Fluorescence depolarization method

The fluorescence depolarization technique excites a fluorescent dye by linearly polarized light and measures the polarization anisotropy of the fluorescence emission. The fluorescence anisotropy, r, is defined as 

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Aoki, H., Kitamura, M. and Ito, S. (2008) Nanosecond dynamics of poly(methyl methacrylate) brushes in solvents studied by fluorescence depolarization method. Macromolecules, 41, 285-287. 

Horinaka, J., Ono, K. and Yamamoto, M. (1995) Local chain dynamics of syndiotactic poly(methyl methacrylate) studied by the fluorescence depolarization method. Polym. J., 27, 429-435. 

An important advantage of the depolarization technique is that it allows one to measure the molecular ordering, as well as the motional parameters. For this purpose, it is necessary to detect the time dependence of the anisotropy. In the presence of ordering constraints, the r value does not decay to zero, but to some limiting value foo r = (ro — roo)e / c - - poo. The rate of decay defines a rotational correlation time, and Poo is a direct measure of the order parameter through the following relation s = Poo/ o (29). The fluorescence depolarization method works well as long as fluorescence lifetimes, which are typically 10 s, are not too different from the rotation relaxation times to be measured. When the rotational correlation time... 

The measurement of the time-dependent depolarization of the fluorescence from molecules rotating on a time-scale comparable to the fluorescence decay time, enables information to be derived concerning the molecular reorientation motion. A review of these techniques has been published. A method involving an optical delay line has been used to record time-resolved fluorescence depolarization methods using only 1 photodetector, and thus some of the possible instrumental distortions are removed. ... 

T. Sasaki, M. Yamamoto, and Y. Nishijima. Chain dynamics of poly(methyl methacrylate) in dilute solutions studied by the fluorescence depolarization method. Macromolecules, 21 (1988), 610-616. 

J-i. Horinaka, S. Amano, H. Funada, S. Ito, and M. Yamamoto. Local chain dynamics of poly(oxyethylene) studied by the fluorescence depolarization method. Macromolecules, 31 (1998), 1197-1201. 

PDS, and 6-PDS using the time-resolved fluorescence depolarization method involving two dissimilar probes, 2,5-dimethyl-l,4-dioxo-3,6-diphenylpyr-rolo[3,4-c]pyrrole (DMDPP) and coumarin 6 (C6). The decay of anisotropy for both probes in all the six micelles has been rationalized on the basis of a two-step model consisting of fast-restricted rotation of the probe and slow lateral diffusion of the probe in the micelle that are coupled to the overall rotation of the micelle. On the basis of the assumption that the fast and slow motions are separable, the experimentally obtained slow and fast reorientation times and Xfajt) are related to the time constants for lateral diffusion (xj, wobbling motion (Xw), and rotation of the micelle as a whole (Xm) by the following relationships ... 

This chapter presents new information about the physical properties of humic acid fractions from the Okefenokee Swamp, Georgia. Specialized techniques of fluorescence depolarization spectroscopy and phase-shift fluorometry allow the nondestructive determination of molar volume and shape in aqueous solutions. The techniques also provide sufficient data to make a reliable estimate of the number of different fluorophores in the molecule their respective excitation and emission spectra, and their phase-resolved emission spectra. These measurements are possible even in instances where two fluorophores have nearly identical emission specta. The general theoretical background of each method is presented first, followed by the specific results of our measurements. Parts of the theoretical treatment of depolarization and phase-shift fluorometry given here are more fully expanded upon in (5,9-ll). Recent work and reviews of these techniques are given by Warner and McGown (72). 

In the present context, fluorescence is an important tool for the rapid comparison of a protein with a standard sample of the recombinant or the authentic protein, as part of the process of characterizing and authenticating a recombinant protein. Coupled with measurements of the depolarization of fluorescence, the method can provide information on the dynamics of a protein and on the change in dynamics in response to, for example, binding of ligands. 

The theory of rotation effects on prolate luminescent molecules in solution and its experimental verification have been developed and compared. Generalized diffusion equations for the rotational motion of an asymmetric rigid motor have been used to given an expression for steady-state fluorescence depolarization. " The radiationless transition from the first excited singlet state of Eosin has been measured by optoacoustic relaxation, and the absolute fluorescence quantum yields of organic dyes in poly(vinyl alcohol) have also been measured by the photoacoustic method. The accuracy of the method has been discussed in the latter paper. Actinometry in flash photolysis experiments has been assisted by new measurements on the extinction coefficient of triplet benzophenone. Matrix-isolation fluorescence spectrometry has been used to detect polycyclic aromatic hydrocarbons from gas chromatography. ... 

Time-resolved fluorescence depolarization studies have, over the past decade, provided an interesting method for monitoring molecular reorientational motions in solution. The technique has been applied to investigations of both nthetic polymers and macromolecules of biological interest, and a selection of the results of these are discussed here. However, until recently, the relatively pc r quality of much of the data available from these measurements has precluded detafled quantitative interpretations of the results. With the advent of improved experimental techniques for fluorescence decay time determinations due in part to the availability of pulsed lasers for sample excitation and more accurate data analysis procedures, it is envisaged that interest in the technique may be revived. We will present here a short recapitulation of the background to these experiments, following from Sect. A. V. 

Steady-state depolarization measurements of the fluorescence of probe molecules dispersed in the hydrocarbon interior of a lipH bilayer have been used extensively as a probe of the lipid microviscosity. This applkation has been reviewed Two inherent assumptions in the method, however, cast some doubt on the results obtained. Firstly the probe molecule is assumed to be a rigid spherical body rotating isotropically, a situation unlikely considering the heterogeneous nature of the system. Secondly, it is amimed that the hydrocarbon interior can be modelled using homogeneous solvents. These problems can be lai ely overcome with the use of time-resolved fluorescence depolarization measurements. 

Many methods for detennination of molecular order can only measure P2) [Mcbl, Warl]. Among them are measurements of the infrared dichroism and of the refractive index. P2) and P4) can be determined by Raman fluorescence depolarization and from wideline NMR spectra. However, X-ray diffraction and lineshape analysis of resonances from isolated NMR interactions can also provide higher moments. 

This same type of analysis is useful in connection with other experimental methods for determining orientational correlation functions. As an example we consider fluorescence depolarization experiments. In a fluorescence experiment, the following steps are followed ... 

MicroBrownian dynamics of microemulsions can be studied by various techniques including dynamic-mechanical, dielectric, ultrasonic and NMR relaxation, ESR, volume, enthalpy and specific heat relaxation, quasielastic light and neutron scadering, fluorescence-depolarization experiments, and many other methods (90, 102-107). The information thus acquired provides an opportunity to clarify... 

In conclusion, the method of fluorescence depolarization measurements on particles in compressed gels can be performed on chlorosomes and they lead to results, which are in good agreement with the theoretical predictions from [1], and results fromt linear dichroism measurements [2]. The results support the further use of the expressions in... 

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