Emission anisotropy measurement

Ultrafast emission measurements are possible with the dendrimer metal nanocomposites. The gold and silver internal dendrimer nanocomposites showed a fast emission decay of approximately 0.5 ps, which was followed by a slower decay process. The fast decay emission is attributed to decay processes of the gold (or silver) metal nanoparticles. Ultrafast emission anisotropy measure-... 

Quantitative information can be obtained only if the time-scale of rotational motions is of the order of the excited-state lifetime r. In fact, if the motions are slow with respect to r(r ro) or rapid (r 0), no information on motions can be obtained from emission anisotropy measurements because these motions occur out of the experimental time window. 

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

In Chapter 5, devoted to fluorescence polarization, it was shown that information on the rotational motions of a fluorophore can be obtained from emission anisotropy measurements. Application to the evaluation of the fluidity of a medium, or molecular mobility, is presented below. 

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

Partial orientation is much easier to achieve and generally sufficient to determine the direction of the transition moments, particularly with respect to any axes of symmetry that the molecules may have. Partial orientation can be achieved by photoselection using a linearly polarized excitation source, by application of an electric field or by dissolving the solute in a transparent anisotropic medium (liquid crystals, stretched polymers).182 Photoselection is the basis of emission anisotropy measurements discussed below. It is also used for molecules that can be either generated or destroyed photochemically in a rigid medium such as poly(methyl methacrylate) or glassy solvents at low temperature. Preferential alignments of dipolar molecules that are achievable by electrostatic fields are unfortunately fairly small. 

Definition and Uses of Standards. In the context of this paper, the term "standard" denotes a well-characterized material for which a physical parameter or concentration of chemical constituent has been determined with a known precision and accuracy. These standards can be used to check or determine (a) instrumental parameters such as wavelength accuracy, detection-system spectral responsivity, and stability (b) the instrument response to specific fluorescent species and (c) the accuracy of measurements made by specific Instruments or measurement procedures (assess whether the analytical measurement process is in statistical control and whether it exhibits bias). Once the luminescence instrumentation has been calibrated, it can be used to measure the luminescence characteristics of chemical systems, including corrected excitation and emission spectra, quantum yields, decay times, emission anisotropies, energy transfer, and, with appropriate standards, the concentrations of chemical constituents in complex S2unples. 

The chromophore environment can affect the spectral position of the absorption and emission bands, the absorption and emission intensity (eM, r), and the fluorescence lifetime as well as the emission anisotropy, e.g., in the case of rigid matrices or hydrogen bonding. Changes in temperature typically result only in small spectral shifts, yet in considerable changes in the fluorescence quantum yield and lifetime. This sensitivity can be favorably exploited for the design of fluorescent sensors and probes [24, 51], though it can unfortunately also hamper quantification from simple measurements of fluorescence intensity [116], The latter can be, e.g., circumvented by ratiometric measurements [24, 115],... 

When the incident light is horizontally polarized, the horizontal Ox axis is an axis of symmetry for the fluorescence intensity Iy = Iz. The fluorescence observed in the direction of this axis (i.e. at 90° in a horizontal plane) should thus be unpolarized (Figure 5.3). This configuration is of practical interest in checking the possible residual polarization due to imperfect optical tuning. When a monochromator is used for observation, the polarization observed is due to the dependence of its transmission efficiency on the polarization of light. Then, measurement of the polarization with a horizontally polarized incident beam permits correction to get the true emission anisotropy (see Section 6.1.6). 

This equation shows that, at time t, each anisotropy term is weighted by a factor that depends on the relative contribution to the total fluorescence intensity at that time. This is surprising at first sight, but simply results from the definition used for the emission anisotropy, which is based on the practical measurement of the overall ly and I components. A noticeable consequence is that the emission anisotropy of a mixture may not decay monotonously, depending of the values of r, and Ti for each species. Thus, r(t) should be viewed as an apparent or a technical anisotropy because it does not reflect the overall orientation relaxation after photoselection, as in the case of a single population of fluorophores. 

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 ... 

It should first be noted that the measurement of emission anisotropy is difficult, and instrumental artefacts such as large cone angles of the incident and/or observation beams, imperfect or misaligned polarizers, re-absorption of fluorescence, optical rotation, birefringence, etc., might be partly responsible for the difference between the fundamental and limiting anisotropies. 

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. 

Measurement of steady-state emission anisotropy. Polarization spectra... 

The case of several populations of fluorophores having their own fluorescence decay i (t) and time constants characterizing r (t) deserves particular attention. In Section 5.3, it was concluded that an apparent or a technical emission anisotropy r(t) can be obtained by considering that the measured polarized components, I(t) and I (t), are the sums of the individual components (i.e. of each population) and by using Eq. (6.43). Hence... 

It is worth pointing out that many artifacts can alter the measurements of emission anisotropy. It is necessary to control the instrument with a scattering non-fluorescent solution (r close to 1) and with a solution of a fluorophore with a long lifetime in a solvent of low viscosity (r x 0). It is also recommended that the probe concentration is kept low enough to avoid interaction between probes. 

Fluorescence polarization is the subject of Chapter 5. Factors affecting the polarization of fluorescence are described and it is shown how the measurement of emission anisotropy can provide information on fluidity and order parameters. 

Additionally, since the acceptor is excited as a result of FRET, those acceptors that are fluorescent will emit photons (proportional to their quantum efficiency) also when FRET occurs. This is called sensitized emission and can also be a good measure of FRET (see Fig. 1). To quantitate FRET efficiency in practice, several approaches have been evolved so far. In flow cytometric FRET (7), we can obtain cell-averaged statistics for large cell populations, while the subcellular details can be investigated with various microscopic approaches. Jares-Erijman and Jovin have classified 22 different approaches that can be used to quantify energy transfer (8). Most of them are based on donor quenching and/or acceptor sensitization, and a few on measuring emission anisotropy of either the donor or the acceptor. Some of these methods can be combined to extend the information content of the measurement, for example two-sided FRET (9) involves both acceptor depletion (10) and... 

This problem does not exist with time-dependent fluorescence polarization measurements where the decay of the emission anisotropy r(t) is obtained by determining the decay of Iz and Ix according to eq 12. 

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... 

There are many uncertainties in this procedure, and it seems clear that direct measurement of the time dependence of emission anisotropy throu l (t) and Ix(t) is preferable. Such measurements are described in a later section. 

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