Steady-state anisotropy

The measurement of steady-state anisotropy r is simple and needs two polarizers, one in excitation and the other in emission beams. When the sample is excited... 

Summarizing, we stress that the anisotropy and the fluorescence decay functions change in a complex way as a function of target concentration. Species that fluoresce more intensely contribute disproportionably stronger to the measured parameters. Simultaneous measurements of steady-state intensities allow accounting this effect. 

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] ... 

High or low anisotropy values, which are more desirable A low steady-state anisotropy of the donor is generally attributed to rapid... 

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... 

On continuous illumination (i.e. when the incident light intensity is constant), the measured anisotropy is called steady-state anisotropy r. Using the general definition of an averaged quantity, with the total normalized fluorescence intensity as the probability law, we obtain... 

In the case of a single exponential decay with time constant % (excited-state lifetime), the steady-state anisotropy is given by... 

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 ... 

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) ... 

Steady-state anisotropy measurements are then insufficient for fully characterizing rotational motions and time-resolved experiments are required. 

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

Fig. 6.4. Schematic diagram showing how the four intensity components are measured for determination of the steady-state anisotropy.

At low frequency, r(co) tends towards the steady-state anisotropy, and at high frequency r(co) approaches ro, the emission anisotropy in the absence of rotational motions. 

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... 

For isotropic motions in an isotropic medium, the values of the instantaneous and steady-state emission anisotropies are linked to the rotational diffusion coefficient Dr by the following relations (see Chapter 5) ... 

From a practical point of view, the steady-state technique (continuous illumination) is far simpler than the time-resolved technique, but it can only be used in the case of isotropic rotations in isotropic media (Eqs 8.26 and 8.28) provided that the probe lifetime is known. Attention should be paid to the fact that the variations in steady-state anisotropy resulting from an external perturbation (e.g. temperature) may not be due only to changes in rotational rate, because this perturbation may also affect the lifetime. 

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

As expected from the anisotropy of chemical etching of Si in alkaline solutions, the electrochemical dissolution reaction shows a strong dependence on crystal orientation. For all crystal orientations except (111) a sweep rate independent anodic steady-state current density is observed for potentials below PP. For (111) silicon electrodes the passivation peak becomes sweep rate dependent and corresponds to a constant charge of 2.4 0.5 mCcm-2 [Sm6]. OCP and PP show a slight shift to more anodic potentials for (111) silicon if compared to (100) substrates, as shown in Fig. 3.4. 

In this section we will review the application of near-IR system instrumentation to the most commonly encountered fluorescence measurements such as steady-state spectra, excited state lifetimes, anisotropy, microscopy, multiplexing, high-performance liquid chromatography (HPLC), and sensors. 

The physical dimensions and dynamics of calmodulin have also been investigated by tyrosine fluorescence. To learn about the internal mobility of calmodulin, Lambooy et al 1 and Steiner et al measured the steady-state fluorescence anisotropy of the tyrosine. Since the average correlation... 

The mobility of tyrosine in Leu3 enkephalin was examined by Lakowicz and Maliwal/17 ) who used oxygen quenching to measure lifetime-resolved steady-state anisotropies of a series of tyrosine-containing peptides. They measured a phase lifetime of 1.4 ns (30-MHz modulation frequency) without quenching, and they obtained apparent rotational correlation times of 0.18 ns and 0.33 ns, for Tyr1 and the peptide. Their data analysis assumed a simple model in which the decays of the anisotropy due to the overall motion of the peptide and the independent motion of the aromatic residue are single exponentials and these motions are independent of each other. 

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. 

Of considerable interest is the fact that not only the steady-state anisotropy but also its kinetics depend on the excitation wavelength. In this case another red-edge effect connected with site photoselection may be observed. Dipole-orientational relaxation may occur not only by rotation of the dipoles surrounding the fluorophore but also by rotation of the aromatic group itself. If red-edge excitation results in the photoselection of fluorophores whose energy of interaction with the environment already corresponds to that in the excited state, then the relaxation-associated rotation should not be observed and the rotation that occurs should be completely Brownian in character.(22)... 

G. B. Strambini and W. C. Galley, Detection of slow rotational motions of proteins by steady-state phosphorescence anisotropy, Nature 260, 554-555 (1976). 

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