Fluorescence polarization measurements

Situation with H-bonding also demands to take into account the fact that alcohols have ability to form various associates or even clusters at normal conditions. The most efficient method for determination of inhomogeneity in the excited states is fluorescence polarization measurements. These methods also frequently applied for studying of solvent viscosity, they may be provided in two variants steady state and time-resolved. Relations for time-resolved and steady state fluorescence anisotropy may be given as [1, 2, 75] ... 

Cohen-Kashi, M., Namer, Y. and Deutsch, M. (2006). Fluorescence resonance energy transfer imaging via fluorescence polarization measurement. J. Biomed. Opt. 11, 34015. 

Fluorescence polarization measurements can thus provide useful information on molecular mobility, size, shape and flexibility of molecules, fluidity of a medium, and order parameters (e.g. in a lipid bi layer)2 . 

Dr can be determined by time-resolved fluorescence polarization measurements, either by pulse fluorometry from the recorded decays of the polarized components I l and 11, or by phase fluorometry from the variations in the phase shift between J and I as a function of frequency (see Chapter 6). If the excited-state lifetime is unique and determined separately, steady-state anisotropy measurements allow us to determine Dr from the following equation, which results from Eqs (5.10) and (5.41) ... 

Once Dr is determined by fluorescence polarization measurements, the Stokes-Einstein relation can be used ... 

This relation shows that the rotational correlation time is uncoupled from the excited-state lifetime, in contrast to classical steady-state or time-resolved fluorescence polarization measurements (see Chapter 5). The important consequence is the possibility of observing slow rotations with fluorophores of short lifetime. This is the case for biological macromolecules labeled with fluorophores (e.g. rhodamine) whose lifetime is of a few nanoseconds. 

This problem does not exist with time-dependent fluorescence polarization measurements where the decay of the emission anisotropy r(t) is obtained by determining the decay of Iz and Ix according to eq 12. 

In order to evaluate microscopic viscosity around the guest incorporated into the hybrid assemblies, the steady-state fluorescence polarization measurements were performed for ANS and TNS (Table 3). The P value for ANS... 

Time-resolved fluorescence polarization measurements for 1 and 2 in n-heptane showed that after pulsed excitation, the initial value of the fluorescence anisotropy is about 0.25. This... 

Protein-ligand interactions can not only be secreened or selected in vitro, but also can be directly characterized for particular interaction partners. Nemoto et al. (1999) applied the mRNA-peptide fusion technology to fluorescently label the displayed proteins in order to study protein-protein interactions by fluorescence polarization measurements. 

Marks, B.D. et al. 2005. Multiparameter analysis of a screen for progesterone receptor ligands comparing fluorescence lifetime and fluorescence polarization measurements. Assay Drug Devel. Technol. 3, 613-622. 

The changes have been used to provide information about the enviromnent of the fluorescent probe and to follow changes in conformation of the macromolecule. In other work the study of the fluorescence polarization properties of the attached probe under steady state illumination and the application of Perrin s equation enable calcu-latnn of the rotary Brownian motion of the polymer. This technique has been extended by Jablonski and Wahl to the use of time-resolved fluorescence polarization measurements to calculate rotational relaxation times of molecules These experiments are discussed fiilly in the fdlowing section of this review. 

To characterize the binding and the motions of the placed single T4 lysozyme molecules on cell walls, we used single-molecule fluorescence polarization measurements. The orientation of the single-molecule transition dipole can be probed by either linear polarized excitation or linear polarized emission. In this work, the excitation light was unpolarized. The emission was split into orthogonal (s polarized /i and p polarized I2) polarizations and detected by two photon detectors [22]. The intensity trajectories probed at the two orthogonal polarizations are shown in Fig. 24.9c (upper panel). The polarization P is defined as ... 

As indicated, P is the difference between the two observed intensities divided by their sum. Fluorescence polarization is measured by placing a mechanically or electrically driven polarizer between the sample cuvet and the detector. A diagram of a fluorescence polarization measurement system is shown in Figure 3-16. In the normal instrumentation mode, the sample is excited with polarized hght to obtain maximum sensitivity. The polarization analyzer is positioned first to measure the intensity of the emitted fluorescence hght in the vertical plane ly), and then the polarization analyzer is rotated 90° to measure the emitted fluorescence hght intensity in the horizontal plane (Ij,). P is then calculated manually or automatically by use of equation (13). 

Fig. 10 Principle of a fluorescence polarization measurement. Polarized excitation light is used to detect changes in the rotational mobility of fluorophores. A small fluorescent tracer rotates quickly, resulting in a low polarization value if the tracer is bound the fluorescence emission is polarized.

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). 

The steady-state fluorescence polarization measured for 9,10-DMA attached to PAA and PMA at degrees of neutralization (o) in the range... 

Andrich, M.P., and Vanderkooi, J.M., Temperature dependence of 1,6-diphenyl-1,3,5-hexa-triene fluorescence in phospholipid artificial membranes, Biochemistry, 15, 1257, 1976. Blitterswijk, W.J.V., Hoeven, R.P.V., and Dermeer, B.W.V., Lipid structural order parameters (reciprocal of fluidity) in biomembranes derived from steady-state fluorescence polarization measurements, Biochem. Biophys. Acta, 644, 323, 1981. 

L.A. Kelly, J.G. Trunk, J.C. Sutherland, Time-resolved fluorescence polarization measurements for entire emission spectra with a resisitive-anode, single-photon-counting detector The fluoreseence omnilizer, Rev. Sci. Instrum. 68, 2279-2286 (1997)... 

Fluorescence polarization measurements were performed with an apparatus built in the laboratory and connected to a minicomputer (Digital LSI II). This was, in turn, connected to a Vax 11/780, thus allowing complete automatization of the measurements. Diphenylhexa-triene (DPH) in tetrahydrofuran (6 mM) was added to phospholipids in amounts never exceeding 1 %. 

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