Anisotropy decays data analysis

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)... 

Beechem JM, Gratton E, Ameloot M, Knutson JR, Brand L (1991) The global analysis of fluorescence intensity and anisotropy decay data second-generation theory and programs. In Lakowicz JR (ed) Topics in fluorescence spectroscopy, vol 2 principles. Plenum Press, New York... 

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... 

The mobility of tyrosine in Leu3 enkephalin was examined by Lakowicz and Maliwal/17 ) who used oxygen quenching to measure lifetime-resolved steady-state anisotropies of a series of tyrosine-containing peptides. They measured a phase lifetime of 1.4 ns (30-MHz modulation frequency) without quenching, and they obtained apparent rotational correlation times of 0.18 ns and 0.33 ns, for Tyr1 and the peptide. Their data analysis assumed a simple model in which the decays of the anisotropy due to the overall motion of the peptide and the independent motion of the aromatic residue are single exponentials and these motions are independent of each other. 

The rotational relaxation of DNA from 1 to 150 ns is due mainly to Brownian torsional (twisting) deformations of the elastic filament. Partial relaxation of the FPA on a 30-ns time scale was observed and qualitatively attributed to torsional deformations already in 1970.(15) However, our quantitative understanding of DNA motions in the 0- to 150-ns time range has come from more accurate time-resolved measurements of the FPA in conjunction with new theory and has developed entirely since 1979. In that year, the first theoretical treatments of FPA relaxation by spontaneous torsional deformations appeared. 16 171 and the first commercial synch-pump dye laser systems were delivered. Experimental confirmation of the predicted FPA decay function and determination of the torsional rigidity of DNA were first reported in 1980.(18) Other labs 19 21" subsequently reported similar results, although their anisotropy formulas were not entirely correct, and they did not so rigorously test the predicted decay function or attempt to fit likely alternatives. The development of new instrumentation, new data analysis techniques, and new theory and their application to different DNAs in various circumstances have continued to advance this field up to the present time. 

In the FD analysis, the rolation- ee intensity deaqr is measured in a separate experiment using magic-anp polarizer conditions. The parameter vahiea, typically 0 and t fur the midtiexpaoential model, are held constant during the calculation of xJ Bq. [11.34]. In principle, one could measure the phase and modulation of the polarized components and use these data to recover Jff) and fft). This would be analogous to the method used for TCSPC data. However, it is believed that the anisotropy decay is better determined by direct measurement of the difference (i .) and ratio (A ) values. 

For rapid single-exponential anisotropy dec s, analysis of data from the most quenched sample is adequate. However, the usual goal is to improve resolution of the entire anisolropy decay. This can be accom dished by simultaneous analysis of data from the unquenched and several quenched samples. The data from the unquenched sample contribute most to the deleiminalion of the longer correlation limes, and the data from the quenched samples contribute to resolution of the faster motions. 

ISS data have been recorded in many pure and mixed molecular liquids [34,49, 75, 83, 83-85], In most cases, the data are not described precisely by Eq. (27). Rather, an additional decay component appears at intermediate times (decay times 500 fs). This has been interpreted [49, 84] in terms of higher order polarizability contributions to C (t) which represent translational motions, an interpretation supported by observations in CCI4 (whose single-molecule polarizability anisotropy vanishes by symmetry). This interpretation is not consistent with several molecular dynamics simulations of CSj [71, 86]. An alternative analysis has been presented [82] that incorporates theoretical results showing that even the single-molecule orientational correlation function C (t) should in fact show decay on the 0.5-ps time scale of cage fluctuations [87, 88]. 

At present, there is widespread interest in directly measuring the time-dependent processes. This is because considerably more information is av lilable from the time-dependent data. For example, the time-dependent decays of protein fluorescence can occasionally be used to recover the emission spectra of individual tryptophan residues in a protein. The time-resolved anisotropies can reveal the shape of the protein and/or the extent of residue mobility within the protein. The time-resolved energy transfer can reveal not only the distance between the donor and acceptor, but also the distribution of these distances. The acquisition of such detailed information requires high resolution instrumentation and careful data acquisition and analysis. 

The advances in time resolved techniques have fostered a reexamination of theories of the rotational motions of molecules in liquids. Models considered include the anisotropic motion of unsymmetrical fluorophores the internal motions of probes relative to the overall movement with respect to their surroundings, the restricted motion of molecules within membranes (e.g., wobbling within a cone), and the segmental motion of synthetic macromolecules [8]. Analyses of these models point to experimental situations in which the anisotropy can show both multi-exponential and none-exponential decay. Current experimental techniques are capable in principle of distinguishing between these different models. It should be emphasized, however, that to extract a single average rotational correlation time demands the same precision of data and analysis as fluorescence decay experiments which exhibit dual exponential decays. Multiple or non-exponential anisotropy experiments are thus near the limits of present capabilities, and generally demand favourable combinations of fluorescence and rotational diffusion times [48]. 

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