Fluorescence Anisotropy Decay Time

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. 

In the time-decay method, fluorescence anisotropy can be calculated as follows  

Vv and Vh are fluorescence intensities of vertically and horizontally polarized components, respectively, obtained with vertically polarized light, g is the correction factor and is equal 

For a fluorophore bound tightly to a protein, anisotropy decays as a single exponential, 

When a fluorophore exhibits segmental motions, time-resolved anisotropy decay must be analyzed as the sum of exponential decays  

Vv and Vh are the intensities of the vertically and horizontally polarized components, respectively, of the fluorescence elicited by vertically polarized light, g is the correction factor and is equal to Hy / Hh where Hy and Hh ai e the verticaljy and horizontaljy polarized components, respectively, of the fluorescence elicited by horizontaljy polarized light. 

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Cross A J and Fleming G R 1984 Analysis of time-resolved fluorescence anisotropy decays Blophys. J. 46 45-56... 

Figure 4.9 Correlation time of the fluorescence anisotropy decay for the PMMA brush. The open and closed circles indicate the correlation times for the brush in acetonitrile and benzene, respectively.

Figure 5.2. Simulated fluorescence anisotropy decays for (a) an isotropic system, (b) a system such as a lipid bilayer with short and long rotational correlation times, and (c) a system in which one of the rotational correlation times is infinite, and there is therefore a residual anisotropy or r .

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)... 

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... 

If a collisional quencher of the fluorophore is also incorporated into the membrane, the lifetime will be shortened. The time resolution of the fluorescence anisotropy decay is then increased,(63) providing the collisional quenching itself does not alter the anisotropy decay. If the latter condition does not hold, this will be indicated by an inability to simultaneously fit the data measured at several different quencher concentrations to a single anisotropy decay process. This method has so far been applied to the case of tryptophans in proteins(63) but could potentially be extended to lipid-bound fluorophores in membranes. If the quencher distribution in the membrane differed from that of the fluorophore, it would also be possible to extract information on selected populations of fluorophores possibly locating in different membrane environments. 

The anisotropy decay of the tryptophan fluorescence of both model peptides and biologically active peptides containing a single tryptophan residue has been determined in various studies. Even in the case of the tripeptide H-Gly-Trp-Gly-OH quenched by acrylamide the anisotropy decay displayed two correlation times with values of 39 and 135 ps. 44 The shorter correlation time was thought to be due to motions of the indole ring relative to the tripeptide. In the case of ACTH(l-24) the fluorescence anisotropy decay of the single tryptophan residue in position 9 of the peptide sequence obtained in phosphate buffer (pH 7, 3.5 °C) was also double-exponential. 29 The shorter rotational correlation time (0 = 92ps)... 

The ultrafast excitation-energy transfer of the J-aggregates on the octahedral AgBr is studied by the time-resolved fluorescence-anisotropy decay (r(t)) measurements [9]. They are biphasic with two time constants of -0.15 ps and 2-7 ps as shown in Fig. 6. Each phase should reflect some difference in the orientation of the dye molecules of the J-aggregates. 

The above described model sequences have been studied both as oligomers [7,8,11-13,19] and as polymers [9,11,20]. An increase in the size of the helix is known to reinforce its stability, as revealed by their melting curves [18] and attested by X-ray diffraction measurements in solution [21]. Therefore, in this chapter we focus on the polymeric duplexes poly(dGdC).poly(dGdC) [= 1000 base-pairs], poly(dAdT).poly(dAdT) [= 200-400 base-pairs] and poly(dA).poly(dT) [= 2000 base-pairs] studied by us. First we discuss the absorption spectra, which reflect the properties of Franck-Condon states, in connection with theoretical studies. Then we turn to fluorescence properties fluorescence intensity decays (hereafter called simply fluorescence decays ), fluorescence anisotropy decays and time-resolved fluorescence spectra. We... 

The ensemble of the experimental results briefly reviewed here, e.g. steady-state absorption and fluorescence spectra, fluorescence decays, fluorescence anisotropy decays and time-resolved fluorescence spectra, allow us to draw a qualitative picture regarding the excited state relaxation in the examined polymeric duplexes. Our interpretation is guided by the theoretical calculation of the Franck-Condon excited states of shorter oligomers with the same base sequence. 

Fluorescence Polarization under continuous excitation are presently developed. But these experiments need the a priori choice of a model of motion to be interpreted, and such models do not exist so far for the local dynamics in bulk polymers. This limitation is very troublesome, since experiments carried out on polymers in solution have shown that varying the choice of the model used in data treatment could lead to important discrepancies in the derived correlation times or activation energies. In the following, we will show how Fluorescence Anisotropy Decay may help to overcome this difficulty, and we will give some examples of original information that can be obtained using this technique in conjunction with the powerful synchrotron light source. 

Order parameters are used to interpret data on order and fluidity of a number of probes in lipid membranes obtained by measurements of fluorescence anisotropy decay 32 Ambiguities in the interpretation of time resolved fluorescence anisotropy measurements in lipid vesicle systems with DPH or TMA-DPH probes are attributed to the unsatisfactory models being used to interpret the data . The solubilisation of diphenylpolyenes in lipid bilayers has been critically examined33. It is concluded that such probes are satisfactory if used at low concentrations. 

From time-resolved fluorescence depolarization measurements, the anisotropy decay times (0) and the associated anisotropy ([>) have been determined for all first generation dendrimers using Eq.(l) ... 

In this paper we have investigated the segmental motions of bulk polybutadiene, in a temperature range hig r than (Tg + 60K), by using fluorescence anisotropy decay, and C spin-lattice magnetic relaxation time, Tp... 

The relationship between the structure of a polymer chain and it dynamics has long been a focus for work in polymer science. It is on the local level that the dynamics of a polymer chain are most directly linked to the monomer structure. The techniques of time-resolved optical spectroscopy provide a uniquely detailed picture of local segmental motions. This is accomplished through the direct observation of the time dependence of the orientation autocorrelation function of a bond in the polymer chain. Optical techniques include fluorescence anisotropy decay experiments (J ) and transient absorption measurements(7 ). A common feature of these methods is the use of polymer chains with chromophore labels attached. The transition dipole of the attached chromophore defines the vector whose reorientation is observed in the experiment. A common labeling scheme is to bond the chromophore into the polymer chain such that the transition dipole is rigidly affixed either para 1 lei (1-7) or perpendicular(8,9) to the chain backbone. 

Time-resolved optical experiments rely on a short pulse of polarized light from a laser, synchrotron, or flash lamp to photoselect chromophores which have their transition dipoles oriented in the same direction as the polarization of the exciting light. This non-random orientational distribution of excited state transition dipoles will randomize in time due to motions of the polymer chains to which the chromophores are attached. The precise manner in which the oriented distribution randomizes depends upon the detailed character of the molecular motions taking place and is described by the orientation autocorrelation function. This randomization of the orientational distribution can be observed either through time-resolved polarized fluorescence (as in fluorescence anisotropy decay experiments) or through time-resolved polarized absorption. 

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. 

In biological physics studies, we have measured the excitation intensity dependence of chlorophyll a/b fluorescence (17), anisotropy decay times of tryptophan in various environments (18). energy transfer in spinach ehloroplasts (19), and decay kinetics of hematoporphyrin derivative (20). 

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. 

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