Fluorescence depolarization technique

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... 

The fluorescence depolarization technique for mobility and ordering is based on the fact that the probability of absorption and emission is directional. Light polarized along a certain axis will preferably excite molecules oriented with their transition dipole moment in the same direction. The probability varies with cos 0, where 0 is the angle between the transition dipole moment and the electric field vector of the light. Emission of a photon obeys the same cos 0 (28) rule. That means that a molecule oriented with its transition dipole moment along the Z-axis will be likely to emit a photon with the same polarization. In the depolarization technique, polarizers are used to quantify the intensity of the parallel (ly) and perpendicular (Ij.) components to the original direction of polarization. 

The use of both singlet and triplet states as molecular reporters permits access to an extremely wide range of effective test frequencies. Fluorescence depolarization techniques have been employed extensively to study the relaxation behaviour of macromolecules in solution (cf. references (1 - 3) and references therein). Such anisotropy measurements have also been used, to a much lesser extent, to study high frequency relaxation processes in the polymeric... 

Earlier experiments have shown the utility of excitation transport measurements in providing relative information regarding coll size in pol)rmer blends (18). Here, we will summarize the results of recent experiments (28) which demonstrate that monitoring excitation transport on isolated colls in solid blends through time-resolved fluorescence depolarization techniques provides a quantitative measure of for the guest pol3mier. 

The effect of added alcohols upon the microviscosity of micelles was studied by Turro et al. [76]. The low-molecular alcohols reduce the microviscosity of CTAB micelles. The high-molecular alcohols do not influence the microviscosity. Schinitzky et al. observed a phase transition of a bilayer membrane by the fluorescence depolarization technique [75]. 

The fluorescence decay times of excited states are such that the fluorescence depolarization technique may only be used to examine relatively high frequency relaxation processes of polymers. Consequently fluorescence depolarization has been primarily limited to the study of relaxation processes of polymers in solution. The anisotropy of a system, r t), is derived from measurements of the fluorescence decays with polarizations parallel and perpendicular to the polarization of excitation ... 

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). 

Physico-chemical properties. In the fifties and sixties, several studies on the conformation of ACTH in solution were carried out. Among the used techniques were ORD, CD, fluorescence depolarization studies and kinetics of deuterium hydrogen exchange (for a review see ref. 2). The results pointed to a highly flexible random coil in solution however, Eisinger (40) found that the distance between Tyr and Trp [in ACTH-(1-24)] as measured by excitation spectroscopy, was in better agreement with some form of loop or helical structure. In addition. Squire and Bewley noted 11-15% helical content, located in the N-terminal 1-11 part of the molecule, when measuring the ORD of ACTH at pH 8.1 (41) (a random coil was found at neutral and acidic pH values, 2). 

The nmr data for this type of motion are direct and the motion clearly involves rotation about bonds in the millisecond time scale range. However. less direct evidence for motion comes from other techniques such as fluorescence depolarization, 02 diffusion, hydrogen exchange kinetics, and nmr relaxation times (see Ref. 4). The extent of this motion is not yet easy to define, but this evidence points to motion in the nanosecond time scale range. It is tempting to see the motion in this time scale as bond oscillations rather than rotations. To put it in a different way, on this time scale the side chains have some freedom to move with respect to each other but not normally to undergo substantial bond rotation. Table IV summarizes some references for motion of different types. Additionally, nmr relaxation studies suggest that the backbone or main chain of a protein is more restricted than that of the side chains. 

Secondly, the technique of fluorescent depolarization which was introduced into protein chemistry by Weber in 1952... 

The technique of fluorescence depolarization uses in principle fluorescent dyes covalently bound to proteins. The label is excited by polarized light the fluorescence is also polarized to a degree that is inversely related to the amount of Brownian motion occurring during the interval between absorption and emission of the light. The information about the amount of motion helps to form an idea on the size and shape of the protein being investigated. 

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 plane-polarized light pulses characteristic of mode-locked lasers also provide an ideal excitation source for time-dependent fluorescence depolarization studies although conventional excitation sources can be used. If the rotational relaxation time of the excited molecule is comparable to its fluorescence decay time, then the vertical (I ) and horizontal (Ix) components of the fluorescence decay observed through suitable polarizers following excitation by polarized li t pulses, may be analysed to provide information concerning the size and motion of die molecule and Sect. 5. However, if only the true fluorescence decay characteristics are of interest it is necessary to compensate for these emission anisotropy effects Perhaps the simplest technique is to analyse only that component of fluorescence emitted at 54.7° to the direction of pdarization of the excitation source, the so-called magic-angle ... 

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. 

The flexibility was more restricted in the nurse shark IgM. The above results, together with a steady-state fluorescence depolarization study on the flexibility of immunoglobulins from amphibia and reptiles has suggested that the degree of flexibility decreases with the level of phytogeny. Thus, time-resolved fluorescence depolarization measurements may provide one of the first techniques capable of indicating... 

In the current paper we extend previous work on poly(methyl acrylate) (9 - 10) and poly(methyl methacrylate) (9 - 11) in which the phosphorescence depolarization technique was shown to provide data which were consistent with reported dielectric and mechanical relaxation experiments, to the study of the molecular behaviour of poly(n-butyl methacrylate). This polymer, whilst of technological application, has received much lesser attention using conventional dynamic relaxation techniques than has been devoted to PMA and PMMA. In addition, fluorescence depolarization measurements have been employed in an attempt to provide complementary information regarding the higher frequency behaviour of the polymer. 

These experiments, in conjunction with the theory described here, demonstrate the utility of excitation transport induced fluorescence depolarization in the study of pol3rmer blends. The technique allows quantitative determination of for... 

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