Anisotropy decay times

Energy hopping among the peryleneimide chromophores, revealed by anisotropy decay times [30], occurs with a rate constant of 4.6x10 s E When three peryleneimide and one terryleneimide chromophores are attached to the dendrimer rim, energy transfer from the former to the latter units takes place with... 

From time-resolved fluorescence depolarization measurements, the anisotropy decay times (0) and the associated anisotropy ([>) have been determined for all first generation dendrimers using Eq.(l) ... 

Information about the rate constant of hopping ( hopp) through excitation energy transfer can be derived from the fast anisotropy decay time (02)- In order to take into account the possibility of multiple energy transfer channels in the case of a multichromophoric system containing identical chromophores, among which efficient dipole-dipole interactions occur, the measured anisotropy decay time 02 can be related to fchopp by Eq. (5), where the value of i represents the number... 

In biological physics studies, we have measured the excitation intensity dependence of chlorophyll a/b fluorescence (17), anisotropy decay times of tryptophan in various environments (18). energy transfer in spinach ehloroplasts (19), and decay kinetics of hematoporphyrin derivative (20). 

Cross A J and Fleming G R 1984 Analysis of time-resolved fluorescence anisotropy decays Blophys. J. 46 45-56... 

Definition and Uses of Standards. In the context of this paper, the term "standard" denotes a well-characterized material for which a physical parameter or concentration of chemical constituent has been determined with a known precision and accuracy. These standards can be used to check or determine (a) instrumental parameters such as wavelength accuracy, detection-system spectral responsivity, and stability (b) the instrument response to specific fluorescent species and (c) the accuracy of measurements made by specific Instruments or measurement procedures (assess whether the analytical measurement process is in statistical control and whether it exhibits bias). Once the luminescence instrumentation has been calibrated, it can be used to measure the luminescence characteristics of chemical systems, including corrected excitation and emission spectra, quantum yields, decay times, emission anisotropies, energy transfer, and, with appropriate standards, the concentrations of chemical constituents in complex S2unples. 

Figure 4.9 Correlation time of the fluorescence anisotropy decay for the PMMA brush. The open and closed circles indicate the correlation times for the brush in acetonitrile and benzene, respectively.

Theoretical calculations for ultrafast neat water spectroscopy are difficult to perform and difficult to interpret (because of the near-resonant OH stretch coupling). One classical calculation of the 2DIR spectrum even preceded the experiments [163] Torii has calculated the anisotropy decay [97], finding reasonable agreement with the experimental time scale. Mixed quantum/ classical calculations of nonlinear spectroscopy for many coupled chromo-phores is a daunting task. We developed the TAA for linear spectroscopy, and Jansen has very recently extended it to nonlinear spectroscopy [164]. We hope that this will allow for mixed quantum/classical calculations of the 2DIR spectrum for neat water and that this will provide the context for a molecular-level interpretation of these complex but fascinating experiments. 

Fast librational motions of the fluorophore within the solvation shell should also be consideredd). The estimated characteristic time for perylene in paraffin is about 1 ps, which is not detectable by time-resolved anisotropy decay measurement. An apparent value of the emission anisotropy is thus measured, which is smaller than in the absence of libration. Such an explanation is consistent with the fact that fluorescein bound to a large molecule (e.g. polyacrylamide or monoglucoronide) exhibits a larger limiting anisotropy than free fluorescein in aqueous glycerolic solutions. However, the absorption and fluorescence spectra are different for free and bound fluorescein the question then arises as to whether r0 could be an intrinsic property of the fluorophore. 

A distinction should be made between free rotation and hindered rotation. In the case of free rotation, after a (5-pulse excitation the emission anisotropy decays from ro to 0 because the rotational motions of the molecules lead to a random orientation at long times. In the case of hindered rotations, the molecules cannot become randomly oriented at long times, and the emission anisotropy does not decay to zero but to a steady value, r (Figure 5.10). These two cases of free and hindered rotations will now be discussed. 

In principle, the shape parameters of asymmetric rotors can be estimated from time-resolved anisotropy decay measurements, but in practice it is difficult to obtain accurate anisotropy decay curves over much more than one decade, which is often insufficient to determine more than two rotational correlation times. 

The emission anisotropy can be calculated from the experimental decays of the polarized components by means of Eq. (6.43), but only if the decay times are much larger than the width of the excitation pulse. Otherwise, Eq. (6.43) cannot be used because the responses R(t) (= E(t)I(t)) and R (t) (= E(t) 0 I (t)) must be deconvoluted. Several methods can then be used to recover the parameters ... 

With the development of multifrequency phase-modulation technology, Lakowicz and co-workers(171) were able to examine the time dependence of the anisotropy decay of BPTI. They noted that the intensity decay of the fluorescence is best fit by a biexponential decay law and that the anisotropy decay is also complex. At 25 °C and pH 6.5, correlation times of 39 ps and 2.25 ns were recovered from analysis of data obtained over the range 20 MHz to 2 GHz. The longer correlation time is close to that predicted for the overall rotational motion of a molecule of the size of BPTI. They indicated, however, that additional experiments need to be done to resolve whether the 39-ps... 

