Fluorescence anisotropy decay

Steady state anisotropy measurements are usually performed using a continuous excitation source. Such measurements are quite simple and require inexpensive instrumentation. Nevertheless SS techniques do not allow actually to follow the 

The two components can be recorded with a pump and probe system or with a time correlated single photon counting (TCSPC) device. As in the case of SS FA the correction due to the G factor must be taken into consideration. 

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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 4.7 Fluorescence anisotropy decay curves for the PMMA brush swollen in benzene (filled circles) and the free PMMA chain in benzene solution at concentrations of 0.33 (triangles) and 2.9 X 10 g (open circles). The graft density of the brush is 0.46 chains nm . The solid curve indicates the instrument response function. Reproduced with permission from the American Chemical Society.

Figure 4.8 shows the fluorescence anisotropy decay curves for PMMA brushes with various graft densities swollen in benzene and acetonitrile. Benzene and acetonitrile are good and 0 solvents for PMMA. As clearly shown in this figure. 

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 4.12 Fluorescence image of PMMA brush layer (a) and schematic drawing of the brush chain (b). The dark region (a) corresponds to the substrate surface exposed by scratching off the brush layer. The filled and open circles indicate the points where the fluorescence anisotropy decay was acquired.

FIG. 11 Order parameter variation along acyl chains in red cell ghosts ( ), small unilamellar vesicles of egg phosphatidylcholine (V), and paraffin oil (+), as determined by the fluorescence anisotropy decay of the w-anthroyloxy fatty acid probes. (Reprinted by permission from Ref. 12.)... 

Tleugabulova, D., Duft, A.M., Zhang, Z., Chen, Y., Brook, M.A. and Brennan, J.D. (2004) Evaluating formation and growth mechanisms of silica particles using fluorescence anisotropy decay analysis. Langmuir, 20, 5924—5932. 

The diffusion constant D with the underlying microviscosity , and the two order parameters , <(P4> reflecting the degree of orientational constraint have been successfully determined from the fluorescence anisotropy decay in... 

Fig. B8.3.1. Fluorescence anisotropy decays at 4 °C of PPL A in phospholipid vesicles (PC PI, 95 5 mol %). B in Torpedo membranes. From the best fit of the /(t) and l (t) components, and by using the wobble-in-cone model, the...

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. 

M. Straume and B. J. Litman, Influence of cholesterol on equilibrium and dynamic bilayer structure of unsaturated acyl chain phosphatidylcholine vesicles as determined from higher order analysis of fluorescence anisotropy decay, Biochemistry 26, 5121-5126 (1987). 

J. R. Lakowicz, H. Cherek, I. Gryczynski, N. Joshi, and M. L. Johnson, Enhanced resolution of fluorescence anisotropy decays by simultaneous analysis of progressively quenched samples, Biophys. J. 51, 755-768 (1987). 

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). 

Fluorescence anisotropy decay measurements, which are based on the excitation of probes with polarized light and subsequent polarized fluorescence emission, can... 

Steady-state fluorescence polarization studies have been carried out with a number of peptides, including model peptides, ACTH, glucagon, melittin, and thyrocalcitonin. This work has been reviewed 5 and will not be discussed in the present article. More recently, interesting information on the rotational behavior and structural flexibility of various peptides has been obtained from fluorescence anisotropy decay measurements. 

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)... 

In the present study we investigated energy transfer between the Zn-porphyrin units in a sequence of dendrimers varying in size from 4 to 64 porphyrin units (Fig. 1). Reference measurements were performed on the monomer, P1D1. In order to follow energy transfer within the dendrimers, the fluorescence anisotropy decay were analysed. To determine the lifetime of the dendrimers, additional analysis of the kinetics measured at magic angle was performed. The fluorescence anisotropy is defined by... 

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... 

As we explained in the previous section, fluorescence decays do not bring any direct evidence about energy transfer among DNA bases within a helix. In contrast, fluorescence anisotropy decays can provide this type of information. Such a possibility is based on the correlation of macroscopic observables to molecular parameters. On the molecular scale, r is related to the angle 6 formed between the transition dipoles associated to photon absorption and photon emission ... 

Fig. 10 Fluorescence anisotropy decays obtained for poly(dGdC).poly(dGdC) (green), poly(dAdT).poly(dAdT) (blue) and poly(dA).poly(dT) (red) in phosphate buffer. Black signals correspond to an equimolar mixture of nucleotides (left dGMP and dCMP center and right dAMP and TMP). Excitation wavelength 267 nm. Emission wavelength 330 nm.

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. 

This article shows how the recent developments in the Fluorescence Anisotropy Decay technique permitted by Synchrotron Radiation can be used to study local polymer dynamics both in dilute solutions and in bulk polymers. 

In section 2, the different theoretical models for local dynamics are briefly reviewed, and their connection with spectroscopic experiments is recalled. The Fluorescence Anisotropy Decay technique and the synchrotron source are presented in section 3. The fourth section is concerned with two typical examples. Using a series of experiments performed on polystyrene dilute solutions and another one performed on melt poly butadiene, we show how the different theoretical models can be told apart, and we present new information about the processes responsible of backbone rearrangement which has been obtained using the cyclosynchrotron LURE-ACO at Orsay (France). 

Recent Progress in the Fluorescence Anisotropy Decay Technique.105... 

Use of Fluorescence Anisotropy Decay for Studying the OACF of Polymers. . . 110... 

In the case of dilute solutions of polymers, numerous studies using spectroscopic methods able to probe motions at a molecular level, such as NMR, ESR or Fluorescence Anisotropy Decay (FAD) revealed some molecular aspects of the dynamics, in particular the influence of chain connectivity on local processes This knowledge was recently improved thanks to the application of Synchrotron Radiation to FAD This technique provides a quantitative tool for discussing the numerous theoretical models proposed to account for single chain polymer dynamics, as shown in the following. 

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. 

Fig. 1. Block diagram of the Fluorescence Anisotropy Decay experiment...

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