Time-resolved fluorescence intensity decay

Time-Resolved Fluorescence. To improve our understanding of the photophysics of the PRODAN-CF3H system we performed a series of time-resolved fluorescence experiments at pr = 1.25 as a function of temperature. Traditionally, one describes the time-resolved fluorescence intensity decay (I(t)) by a sum of (n) discrete exponentials of the form (43,46) ... 

The most important results can be summarized as follows The time-resolved fluorescence intensity decays measured in solutions of several PMA samples in a wide range of pH and ionic strength were always double-exponential. Both the short (units of nanosecond) and the long (tens of nanoseconds) fluorescence lifetimes increase with decreasing pH. Typical data are shown in Fig. 6a. The Tp,i and Tp,2 dependences vs. pH (curves 1 and 2, respectively) exhibit a pronounced sigmoid shape, with the inflection point close to pH 5.5. The relative pre-exponential factor of the short-living fluorescence component, Ai, increases with increasing pH. The A dependence on pH is depicted in Fig. 6b. 

Reference (25) details a study in which time-resolved fluorescence intensity and anisotropy decay measurements were combined with stopped-flow mixing to monitor the local and global dynamics of E. coli dihydrofolate reductase during refolding. A general review of such (so-called) double kinetic experiments is given in (26). 

Recent contributions that both time-resolved fluorescence intensity and anisotropy decay studies have made to om understanding of the structure and d3mamics of proteins and nucleic acids are reviewed in (27). 

We have already discussed quantum-beat spectroscopy (QBS) in connection with beam-foil excitation (Fig.6.6). There the case of abrupt excitation upon passage through a foil was discussed. Here we will consider the much more well-defined case of a pulsed optical excitation. If two close-lying levels are populated simultaneously by a short laser pulse, the time-resolved fluorescence intensity will decay exponentially with a superimposed modulation, as illustrated in Fig. 6.6. The modulation, or the quantum beat phenomenon, is due to interference between the transition amplitudes from these coherently excited states. Consider the simultaneous excitation, by a laser pulse, of two eigenstates, 1 and 2, from a common initial state i. In order to achieve coherent excitation of both states by a pulse of duration At, the Fourier-limited spectral bandwidth Au 1/At must be larger than the frequency separation ( - 2)/ = the pulsed excitation occurs at... 

The time-resolved fluorescence intensities, Fm(0 and Fd(0, are proportional to the instantaneous concentrations of the excited monomers and excimers, respectively. The monomer decay is a sum of two exponentials, which means that it decreases faster than the unquenched monomer decay. The excimer is not present immediately after excitation and is formed by a diffusion-controlled process, i.e., the emission increases at early times, passes maximum, and at later times decays more slowly than the monomer fluorescence. The realistic (experimental) decays, F,(0exp> are schematically shown in Fig. 11. 

Homogeneous Time Resolved Fluorescence (HTRF) (Cisbio International) is an assay based on the proximity of a lanthanide cryptate donor and a fluorescent acceptor molecule whose excitation wavelength overlaps that of the cryptate s emission. The utility of this technique is based on the time resolved fluorescence properties of lanthanides. Lanthanides are unique in the increased lifetime of their fluorescence decay relative to other atoms, so a delay in collection of the emission intensity removes the background from other fluorescent molecules. An example of the HTRF assay is a generic protein-protein interaction assay shown in Fig. 2. 

There should exist a correlation between the two time-resolved functions the decay of the fluorescence intensity and the decay of the emission anisotropy. If the fluorophore undergoes intramolecular rotation with some potential energy and the quenching of its emission has an angular dependence, then the intensity decay function is predicted to be strongly dependent on the rotational diffusion coefficient of the fluorophore.(112) It is expected to be single-exponential only in the case when the internal rotation is fast as compared with an averaged decay rate. As the internal rotation becomes slower, the intensity decay function should exhibit nonexponential behavior. 

The above described model sequences have been studied both as oligomers [7,8,11-13,19] and as polymers [9,11,20]. An increase in the size of the helix is known to reinforce its stability, as revealed by their melting curves [18] and attested by X-ray diffraction measurements in solution [21]. Therefore, in this chapter we focus on the polymeric duplexes poly(dGdC).poly(dGdC) [= 1000 base-pairs], poly(dAdT).poly(dAdT) [= 200-400 base-pairs] and poly(dA).poly(dT) [= 2000 base-pairs] studied by us. First we discuss the absorption spectra, which reflect the properties of Franck-Condon states, in connection with theoretical studies. Then we turn to fluorescence properties fluorescence intensity decays (hereafter called simply fluorescence decays ), fluorescence anisotropy decays and time-resolved fluorescence spectra. We... 

This chapter might be viewed as a plea for the measurement of time-resolved fluorescence of 2AP when it is introduced into an RNA. The resulting picture of the flexibility of the nucleobase in different chemical and physical contexts would be extremely useful as we build our understanding of the dynamics of RNAs. Measurements of fluorescence intensity can be excellent reporters of RNA folding, as the above examples show, but especially when fluorescence is quenched, the most popular interpretation is that the base is stacked with the attendant implication that its position is static. However, if 2AP remains flexible but spends part of its time stacked with another nucleobase, the solution average fluorescence intensity will still be reduced. Only by measurement of the decay lifetimes of 2AP will its true dynamics be discerned. 

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. 

We have used the spectral reconstruction method to obtain a time-resolved fluorescence spectrum [13] When the fluorescence up-conversion method is used, the relative intensity between each wavelength becomes uncertain because the angle of the nonlinear crystal has to be tuned at each wavelength of observation. However, the intensity of the fluorescence /(A, (), at a given time t and wavelength A, can be obtained from the normalized fitted decay series D(t, A) and intensity of the steady-state fluorescence /0(A) ... 

The principles of time-resolved fluorometry are illustrated in Fig. 7.4. The d-pulse response of a fluorescent sample (i.e. the fluorescence intensity decay in response to an infinitely short light pulse mathematically represented by the Dirac function <5(t) delta excitation) is, in the simplest case, a single exponential whose time constant is the excited-state lifetime, but more frequently it is a sum of discrete exponentials, or a more complicated function sometimes, the system is characterized by a distribution of decay times. For any excitation function E(t), the response R(t) of the sample is the convolution product of this function by the <5-pulse response ... 

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