Decay associated spectra

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... 

Figure 9. Time-resolved decay-associated spectra of the fluorescence components with the relative...

Figure 13. Time-resolved decay-associated spectra of the UV (protein) fluorescence components i, rp)i - 4 of Pr phytochrome (124 kDa) and of the red-light adapted Pr + Pfr mixture obtained by global analysis. The dashed line corresponds to the stationary fluorescence spectrum obtained by A c = 295 nm (cf. Figure 11). The amplitudes of the two sets of spectra can be compared on an absolute basis (Holzwarth et al. [108]).

Figure 30. Decay associated spectra for adenine (dashed lines) and 9-methyl adenine (solid lines), extracted from the 2D TRPES spectra using global fitting procedures. Both molecules were fit by the same two time constants ij <0.1 and 12 1.1 ps, agreeing quantitatively with previous results. The spectra, however, are very different for adenine as compared to 9-methyl adenine. For details, see the text. See color insert.

Although the time constants for adenine and 9-methyl adenine are very similar, the associated photoelectron spectra reveal important differences that are obscured in ion-yield measurements. The decay associated spectra obtained from the fitting algorithm are shown in Fig. 30. The spectra of the fast ( < 0.1 ps) components are shown for adenine (dashed green) and 9-methyl adenine (solid blue). Likewise, the spectra of the 1.1 ps components for adenine (dashed red) and 9-methyl adenine (solid black) are given. The electronic states of the cations are Do(ti 1), D (n x), and D2 (ti ). The expected Koopmans correlations would therefore be 7171 — Do(7i 1), D2(ti 1), and nn —> As... 

Figure 31. Decay associated spectra of the short-lived state compared with calculated FC spectra for 9-methyl adenine (a) and adenine (b). In 9-methyl adenine, the nn — >o(ti-1),D2(ti-1) transitions leave a FC gap. In adenine, this gap is filled by the Tier ionizing transitions. See color insert.

Analysis of the decay associated spectra (DAS) with 10-nm resolution confirms the physical picture of the various time scales. The shortest time scales correspond to energy flow out of highest-energy Chls. The 0.3-ps component appears as a decay in the 650- to 670-nm windows and as a major rise at 680-700 nm (680 nm is the maximum of the absorption spectrum). The 2- to 3-ps... 

Decay associated spectra (Fig. 7.S) show that fluorescence lifetime components display a characteristic emission spectrum with a maximum located at 325,332 and 340 nm, respectively. The results reveal that a very weak emission spectrum crare xnids to the short fifetime con xment... 

Figure 7.10. Decay-associated spectra obtained global analysis of heterogeneous fluorescence kinetics of tryptophan in water and of indole n a water/glycerol mixture (3/2. V/V) measured at lO C. Two components in the case of tryptophan (lelt) are related to rotanieric forms. The structural relaxation in indole s environment m tfie abseiKe of any rolamers, can lead to an apparently similar heterogeneHy (r ht). Source LadoMiiru A. S. and White, S. H 2001, Biopl sical Journal. 181,1825 1827. Authorization of reprint accorded the American Biophysica] Sooe. ...

A very common method to put into evidence the fluorescence spectrum of each component is the decay associated spectra. This method allows combining the dynamic time-resolved fluorescence data with the steady-state emission spectrum. The fluorescence decays are globally or individually fitted to the n-exponential function (n being the number of species, tryptophan or tyrosine residues for example), and the decay associated spectra are constructed (Krishna and Periasamy, 1997). Figure 8.27 displays the decay associated spectra of Trp residues of ai-acid glycoprotein performed by Hof et al (1996). The authors found four fluorescence lifetimes instead of the three we obtained. They attributed the peak of 337 nm to Trp-122 with a location between the surface of the protein and its core. The determination of the degree of hydrophobicity around the Trp residues showed that Trp-25 residue is located in a hydrophobic environment and Trp-160 is the most exposed to the solvent. 

T = 0.7 msec (O) and t2 = 2.2 msec ( ). The corresponding decay-associated spectra A,(X) and A2spectral characteristics of bleached absorption bands [centered at 683... 

Fig. 4. Decay-associated spectra showing the exciton equilibration (12 psec component with positive and negative amplitudes) in the spectrally inhomogeneous antenna of photosystem I from the cyanobacterium Synechococcus sp. [From A. R. Holzwarth, G. Schatz, H. Brock, and E. Bittersmann, Biophys. J. 64, 1813 (1993).]...

The/(X) values recovered from the two-decay-time analysis were used to calculate die decay-associated spectra (DAS). The DAS represent the emission spectrum associated with each lifetime. These spectra (Figure 5.53) show a weak conqionent centered at 340 nm, which is in agreement with the results of TCSPC experiments. 

Decay. 154-155, 169 see also Intensity decay Decay-associated spectra (DAS), 177, 499-501... 

Due to space limitations, we can only make brief mention of DAS and lifetime distributions. DAS (decay associated spectra) are the emission spectra associated with the different decay species and are described in (11). Examples of applications may be found in (12-14). The treatment of fluorescence lifetimes as distributions (rather than as the discrete values implied in Section 2.1) is discussed in (15-18). Commercially available software packages (see Section 2.6.1) are typically able to carry out the necessary analysis to obtain DAS ftom decay measurements made at multiple emission wavelengths, or to obtain lifetime distributions. 

In this section, we have shown the existence of a decay pathway connecting the FC region on the 27r7r state to a Cl with the ground state in a barrierless manner. This pathway crosses a probable three-state Cl involving the Itttt, iTra and 27r7r states. Experimentally, a time constant T4 of approximately 50 fs was extracted from the photoelectron spectra recorded after excitation at A = 238 nm [1, 2, 70]. In [1, 2], the analysis of the decay associated spectra did not show evidence of arise... 

Lofroth, J.E. (1986) Time-resolved emission spectra, decay-associated spectra, and species-associated spectra./. Phys. Chem., 90 (6), 1160-1168. 

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