Nanosecond anisotropy

Single-Molecule Photon-Stamping Spectroscopy Probing spFRET and Nanosecond Anisotropy... 

In principle, pulsed excitation measurements can provide direct observation of time-resolved polarization decays and permit the single-exponential or multiexponential nature of the decay curves to be measured. In practice, however, accurate quantification of a multiexponential curve often requires that the emission decay be measured down to low intensity values, where obtaining a satisfactory signal -to-noise ratio can be a time-consuming process. In addition, the accuracy of rotational rate measurements close to a nanosecond or less are severely limited by tbe pulse width of the flash lamps. As a result, pulsed-excitation polarization measurements are not commonly used for short rotational periods or for careful measurements of rotational anisotropy. 

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)... 

The elucidation of the intramolecular dynamics of tryptophan residues became possible due to anisotropy studies with nanosecond time resolution. Two approaches have been taken direct observation of the anisotropy kinetics on the nanosecond time scale using time-resolved(28) or frequency-domain fluorometry, and studies of steady-state anisotropy for xFvarying within wide ranges (lifetime-resolved anisotropy). The latter approach involves the application of collisional quenchers, oxygen(29,71) or acrylamide.(30) The shortening of xF by the quencher decreases the mean time available for rotations of aromatic groups prior to emission. 

J. R. Lakowicz and G. Weber, Nanosecond segmental mobilities of tryptophan residues in proteins observed by lifetime-resolved fluorescence anisotropies, Biophys. J. 32, 591-601 (1980). 

M. Vincent, B. de Foresta, J. Gallay, and A. Alfsen, Nanosecond fluorescence anisotropy decays of n-(9-anthroyloxy) fatty acids in dipalmitoylphosphatidylcholine vesicles with regard to isotropic solvents, Biochemistry 21, 708-716 (1982). 

Numerical micromagnetics, which may be based either on the finite difference or finite element method, resolve the local arrangement of the magnetization which arises from the interaction between intrinsic magnetic properties such as the magnetocrystalline anisotropy and the physical and chemical microstructure of the material. The numerical solution of the equation of motion also provides information on how the magnetization evolves in time. The time and space resolution of numerical micromagnetic simulations is in the order of nanometers and nanoseconds, respectively. 

Wahl, Ph. Itecay fluorescence anisotropy (in Ref. p. 1 Wahl Ph. Nanosecond pulse fluorometry. In New Techniques in Biophysics and Cell Biology, Vol 2. New York ... 

Fig. 51. Nanosecond emission anisotropy kinetics of diphenylhexatriene in liposomes consisting of di-(dihydrosterculoyl) phosphatidyl choline and varying amounts of cholesterol (0.20 and,...

Viovy, Monnerie, and Brochon have performed fluorescence anisotropy decay measurements on the nanosecond time scale on dilute solutions of anthracene-labeled polystyrene( ). In contrast to our results on labeled polyisoprene, Viovy, et al. reported that their Generalized Diffusion and Loss model (see Table I) fit their results better than the Hall-Helfand or Bendler-Yaris models. This conclusion is similar to that recently reached by Sasaki, Yamamoto, and Nishijima 3 ) after performing fluorescence measurements on anthracene-labeled polyCmethyl methacrylate). These differences in the observed correlation function shapes could be taken either to reflect the non-universal character of local motions, or to indicate a significant difference between chains of moderate flexibility and high flexibility. Further investigations will shed light on this point. 

An Applied Photophysics Model SP 2X nanosecond spectrometer incorporating an alternating polarization rotation unit ( ) was used for the time-resolved fluorescence anisotropy measurements. An excitation wavelength of 365 nm was employed for excitation of the anthracene end-groups and emission above 400 nm was isolated with a Schott GG 400 filter. 

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