Time-resolved fluorescence depolarization

Fluorescence depolarization time courses contain information about rotational diffusion (26). The main difficulty with streak camera measurements of time-resolved fluorescence depolarization is limited signal-to-noise ratio of single shot records. The fluorescence anisotropy parameter is defined as s... 

Time Resolved Fluorescence Depolarization. In Equation 3, it is assumed that the polarization decays to zero as a single exponential function, which is equivalent to assuming that the molecular shape is spherical with isotropic rotational motion. Multiexponential decays arise from anisotropic rotational motion, which might indicate a nonspherical molecule, a molecule rotating in a nonuniform environment, a fluorophore bound to tbe molecule in a manner that binders its motion, or a mixture of fluorophores with different rotational rates. 

The present experiments are mute as to the timescale on which delocalization may occur. EPR results on Ru(bpy)"5 demonstrate localization of the bpy electron density in this Ru(II)(bpy)2 (bpy )+ species on the EPR timescale, but suggest that delocalization may occur on a timescale only slightly longer. It is possible that either time-resolved EPR or temperature dependent fluorescence depolarization experiments may establish the time-scale of localization in Ru(bpy) +. 

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... 

Figure 4.1. Time scales for rotational motions of long DNAs that contribute to the relaxation of the optical anisotropy r(t). Experimental methods used to study these motions in different time ranges are also indicated along with the authors and dates of some early work in each case. FPA, Fluorescence polarization anisotropy (Refs. 15, 18-20, and 87) TPD, transient photodichroism (Refs. 28 and 62) TEB, transient electric birefringence (Refs. 26 and 27) DDLS, depolarized dynamic light scattering (Ref. 116) TED, transient electric dichroism (Refs. 25, 115, and 130) Microscopy, time-resolved fluorescent microscopy (Ref. 176).

Thus, the rs is a complex term which embodies the fluorescence lifetime, rotational correlation time (0), and also r0 (r1 in the absence of depolarizing motion). The most common type of experiment involves a comparison of rs for two experimental conditions however, such a comparison of rs ignores possible changes in x, , and r0. Nevertheless, for many cases a comparison of rf values alone may be satisfactory although a more rigorous analysis requires a time-resolved measurement. A comparison of the effects of changes in common membrane properties on time-resolved fluorescence parameters is shown in Table 5.3. 

R. E. Dale, Membrane structure and dynamics by fluorescence probe depolarization kinetics, in Time-Resolved Fluorescence Spectroscopy in Biochemistry and Biology (R. B. Cundall and R. E. Dale, eds.), pp. 555-612, Plenum, New York (1984). 

Doxombicin binds readily to ceU membranes, changing their structure and function. The targets of doxombicin binding are compounds with a negative charge, of which the most extensively studied is the phospholipid cardiolipin (Pollakis et ah, 1983). Cardiolipin occurs in high concentrations in the inner mitochondrial membrane, where it is required for full activity of cytochrome c oxidase. In a recent study (Das and Mazumdar, 2000), the interaction between cytochrome c oxidase and cardiohpin in the presence of doxombicin was analysed, and the results of pico-second time-resolved fluorescence depolarization showed that the cardiohpin layer was depleted due to complexation with the dmg. 

Das, T.K. and Mazumdar, S., 2000, Effect of Adriamycin on the boundary hpid structure of cytochrome c oxidase pico-second time-resolved fluorescence depolarization studies. Biophys. Chem. 86 15-28... 

For chromophores that are part of small molecules, or that are located flexibly on large molecules, the depolarization is complete—i.e., P = 0. A protein of Mr = 25 kDa, however, has a rotational diffusion coefficient such that only limited rotation occurs before emission of fluorescence and only partial depolarization occurs, measured as 1 > P > 0. The depolarization can therefore provide access to the rotational diffusion coefficient and hence the asymmetry and/or degree of expansion of the protein molecule, its state of association, and its major conformational changes. This holds provided that the chromo-phore is firmly bound within the protein and not able to rotate independently. Chromophores can be either intrinsic—e.g., tryptophan—or extrinsic covalently bound fluorophores—e.g., the dansyl (5-dimethylamino-1-naphthalenesulfonyl) group. More detailed information can be obtained from time-resolved measurements of depolarization, in which the kinetics of rotation, rather than the average degree of rotation, are measured. For further details, see Lakowicz (1983) and Campbell and Dwek (1984). 

Nishimoto, E., Yamashita, S., Szabo, A. G., and Imoto, T. (1998) Internal motion of lyzozyme studied by time-resolved fluorescence depolarization of tryptophan residues, Biochemistry 37, 5599-5607. 

The rotational reorientation times of the sample in several solvents at room temperature were measured by picosecond time-resolved fluorescence and absorption depolarization spectroscopy. Details of our experimental setups were described elsewhere. For the time-correlated single photon counting measurement of which the response time is a ut 40 ps, the sample solution was excited with a second harmonics of a femtosecond Ti sapphire laser (370 nm) and the fluorescence polarized parallel and perpendicular to the direction of the excitation pulse polarization as well as the magic angle one were monitored. The second harmonics of the rhodamine-640 dye laser (313 nm 10 ps FWHM) was used to raesisure the polarized transient absorption spectra. The synthesis of the sample is given elsewhere. All the solvents of spectro-grade were used without further purification. 

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. 

It should be noted that the decay curve measured in the absence of an emission lar-izer will be In(t) + Ix(t) whereas inspectfon of the above demonstrates that the true fluorescence decay curve will be proportional to I (t) + 2Ix(t). Thus, for molecules rotating on the same timescale as the fluorescence decay, the fluorescence decay curve measured in the absence of a polarizer, will be distorted, and so time-resolved fluorescence depolarization must be considered even when the only desired measurement is the intrinsic fluorescence decay of the chromophore. To overcome this problem the fluorescence ould be monitored through a polarizer set at 54.7° to the excitation polarization vector ... 

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... 

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... 

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. 

A popular probe molecule which has b n employed for such studies is 1,6 diphenyl 1,3,5 hexatriene (DPH). This molecule has both absorption and emission along the long molecular axis and is thought to dissolve in the hydrocarbon interior. Time-resolved fluorescence depolarization studies with DPH probe molecules have been performed on the following bflayer syrtems dKdihydrosteraculoyl)pho halidyl choline dipalmitoyl phosphatidyl choline L-a-dimyristollecithin residues egg lecithin residues and mouse leukaemic L 1210cells In all reports the time-dependence of the emission anisotropy was found to decay non xponentially indkat-ing either... 

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