Lifetime of the excited state

All the nucleic acid bases absorb UV radiation, as seen in Tables 11-1, 11-2, 11-3, 11-4, and 11-5, making them vulnerable to the UV radiation of sunlight, since the energy of the photons absorbed could lead to photochemical reactions. As already mentioned above, the excited state lifetimes of the natural nucleobases, and their nucleotides, and nucleosides are very short, indicating that ultrafast radiationless decay to the ground state takes place [6], The mechanism for nonradiative decay in all the nucleobases has been investigated with quantum mechanical methods. Below we summarize these studies for each base and make an effort to find common mechanisms if they exist. 

Day-to-day drifts of instrumental characteristics, differences in temperature and sample preparation may affect the recorded lifetimes. For instance, differences in temperature can affect both the excited state lifetime of the fluorophore and instrument properties like noise or delays of wirings and electronics. 

Finally, a nonfluorescent YFP variant has been constructed for use as a FRET acceptor for GFP [89]. This allows the detection of the complete emission spectrum of GFP, while the FRET efficiency is high (R0 = 5.9 nm) due to strong overlap of the GFP fluorescence and YFP absorbance band. The occurrence of FRET was detected by a reduction in the excited state lifetime of the GFP by FLIM. The main disadvantage is that the presence of the acceptor cannot be detected in living cells. 

Figure 2. Principles of reversible luminescence sensing using photochemical quenching processes (electron, energy or proton transfer). Dye = luminescent indicator Q = quencher species dotted arrow non-radiative deactivation processes. The luminescence intensity (and excited state lifetime) of the indicator dye decreases in the presence of the quencher. The indicator dye is typically supported onto a polymer material in contact with the sample. The quencher may he the analyte itself or a third partner species that interacts with the analyte (see text).

Exciton decay When an exciton decays radiatively a photon is emitted. When the excitons form in fluorescent materials radiative decay is limited to singlet excitons and emission occurs close to the recombination region [7] of the OLED due to the relatively short lifetime of the excited state (of the order of 10 ns). For phosphorescent materials, emission can occur from triplet excitons. Due to the longer excited state lifetime (of the order of hundreds of nanoseconds), triplet excitons can diffuse further before decaying. 

It was shown in Section 3.5 that the excited-state lifetime of the Si state is given by ... 

Ro is the Forster critical radius (defined in Section 4.6.3), and rf) is the excited-state lifetime of the donor in the absence of transfer. 

The excited-state lifetime of the uncomplexed fluorophore M is unaffected, in contrast to dynamic quenching. The fluorescence intensity of the solution decreases upon addition of Q, but the fluorescence decay after pulse excitation is unaffected. Quinones, hydroquinones, purines and pyrimidines are well-known examples of molecules responsible for static quenching. 

The time evolution of the fluorescence intensity of the monomer M and the excimer E following a d-pulse excitation can be obtained from the differential equations expressing the evolution of the species. These equations are written according to the kinetic in Scheme 4.5 where kM and kE are reciprocals of the excited-state lifetimes of the monomer and the excimer, respectively, and ki and k i are the rate constants for the excimer formation and dissociation processes, respectively. Note that this scheme is equivalent to Scheme 4.3 where (MQ) = (MM) = E and in which the formation of products is ignored. 

Let us consider the possible events following excitation of an acid AH that is stronger in the excited state than in the ground state (pK < pK). In the simplest case, where there is no geminate proton recombination, the processes are presented in Scheme 4.6, where t0 and Tq are the excited-state lifetimes of the acidic (AH ) and basic (A- ) forms, respectively, and ki and k i are the rate constants for deprotonation and reprotonation, respectively, kj is a pseudo-first order rate constant, whereas k i is a second-order rate constant. The excited-state equilibrium constant is K = k /k 7 ... 

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 ... 

