Emission anisotropy time-resolved

However, time-resolved optical spectroscopy is perhaps the premier method for learning about the dynamics of a complex system, especially on nanosecond or picosecond time scales. Some DNA dynamics data from NMR spectroscopy are presented in Table 4.3. Time-resolved emission decays, time-resolved fluorescence anisotropy, and time-resolved Stokes shifts measurements of probe molecules in DNA have been described (and see below) and fast components in the time decays assigned to various DNA motions. The dynamics as a function of sequence are incompletely mapped and provide an exciting area for future investigations. 

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. 

Homo-FRET is a useful tool to study the interactions in living cells that can be detected by the decrease in anisotropy [106, 107]. Since commonly the donor and acceptor dipoles are not perfectly aligned in space, the energy transfer results in depolarization of acceptor emission. Imaging in polarized light can be provided both in confocal and time-resolved microscopies. However, a decrease of steady-state anisotropy can be observed not only due to homo-FRET, but also due to rotation of the fluorescence emitter. The only possibility of discriminating them in an unknown system is to use the variation of excitation wavelength and apply the... 

The difference between the theoretical value of the emission anisotropy in the absence of motions (fundamental anisotropy) and the experimental value (limiting anisotropy) deserves particular attention. The limiting anisotropy can be determined either by steady-state measurements in a rigid medium (in order to avoid the effects of Brownian motion), or time-resolved measurements by taking the value of the emission anisotropy at time zero, because the instantaneous anisotropy can be written in the following form ... 

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. 

Time-resolved emission anisotropy measurements are more straightforward in pulse fluorometry. 

Fluorescence polarization 1) steady state 2) time-resolved emission anisotropy rotational diffusion of the whole probe simple technique but Perrin s Law often not valid sophisticated technique but very powerful also provides order parameters... 

Time-resolved emission anisotropy experiments provide information not only on the fluidity via the correlation time rc, but also on the order of the medium via the ratio rco/ro. The theoretical aspects are presented in Section 5.5.2, with special attention to the wobble-in-cone model (Kinosita et al., 1977 Lipari and Szabo, 1980). Phospholipid vesicles and natural membranes have been extensively studied by time-resolved fluorescence anisotropy. An illustration is given in Box 8.3. 

Steady-state and time-resolved emission anisotropy measurements also allows distinction of single molecules on the basis of their rotational correlation time. 

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. 

There should exist a correlation between the two time-resolved functions the decay of the fluorescence intensity and the decay of the emission anisotropy. If the fluorophore undergoes intramolecular rotation with some potential energy and the quenching of its emission has an angular dependence, then the intensity decay function is predicted to be strongly dependent on the rotational diffusion coefficient of the fluorophore.(112) It is expected to be single-exponential only in the case when the internal rotation is fast as compared with an averaged decay rate. As the internal rotation becomes slower, the intensity decay function should exhibit nonexponential behavior. 

The limiting values of the fluorescence anisotropy are 0.4 and -0.2, corresponding to parallel and perpendicular absorption and emission dipoles, respectively. For a chromophore undergoing internal conversion, the change in angle between the two vectors can be instantaneous. It may also be caused by energy transfer or physical motion. On the laboratory scale, the time-resolved anisotropy decay is determined as ... 

Anisotropy measurements yield information on molecular motions taking place during the fluorescence lifetime. Thus, measuring the time-dependent decay of fluorescence anisotropy provides information regarding rotational and diffusive motions of macromolecules (Wahl and Weber, 1967). Time-resolved anisotropy is determined by placing polarizers in the excitation and emission channels, and measuring the fluorescence decay of the parallel and perpendicular components of the emission. 

The lipids themselves are highly mobile. Steady state and time resolved spectroscopy (absorption, emission, ir, raman, nmr, epr) and anisotropy measurements have revealed rotational, vibration and segmental motions of the headgroups and the hydrocarbon tails of the lipids. Translocation of a lipid from one half of the bilayer to the other, ("flip-flop ) as well as intermembrane... 

In-plane and out-of-plane rotational dynamics of CigRB at the toluene/water interface was evaluated using time-resolved TIR fluorescence spectroscopy [27]. The known transition dipole moment for the absorption of rhodamine B(RB) at about 530 nm (So Si) is almost parallel to that for the emission at about 570 nm (Si -> So) [28]. Time-resolved in-plane fluorescence anisotropy (r[Pg.213]

Thus, as mentioned earlier, time-resolved depolarization measurements afford a means of recording the time profile of the rotational autocorrelation function. The steady state technique, with continuous sample excitation, produces merely the time average of the emission anisotropy, F. For a rotating chromc hore with a sin e fluorescence decay time Tf, F is related to r(t) by the following expression... 

The three decay constants appearing in the expression for r(t) for an ellipsoid of rotation have been calculated and are own in Table 13. As may be seen, the three relaxation times diverge rapidly with increasing axial ratio for a prolate ellipsoid. However, for an oblate ellipsoid the deviations are small even for hi axial ratios and experimentally it m prove difficult to resolve more than a single, nean relaxation time for r(t) in this case. Thus, three situatrens exist in which the emission anisotropy may decay exponentially and it is not po le, therefore, to distingui ... 

A popular probe molecule which has b n employed for such studies is 1,6 diphenyl 1,3,5 hexatriene (DPH). This molecule has both absorption and emission along the long molecular axis and is thought to dissolve in the hydrocarbon interior. Time-resolved fluorescence depolarization studies with DPH probe molecules have been performed on the following bflayer syrtems dKdihydrosteraculoyl)pho halidyl choline dipalmitoyl phosphatidyl choline L-a-dimyristollecithin residues egg lecithin residues and mouse leukaemic L 1210cells In all reports the time-dependence of the emission anisotropy was found to decay non xponentially indkat-ing either... 

The influence of cholesterol on the fluidity of lipid bilayers is well known and has been investigated using time-resolved fluorescence depolarization . The decays of the emission anisotropy recorded for the probe molecule DPH dispersed in liposome of di-(dihydrosterculoyl) phosphalidyl choline with various added amounts... 

Phase shift and modulation techniques have come into greater prominence largely due to the feasibility of varying the modulation frequency of the exciting light. An authoritative description of recent developments with illustrative examples of multiexponent decays, decay of anisotropy, and time resolved emission spectra has... 

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. 

Fluorescence Polarization Studies of PMA and PAA. Time-resolved fluorescence polarization measurements are potentially a powerful means for studying molecular mobility. The fluorescence anisotropy function r(t) may be generated by monitoring the decay of vertically (Iv(t)) and horizontally (Ijj(t)) polarized components of emission following excitation by vertically polarized light pulses (Equation 1). 

Fluorescence anisotropy measurements can also be used to obtain the rates of the excited state tautomerization. Two variants can be applied. The first is based on the analysis of time-resolved anisotropy curves. These are constructed from measurements of the fluorescence decay recorded with different positions of the polarizers in the excitation and emission channels. The anisotropy decay reflects the movement of the transition moment and thus, the hydrogen exchange. For molecules with a long-lived Sj state, the anisotropy decay can also be caused by rotational diffusion. In order to avoid depolarization effects due to molecular rotation, the experiments should be carried out in rigid media, such as polymers or glasses. When the Sj lifetime is short compared to that of rotational diffusion, tautomerization rates can be determined even in solution. This is the case for lb, for which time-resolved anisotropy measurements have been performed at 293 K, using a... 

---