Fluorescence anisotropy function

Figure 10. The raw experimental fluorescence anisotropy function r(f) derived from the parallel and the perpendicular (with respect to the pump polarization) fluorescence signals from M2192 membrane, a mutant LH1 antenna system devoid of peripheral antenna (LH2) and reaction centers. The membrane is excited at 860 nm and detected at 943 nm. (From Ref. 18.)...

Fluorescence Polarization Studies of PMA and PAA. Time-resolved fluorescence polarization measurements are potentially a powerful means for studying molecular mobility. The fluorescence anisotropy function r(t) may be generated by monitoring the decay of vertically (Iv(t)) and horizontally (Ijj(t)) polarized components of emission following excitation by vertically polarized light pulses (Equation 1). 

For this case the fluorescence anisotropy function r t) is defined by... 

The time-resolved fluorescence anisotropy function, rif), is calculated using Equation 1 in which / (0 and IJJ) are the individual decays collected with the polarization analyzer set parallel and perpendicular to the vertically polarized excitation light. The G factor is included to account for any polarization bias of the detection system. The influence of this term was minimized by arranging the polarization analyzer to be the first element in the detection system and using a polarization pseudo-scrambler (Oriel 28115) immediately prior to the emission monochromator slit. 

Viovy,J.L. and Monnerie, L. Fluorescence Anisotropy Technique Using Synchroton Radiation as a Powerful Means for Studying the Orientation Correlation Functions of Polymer Chains. Vol. 67, pp. 99—122. 

Figure 4.6 shows an apparatus for the fluorescence depolarization measurement. The linearly polarized excitation pulse from a mode-locked Ti-Sapphire laser illuminated a polymer brush sample through a microscope objective. The fluorescence from a specimen was collected by the same objective and input to a polarizing beam splitter to detect 7 and I by photomultipliers (PMTs). The photon signal from the PMT was fed to a time-correlated single photon counting electronics to obtain the time profiles of 7 and I simultaneously. The experimental data of the fluorescence anisotropy was fitted to a double exponential function. 

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.

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. 

Another important linear parameter is the excitation anisotropy function, which is used to determine the spectral positions of the optical transitions and the relative orientation of the transition dipole moments. These measurements can be provided in most commercially available spectrofluorometers and require the use of viscous solvents and low concentrations (cM 1 pM) to avoid depolarization of the fluorescence due to molecular reorientations and reabsorption. The anisotropy value for a given excitation wavelength 1 can be calculated as... 

The two cationic dyes, Py+ as a donor and Ox" as an acceptor, were found to be very versatile for demonstrating photonic antenna functionalities for light harvesting, transport, and capturing, as illustrated in Fig. 7. They can be incorporated into zeolite L by means of ion exchange, where they are present as monomers because of the restricted space. In this form they have a high fluorescence quantum yield and favourable spectral properties. The insertion of the dyes can be visualised by means of fluorescence microscopy. The fluorescence anisotropy of Ox -loaded zeolite L has recently been investigated in detail by conventional and by confocal microscopy techniques [15],... 

Sample preparation was given elsewhere [2]. Femtosecond fluorescence upconversion and picosecond time-correlated single-photon-counting set-ups were employed for the measurement of the fluorescence transients. The system response (FWHM) of the femtosecond fluorescence up-conversion and time-correlated single-photon-counting setups are 280 fs and 16 ps, respectively [3] The measured transients were fitted to multiexponential functions convoluted with the system response function. After deconvolution the time resolution was 100 fs. In the upconversion experiments, excitation was at 350 nm, the transients were measured from 420 nm upto 680 nm. Experiments were performed under magic angle conditions (to remove the fluorescence intensity effects of rotational motions of the probed molecules), as well as under polarization conditions in order to obtain the time evolution of the fluorescence anisotropy. 

Steady-state measurements of the fluorescence anisotropy of fluorescein derivatives form the basis of a sensitive analytical technique also used to detect and quantitate proteins [36], steroids [37-39], therapeutic drugs, and narcotics [40-42], In a different approach, the anisotropy of the fluorescein conjugate is measured as a function of the medium viscosity to determine the segmental mobility of the chains that cover the binding site [43-45],... 

In a more detailed study Caturla et al. [112] investigated the interaction of four catechins with PC and PE model membranes by fluorescence spectroscopy, microcalorimetry, and infrared spectroscopy. DPH fluorescence anisotropy was monitored as a function of temperature and it was observed that catechins either did not change or increased DPH anisotropy. The membrane rigidifying effect of (-)-epicatechin (EC) (37b) was the most... 

Figure 4. Ultrafast hydration correlation function c(t) of tryptophan. The c(t) can be fit with a stretched biexponential model as shown. The inset shows the fluorescence anisotropy dynamics of tryptophan after ultrafast ultraviolet (UV) absorption. The internal conversion between La and Lb states occurs within 80 fs. The free rotation time of tryptophan in bulk water is 46 ps.

The inset shows the correlation functions in the short time range. The hydration correlation function of tryptophan in bulk water is also shown (dashed line) for comparison, (b) fs-resolved fluorescence anisotropy dynamics of Trp probes in the aqueous channels of the cubic phase. For clarity, the anisotropy of TME is not shown. The relaxation of Trp in bulk water (46 ps) is also shown (dashed line) for comparison. 

Figure 33. Hydration correlation functions c(t) probed by W31 in hTrx and two mutants in reduced states. The three functions are strikingly similar, indicating a similar local solvent environment upon mutation. The inset shows the fluorescence anisotropy dynamics of W31. The nearly constant r(t) reflects a very rigid local structure.

Equation (11.9) indicates the possibility of calculating the rotational correlation time of the fluorophore not only by varying the T/q ratio but also by adding a collisional quencher. Interaction between the quencher and fluorophore decreases the fluorescence lifetime and intensity of the fluorophore, and increases its fluorescence anisotropy. Plotting 1 /A as a function of r0 yields a straight line with a slope equal to r. If the fluorophore is tightly bound to the macromolecule and does not exhibit any residual motions, the measured 0r is equal to, and the extrapolated anisotropy is equal to that measured at a low temperature. 

In the case of a water/DCE interface [(c) and (d)], on the other hand, a fitting of the data by a single-exponential function cannot be attained by setting an emission polarizer at 45°, as confirmed by deviations of Re and Cr from the optimum values (c). When the fluorescence decay profile is measured by setting an emission polarizer at 54.7° (d), fluorescence anisotropy can be reasonably fitted by a single-exponential function including the time response in the initial stage of excitation (see also and... 

To the best of our knowledge, this is the first time in which the formation of a metal ion complex in solution is followed by fluorescence anisotropy. Indeed, this has been made possible in low-viscosity solvents because of the profound change of mass and dimension between the [ZnC] complex and the [5ZnC] complex. In the self-assembled structme [5ZnC], the clip may function as a ditopic... 

Azurin has a single buried tryptophan. Fluorescence anisotropy has been measured as a function of hydration level for azurin incorporated in a polymer film (Careri and Gratton, 1986). In the wet film the value of the anisotropy is close to that for azurin in solution at high temperature and low viscosity. At low hydrations and in the dry film, motion of the tryptophan chromophore is frozen. 

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