Time-resolved fluorescence depolarization techniques

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. 

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... 

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. ... 

Although the relation between fluorescence depolarisation and rotational Brownian motion was first identified by Perrin and the development of the theoretical background of the time-resolved fluorescence depolarization experiments was made by Jablonski use of the technique was limited until the advent of improved fluorescence decay time measurements some fifteen years i. An alternative, related technique, involving excitation using a continuous polarised light source, provides only the time average of the correlation function (Eq. 18) and as such, is less useful than the time resolved method. Other disadvantages are that the natural decay time of the chromophore must be determined from a sqrarate experiment and it is necessary to alter the viscosity, and/or temperature of the medium, often withun-... 

Time dependent fluorescence depolarization is influenced by the exciton annihilation which occurs in confined molecular domains . Photoemission results from singlet exciton fusion as shown by the excitation intensity dependence which occurs in anthracene crystals. Reabsorption of excitonic luminescence is an effect which has been shown to occur in pyrene crystals. The dynamics of exciton trapping in p-methylnaphthalene doped naphthalene crystals involves phonon assisted detrapping of electronic energy. Ps time resolved spectroscopy was the experimental technique used in this work. 

Thus, as mentioned earlier, time-resolved depolarization measurements afford a means of recording the time profile of the rotational autocorrelation function. The steady state technique, with continuous sample excitation, produces merely the time average of the emission anisotropy, F. For a rotating chromc hore with a sin e fluorescence decay time Tf, F is related to r(t) by the following expression... 

A useful and common way of describing the reorientation dynamics of molecules in the condensed phase is to use single molecule reorientation correlation functions. These will be described later when we discuss solute molecular reorientational dynamics. Indirect experimental probes of the reorientation dynamics of molecules in neat bulk liquids include techniques such as IR, Raman, and NMR spectroscopy. More direct probes involve a variety of time-resolved methods such as dielectric relaxation, time-resolved absorption and emission spectroscopy, and the optical Kerr effect. The basic idea of time-resolved spectroscopic techniques is that a short polarized laser pulse removes a subset of molecular orientations from the equifibrium orientational distribution. The relaxation of the perturbed distribution is monitored by the absorption of a second time-delayed pulse or by the time-dependent change in the fluorescence depolarization. 

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