Steady State and Time Resolved Fluorescence Anisotropy

3 Steady State and Time Resolved Fluorescence Anisotropy 6.3.1 Introduction 

Fluorescence anisotropy (FA) is a very versatile technique which can be exploited to investigate phenomena of different nature indeed any kind of process that 

The final aim of this section about FA is to discuss the basis of this technique and to give to the reader the basic knowledge of the procedures to be used for spectra and decay recording. A more detailed description of the most sophisticated aspects of FA can be found elsewhere [45]. 

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M. Vincent, J. Gallay, J. de Bony, and J.-F. Tocanne, Steady-state and time-resolved fluorescence anisotropy study of phospholipid molecular motion in the gel phase using l-palmitoyl-2-[9-(2-anthryl)-nonanoyl]-sn-glycero-3-phosphocholine as probe, Ear, J. Biochem. 250, 341-347 (1985). 

Fluorescence anisotropy studies are popular in biological and biochemical research of lipid membranes [16-18], proteins [19-22], etc. and also in polymer science. They have been performed for monitoring the conformations and flexibility of polymer chains in dilute, semidilute and concentrated solutions [23-27], in polymer melts and blends [28-31], and also for studying polymer self-assembly [32-34]. Nowadays, steady-state and time-resolved fluorescence anisotropy are currently used methods in polymer chemistry. 

A detailed evaluation of the structural parameters affecting the photophysical properties was performed for the didodecyloxy-substituted quinquephenyl rigid-flexible polyethers 60. More particularly, the odd-even effect was observed for dilute polymeric solutions by means of steady-state and time-resolved fluorescence anisotropy. The different orientations of the quinquephenyl chromophores, and subsequently of their luminescent dipoles, concerning the polymers with an odd number of methylene units x=7, 9, 11) and those with an even (x = 8, 10, 12) one, were found responsible for the observed strong deviations in frozen and dilute solutions, where the flexible aliphatic chains are forced to adopt a nearly sta ered, lower energy conformation. 

Abstract In the first part of this chapter we will illustrate circular dichroism and we will discuss the optical activity of chemical compounds with respect to light absorption which is at the basis of this technique. Moreover, we will introduce the phenomena that lie behind the technique of optical rotatory dispersion. We thought appropriate to include a brief description of linear dichroism spectroscopy, although this technique has nothing to do with optical activity. In the final part of the chapter we will introduce the basic principles of the luminescence teehniques based on polarized (either circularly or linearly) excitation. The experimental approach to the determination of steady-state and time resolved fluorescence anisotropy will be illustrated. For all the teehniques examined in this chapter the required instrumentation will be schematieally deseribed. A few examples of application of these techniques to molecular and supramolecular systems will also be presented. 

The detection limit of fluorescence techniques is strongly dependent on the efflciency of the fluorescence equipment. The type of information provided by these techniques depends on the fluorescence mode used - either steady-state (steady-state fluorescence) or time-resolved (time-resolved fluorescence) - and also whether the excitation is performed with natural or polarized light (steady-state or time-resolved fluorescence anisotropy) [3],... 

In particular, steady-state and time-resolved fluorescence as well anisotropy are powerful tools for studying function and conformation of sensitizer in vesicular membrane systems, in order to provide structural information on sensitizer-colloid binding or to evaluate protein-ligand interactions. 

Situation with H-bonding also demands to take into account the fact that alcohols have ability to form various associates or even clusters at normal conditions. The most efficient method for determination of inhomogeneity in the excited states is fluorescence polarization measurements. These methods also frequently applied for studying of solvent viscosity, they may be provided in two variants steady state and time-resolved. Relations for time-resolved and steady state fluorescence anisotropy may be given as [1, 2, 75] ... 

Thus, the time-resolved measurement of such membrane probes contains information on the dynamics of the hindered probe rotation, often interpreted as the micro-viscosity, and about the hindrance of this rotation, usually interpreted as the static packing arrangement of the lipids or the so-called membrane order [136, 137]. Fluorescence polarisation studies in membranes, however, exhibit some major limitations the experimentally determined steady-state and time-re-solved anisotropies characterize the motional restrictions of the reporter molecule itself and give therefore only indirect information about the dye environment, with the consequence that, if the probe is bound covalently to the lipid (TMA-DPH), this attachment may dominate the recorded depolarisation behaviour. The membrane order parameters obtained from freely mobile probes like (DPH) result from a broad distribution of localisation within the hydrophobic interior, the detailed characterisation of which reveals inherent ambiguities [138]. 

