Fluorescence decay, global analysis

Over the past 15 years, the Huorescence Blob Model (FBM) in 1999 [36] and the Model Free Analysis (MFA) in 2005 [37] were introduced to, first, deal with the distribution of excimer rate constants that led to the multiexponential character of the fluorescence decays acquired with Py-WSPs and, second, provide a robust analytical framework to analyze the monomer and excimer fluorescence decays globally to yield reliable / gg values. The aim of this chapter is to describe the mathematic rationale underlying the application of the FBM and MFA to retrieve /agg firom the decay analysis and present experimental examples describing the implementation of these robust analytical tools. 

Beecham, J. M. Brand, L. Global analysis of fluorescence decay applications to some unusual experimental and theoretical studies. Photochem. Photobiol. 1986, 44, 323-329. 

Beechem f. M., Ameloot M. and Brand L. (1985) Global Analysis of Fluorescence Decay Surfaces Excited-State Reactions, Chem. Phys. Lett. 120, 466-472. 

J. R. Knutson, J. M. Beechem, and L. Brand, Simultaneous analysis of multiple fluorescence decay curves A global approach, Chem. Phys. Lett. 102, 501-507 (1983). 

In kinetic analysis of complex reactions, 210, 382 fluorescence decay rate distributions, 210, 357 implementation in Laplace de-convolution noniterative method, 210, 293 in multiexponential decays, 210, 296 partial global analysis by simulated annealing methods, 210, 365 spectral resolution, 210, 299. 

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]).

Time-resolved emission spectra were reconstructed from a set of multifrequency phase and modulation traces acquired across the emission spectrum (37). The multifrequency phase and modulation data were modeled with the help of a commercially available global analysis software package (Globals Unlimited). The model which offered the best fits to the data with the least number of fitting parameters was a series of bi-exponential decays in which the individual fluorescence lifetimes were linked across the emission spectrum and the pre-exponential terms were allowed to vary. 

Siemiarezuk and Ware l have reinvestigated the fluorescence decay kinetics of 1,2-di(1-pyrenyl)propane and concluded in contradiction to previous reports, that there is a distribution of short lifetimes in addition to two longer lived fluorescence components. This proposal has produced a strong dissent by Zachariasse and Striker 23 vvho on the basis of a global analysis, maintain that only three decays are observed, namely two excimer emissions and one from the monomer. 

This interpretation was confirmed by time-resolved fluorescence spectroscopy using the single photon timing technique. The pentad as a 1 X 10 M solution in chloroform was excited at 590 nm, and emission decay curves were recorded at 14 wavelengths. All 14 decays were then fit simultaneously to four exponential functions (x = 1.12) using a global analysis technique. The results were used to construct the decay associated spectrum shown in Figure 16. The two major components of the decay had lifetimes of 0.039 and 1.2 ns. (The two minor components represent impur-... 

Figure 16. Decay-associated spectrum resulting from a global analysis of the decay of the porphyrin fluorescence of pentad 22 following excitation of a — lxlO M chloroform solution with a 590-nm laser pulse. Data were obtained using the single photon counting technique, and the instrument response time was 0.035 ns. Decays at 14 different wavelengths were analyzed simultaneously, and the goodness of fit parameter was 1.12.

Fig. 7.8. Fluorescence decay curves of ZnP-PH2P recorded at three wavelengths. The full lines represent the best fit resulting from a global analysis of the three curves with a sum of four exponentials (see text). Zexc 430 nm. The observation wavelengths are indicated in the figure.

In Table 4, data from a simultaneous ("global" (61,62)) analysis of the excimer and monomer fluorescence decays for Py on Si-C g are collected, using the excimer decay curve depicted in Fig. 11B. The decay times found in this procedure are nearly identical to those obtained from the analysis of the excimer decay curve alone. 

Fig. 12. Monomer and excimer fluorescence decay curves of 1,3-di(1-pyrenyl)propane, 1Py(3)1Py, in silica/1-octanol at 25°C, analyzed by the method of "global analysis", see ref. (63) and caption to Fig. 11.

Beechem describes a second generation global analysis program for the recovery of complex inhomogeneous fluorescence decay kinetics. 

Figure 1. Decay-associated fluorescence emission spectrum for ca. 1 X 10 M triad 2 in benzonitrile following excitation at 590 nm. The spectrum was obtained from a global analysis of data at the six indicated wavelengths ( = 1.14) analyzed as four exponentials. The major component has the lifetime of 55 ps ( . ...

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. ...

Krishna, M. M. and Periasamy, N, 1997, Spectrally constrained global analysis of fluorescence decays in biomembrane systems. Analytical Biochemistry 253, 1-7. Kubota, Y., Matado, Y., Shi-Genuare, Y. and Fujisaki, Y, 1979, Fluorescence quenching of 10-methylacridinium chloride by nucleotides. Photochemistry and Photobiology 29, 1099-1106. 

The value of trilinear models is clearest in steady-state fluorescence measurements. They are also valuable in time-resolved fluorescence spectroscopy in situations where the appropriateness of a specific parametric equation for time decay, such as a sum of a few exponentials, is unclear. Although we are unaware of any work using trilinear models with other kinds of excited-state spectroscopy, trilinear models will be a valuable means of achieving component resolution whenever the absence of reliable parametric equations makes global analysis impossible. 

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