Dr / r2 1 the donor and acceptor cannot significantly diffuse during the excited-state lifetime of the donor. This case is called the static limit. Distinction according to the mechanism of tranfer should be made. 

Z>Tp/r2 1 the mean distance diffused by the donor and acceptor relative to each other during the excited-state lifetime of the donor is much larger than the mean distance R between donors and acceptors. Two cases are to be considered... 

The dependence of knr on the value or concentration of a [Parameter], in the vicinity of the excited sensor, determines both the luminescence intensity and the excited state lifetime of the sensor. 

Platinum and palladium porphyrins in silicon rubber resins are typical oxygen sensors and carriers, respectively. An analysis of the characteristics of these types of polymer films to sense oxygen is given in Ref. 34. For the sake of simplicity the luminescence decay of most phosphorescence sensors may be fitted to a double exponential function. The first component gives the excited state lifetime of the sensor phosphorescence while the second component, with a zero lifetime, yields the excitation backscatter seen by the detector. The excitation backscatter is usually about three orders of magnitude more intense in small optical fibers (100 than the sensor luminescence. The use of interference filters reduce the excitation substantially but does not eliminate it. The sine and cosine Fourier transforms of/(f) yield the following results ... 

To study the excited state one may use transient absorption or time-resolved fluorescence techniques. In both cases, DNA poses many problems. Its steady-state spectra are situated in the near ultraviolet spectral region which is not easily accessible by standard spectroscopic methods. Moreover, DNA and its constituents are characterised by extremely low fluorescence quantum yields (<10 4) which renders fluorescence studies particularly difficult. Based on steady-state measurements, it was estimated that the excited state lifetimes of the monomeric constituents are very short, about a picosecond [1]. Indeed, such an ultrafast deactivation of their excited states may reduce their reactivity something which has been referred to as a "natural protection against photodamage. To what extent the situation is the same for the polymeric DNA molecule is not clear, but longer excited state lifetimes on the nanosecond time scale, possibly of excimer like origin, have been reported [2-4],... 

Precise measurements of the excited state lifetimes of the DNA constituents were not available till very recently, mainly due to the limited time resolution of conventional spectroscopic techniques. Studying the DNA nucleosides by transient absorption spectroscopy, Kohler and co-workers observed a very short-lived induced absorption in the visible which they assigned to the first excited state [5,6]. The lifetimes observed were all well below 1 picosecond. The first femtosecond fluorescence studies of DNA constituents were performed using the fluorescence upconversion technique. Peon and Zewail [7] reported that the excited state lifetimes of DNA/RNA nucleosides and nucleotides all fall in the subpicosecond time, thus corroborating the results obtained by transient absorption. 

For supramolecular assemblies, intramolecular processes may quench the emission of A to a degree which depends on the relative efficiency of the process when compared with emission. It is often useful to compare the photophysical and chemical behavior of the supramolecular species, e.g. A -L-B, with an appropriate model compound, for instance, AH, which contains the photochemically active component, A, in the absence of any units capable of interacting with A. For example, from luminescence lifetime measurements, the rate of electron transfer may be estimated by comparing the excited-state lifetime of the mononuclear model complex, tModei, with that of the supramolecular species, rsupra, by using the following equation ... 

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. 

Later in this chapter we will describe a series of recent experiments in which non-racemic excited states were obtained from racemic mixtures by adding an optically active quencher molecule. The chiral quencher changes the excited state lifetimes of the enantiomers, and makes them unequal. We designate the new lifetimes as k+ and k, and relate them to the concentration of chiral quencher, [QaJ, and bimolecular quenching constants, k and k as follows... 

Tmodeiare respectively, excited state lifetimes of the dyad and a model complex which contains the metal complex chromophore but not the electron donor. 

An estimate for the rate constants of the intramolecular singlet energy transfer fcgt can be obtained from the extent of quenching of the OPVn fluorescence in the dyads and the singlet excited state lifetime of the OPVn oligomers [103] by... 

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