The first decision to be made in designing an experiment to measure the motional properties of membrane lipids concerns the type of probe molecule. Too often, this choice is made from the point of view of convenience or tradition rather than suitability, although there is now a considerable range of suitable fluorophores from which to choose. The second consideration is the type of measurement to be made. The most detailed and complete motional information is obtained from a time-resolved fluorescence anisotropy measurement which is able to separate the structural or orientational aspects from the dynamic aspects of fluorophore motion. Steady-state anisotropy measurements, which are much easier to perform, provide a more limited physical parameter relating to both of these aspects. 

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. 

The appeal of fluorescence spectroscopy in the study of biomolecular systems lies in the characteristic time scale of the emission process, the sensitivity of the technique, and its ability to accommodate rapid and facile changes in the solvent milieu under conditions corresponding to thermodynamic equilibrium. The time scale of the emission process invites exploitation in two related manners. First, information on hydrodynamic aspects of the system is available from steady-state or time-resolved measurements. Second, detailed information on local dynamic processes within the biomolecular matrix may be derived. Information on hydrodynamic aspects of a macromolecular system may be used to study binding processes, that is, the association of small ligands with macromolecules or macromolecule-macromolecule interactions. In this chapter we focus on the latter applications of polarization or anisotropy data. We shall also try to clarify aspects of this area that our experience has shown to be occasionally misunderstood by initiates. 

This chapter introduces and discusses different fluorescence techniques that do not require the use of external labeling steady-state fluorescence (including 2D fluorescence), fluorescence anisotropy and time-resolved fluorescence and it provides illustrative examples showing how these techniques may be used for the monitoring of membrane processes. 

Van Blitterswijk et al. [6] used time-resolved fluorescence anisotropy to measure the infinite-time anisotropy (r ) (refer to Fig. 4) and the limiting anisotropy (ro) for a series of biological and artificial membranes, plotted the infinitetime anisotropy (r ) versus the measured steady-state anisotropy (r ), and obtained a linear relationship for the probe 1,6-diphenylhexatriene (DPH) ... 

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... 

Dr can be determined by time-resolved fluorescence polarization measurements, either by pulse fluorometry from the recorded decays of the polarized components I l and 11, or by phase fluorometry from the variations in the phase shift between J and I as a function of frequency (see Chapter 6). If the excited-state lifetime is unique and determined separately, steady-state anisotropy measurements allow us to determine Dr from the following equation, which results from Eqs (5.10) and (5.41) ... 

Keywords Steady-state fluorescence spectra Time-resolved fluorescence decays Fluorescence anisotropy Huorescence quenching Nonradiative excitation energy transfer Solvent relaxation Excimer J and H aggregates... 

The steady-state anisotropy can be related to the molecular motion by time-resolved fluorescence analysis, both reported in Fig. 4 and in Table 2. 

Since steady-state data are much easier to obtain, some effort has been directed to methods for deriving time-resolved anisotropy parameters from the steady-state anisotropy/2 4549-1 A number of relationships have been described, some of which require knowledge of r0 and the fluorescence lifetime (see, e.g., Ref. 48). An example(50) of such an empirical relationship is... 

In the first section, steady-state spectroscopy is used to determine the stoichiometry and association constants of molecular ensembles, emphasize the changes due to light irradiation and provide information on the existence of photoinduced processes. Investigation of the dynamics of photoinduced processes, i.e. the determination of the rate constants for these processes, is best done with time-resolved techniques aiming at determining the temporal evolution of absorbance or fluorescence intensity (or anisotropy). The principles of these techniques (pulse fluorometry, phase-modulation fluorometry, transient absorption spectroscopy) will be described, and in each case pertinent examples of applications in the flelds of supramolecular photophysics and photochemistry will be presented. 

The great sensitivity of fluorescence spectral, intensity, decay and anisotropy measurements has led to their widespread use in synthetic polymer systems, where interpretations of results are based upon order, molecular motion, and electronic energy migration (1). Time-resolved methods down to picosecond time-resolution using a variety of detection methods but principally that of time-correlated single photon counting, can in principle, probe these processes in much finer detail than steady-state techniques, but the complexity of most synthetic polymers poses severe problems in interpretation of results. 

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