponential decay should be characterized by the terms time constant or relaxation time, the time constant for the decay of fluorescence intensity after a flash of light is generally referred to as lifetime in the literature on that subject. It represents the average amount of time a molecule remains in the excited state. The intensity of emission at any one time is proportional to the concentration of molecules in the excited state. This can be compared with the properties of exponentials discussed in section 2.1 and the law of mass action (section 3.1). The latter relates the rate of a reaction to the concentration of the reactant. 

The two methods described above for the study of intramolecular dynamics of chain molecules, i.e., the method of fluorescence decay kinetics and the method of intensity fluctuations, are complementary. The two methods cover different time domains of molecular movements, the first being applicable to the study of changes occurring in the nanosecond time scale, and the second extending to movements slower than the time scale of the lifetime of excited state of the energy donor. 

In the present study we investigated energy transfer between the Zn-porphyrin units in a sequence of dendrimers varying in size from 4 to 64 porphyrin units (Fig. 1). Reference measurements were performed on the monomer, P1D1. In order to follow energy transfer within the dendrimers, the fluorescence anisotropy decay were analysed. To determine the lifetime of the dendrimers, additional analysis of the kinetics measured at magic angle was performed. The fluorescence anisotropy is defined by... 

Bimolecular deactivation (pathway vii, Fig. 1) of electronically excited species can compete with the other pathways available for decay of the energy, including emission of luminescent radiation. Quenching of this kind thus reduces the intensity of fluorescence or phosphorescence. Considerable information about the efficiencies of radiative and radiationless processes can be obtained from a study of the kinetic dependence of emission intensity on concentrations of emitting and quenching species. The intensity of emission corresponds closely to the quantum yield, a concept explored in Sect. 7. In the present section we shall concentrate on the kinetic aspects, and first consider the application of stationary-state methods to fluorescence (or phosphorescence) quenching, and then discuss the lifetimes of luminescent emission under nonstationary conditions. 

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