Lakowicz et al.(]7] VB) examined the intensity and anisotropy decays of the tyrosine fluorescence of oxytocin at pH 7 and 25 °C. They found that the fluorescence decay was best fit by a triple exponential having time constants of 80, 359, and 927 ps with respective amplitudes of 0.29, 0.27, and 0.43. It is difficult to compare these results with those of Ross et al,(68) because of the differences in pH (3 vs. 7) and temperature (5° vs. 25 °C). For example, whereas at pH 3 the amino terminus of oxytocin is fully protonated, at pH 7 it is partially ionized, and since the tyrosine is adjacent to the amino terminal residue, the state of ionization could affect the tyrosine emission. The anisotropy decay at 25 °C was well fit by a double exponential with rotational correlation times of 454 and 29 ps. Following the assumptions described previously for the anisotropy decay of enkephalin, the longer correlation time was ascribed to the overall rotational motion of oxytocin, and the shorter correlation time was ascribed to torsional motion of the tyrosine side chain. 

The major reasons for using intrinsic fluorescence and phosphorescence to study conformation are that these spectroscopies are extremely sensitive, they provide many specific parameters to correlate with physical structure, and they cover a wide time range, from picoseconds to seconds, which allows the study of a variety of different processes. The time scale of tyrosine fluorescence extends from picoseconds to a few nanoseconds, which is a good time window to obtain information about rotational diffusion, intermolecular association reactions, and conformational relaxation in the presence and absence of cofactors and substrates. Moreover, the time dependence of the fluorescence intensity and anisotropy decay can be used to test predictions from molecular dynamics.(167) In using tyrosine to study the dynamics of protein structure, it is particularly important that we begin to understand the basis for the anisotropy decay of tyrosine in terms of the potential motions of the phenol ring.(221) For example, the frequency of flips about the C -C bond of tyrosine appears to cover a time range from milliseconds to nanoseconds.(222)... 

Berger and Vanderkooi(88) studied the depolarization of tryptophan from tobacco mosaic virus. The major subunit of the coat protein contains three tryptophans. The phosphorescence decay is non-single-exponential. At 22°C the lifetime of the long component decays with a time constant of 22 ms, and at 3°C the lifetime is 61 ms. The anisotropy decay is clearly not singleexponential and was consistent with the known geometry of the virus. 

The results from fitting the anisotropy decay support the above conclusions. Wells and Lakowicz(200) resolved two exponential components in the anisotropy decay. They obtained ro = 0.11, t r = 0.3 ns, rj = 0.15, and t = 18.5 ns for the sample with no added Mg2+, and ro = 0.05, t R = 0.4 ns, r J = 0.17, and t"R = 17.4 ns for a sample with 10 mM Mg2+. Here r 0 and r o are the amplitudes of the fast and slow components. The longer rotational relaxation time corresponds to overall tumbling of the tRNA, although its amplitude is reduced by much more rapid local motions. The shorter relaxation time corresponds directly to a rapid local motion. Upon addition of Mg2+, the relative amplitude of die rapid local motion decreases, while that of the overall tumbling increases. This implies that the wyebutine base is held in a more rigid or constrained state, such as a 3 stack, in the presence of Mg2+. In that state, the amplitude of local angular motion is substantially diminished in comparison with that in the alternate state that prevails in the absence of Mg2+. As noted before, the exact nature of these conformation(s) is unresolved. 

Figure 5.2. Simulated fluorescence anisotropy decays for (a) an isotropic system, (b) a system such as a lipid bilayer with short and long rotational correlation times, and (c) a system in which one of the rotational correlation times is infinite, and there is therefore a residual anisotropy or r .

Extensions of the analysis of time-resolved fluorescence anisotropy decay data in terms of two order parameters have also been developed (see, e.g., Refs. 51-54). Thus, the corresponding higher order parameter term is <7%) given by(53)... 

There has been considerable interest in using fluorescence anisotropy to detect multiple environments in membranes as with fluorescence lifetimes (see above). For example, if a fluorophore is located in two environments with long and short lifetimes, then the fluorescence anisotropy decay process at longer times after excitation will be dominated by the long-lived fluorescent species. This occurs with parinaric acids, and this situation has been explored for a number of theoretical cases. 60 A similar situation has been found for DPH in two-phase lipid systems by collecting anisotropy decay-associated spectra at early and late times after excitation. 61 Evidence was found for more than one rotational environment in vesicles of a single lipid of it is at the phase transition temperature. It is important to identify systems showing associated anisotropy decays with more than one correlation time, each of... 

If a collisional quencher of the fluorophore is also incorporated into the membrane, the lifetime will be shortened. The time resolution of the fluorescence anisotropy decay is then increased,(63) providing the collisional quenching itself does not alter the anisotropy decay. If the latter condition does not hold, this will be indicated by an inability to simultaneously fit the data measured at several different quencher concentrations to a single anisotropy decay process. This method has so far been applied to the case of tryptophans in proteins(63) but could potentially be extended to lipid-bound fluorophores in membranes. If the quencher distribution in the membrane differed from that of the fluorophore, it would also be possible to extract information on selected populations of fluorophores possibly locating in different membrane environments. 

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