Fluorescence polarization anisotropy probes

Fluorescence polarization 1) steady state 2) time-resolved emission anisotropy rotational diffusion of the whole probe simple technique but Perrin s Law often not valid sophisticated technique but very powerful also provides order parameters... 

Although the structural differences between the water/CClq and water/DCE interfaces are not so large, the chemical and/or physical nature of the organic phase itself reflects on the photophysical properties of a probe molecule, indicating the novelty of the present experimental approaches. Systematic investigations are important to reveal factors governing structural and physical characteristics of water/oil interfaces. Therefore, we introduced fluorescence dynamic anisotropy and excitation energy transfer measurements to other water/oil interfacial systems the data are summarized in Table 12.3. The results are discussed in terms of the relationship between the interfacial stracture and the polarity at the water/oil interfaces (Section 12.6). 

First, we studied the solvent relaxation in solutions of diblock copolymer micelles. A commercially available polarity-sensitive probe, patman (Fig. 10, structure I), frequently used in phospolipid bilayer studies [123], was added to aqueous solutions of PS-PEO micelles. The probe binds strongly to micelles because its hydrophobic aliphatic chain has a strong affinity to the nonpolar PS core. The positively charged fluorescent headgroup is supposed to be located in the PEO shell close to the core-shell interface. The assumed localization has been supported by time-resolved anisotropy measurements. 

Fluorescence polarization measurements have also been used to detomine the apparent viscosity of the side-chain region (center) of membranes. Such measurements of microviscosity are typically performed using a hydrophobic probe like DPH (Figure 1.22, bottom), which partitions into the membrane. The viscosity of membranes is known to decrease in the presence of unsaturated fatty acid side chains. Hence, an increase in the amount of unsaturated fatty acid is expected to decrease the anisotropy- The apparent microviscosity of the membrane is determined by comparing the polarization of the probe measured in the membrane with that observed in solutions of known viscosity. 

See also Biochemical Applications of Fluorescence Spectroscopy Fluorescence Microscopy, Applications Fluorescent Molecular Probes Fluorescence Polarization and Anisotropy Inorganic Condensed Matter, Applications of Luminescence Spectroscopy UV-Visible Absorption and Fluorescence Spectrometers X-Ray Fluorescence Spectrometers X-Ray Fluorescence Spectroscopy, Applications. 

The kinetic interactions between solvent and solute molecules in free solution determine their rotational and translational diffusion characteristics. Fluorescence polarization is a spectroscopic technique that allows the determination of motional preferences of reporter molecules in fluids with respect to both the rate of motion and the orientational restriction of that motion [1,2], For spherical molecules in isotropic fluids at low concentrations, these motions can be described by the Stokes-Einstein and Perrin relationships, and if these motions have an equal probability of occurring in any dimension they are referred to as isotropic. However, when a fluid displays structure, or anisotropy, the motion of diffusing molecules may be restricted, generally to different extents in different dimensions, and these motions are said to be anisotropic. New approaches must then be taken in order to describe the probe s hydrodynamic behavior. By measuring the hydrodynamic properties of a fluorescent probe in solution, it is possible to extract valuable information on the physical structure and properties of a fluid. Knowledge of the physical structure and properties of food fluids and matrices is essential for solving practical problems in food research. 

Hence, it is possible to construct a standard curve relating viscometric measurements to steady-state anisotropy measurements for a particular fluid and use the quantitative relationship to determine the viscosity of the fluid by fluorescence polarization [4,9-11]. A standard curve in a reference calibration oil such as white paraffin oil can be used to determine the viscosity of another fluid as long as the calibration fluid is similar in dielectric constant and viscosity to the fluid being analyzed. It is important to keep in mind that the same fluorescent probe may display different behavior even in different hydrocarbon calibration oils, hence one must exercise caution when determining absolute values for microvis-... 

Fluorescence anisotropy is generally used to provide information about the dipolar orientational dynamics occurring after excitation of a system. This technique has successfully been used to probe ultrafast dynamics of energy transfer in organic conjugated dendrimers. The detected emission intensities Tar and Ter for parallel and perpendicularly polarized excitation respectively, were used to construct an observable emission anisotropy R(t) in accordance with the equation [121] ... 

Fluorescence anisotropy decay measurements, which are based on the excitation of probes with polarized light and subsequent polarized fluorescence emission, can... 

In order to test further the applicability of 1-pyrene carboxaldehyde as a fluorescent probe, we applied Keh and Valeur s method (4) to determine average micellar sizes of sulfonate A and B micelles. This method is based on the assumption that the motion of a probe molecule is coupled to that of the micelle, and that the micellar hydrodynamic volumes are the same in two apolar solvents of different viscosities. For our purposes, time averaged anisotropies of these systems were measured in two n-alkanes hexane and nonane. The fluorescence lifetime of 1-pyrene carboxaldehyde with the two sulfonates in both these solvents was found to be approximately 5 ns. The micellar sizes (diameter) calculated for sulfonates A and B were 53 5A and 82 lOA, respectively. Since these micelles possesed solid polar cores, they were probably more tightly bound than typical inverted micelles such as those of aerosol OT. Hence, it was expected that the probe molecules would not perturb the micelles to an extent which would substantially affect the micellar sizes measured. 

The internal rotational relaxation times of 1-pyrene carboxaldehyde in sulfonate systems may offer some indication of the extent of probe binding to the inverted micelle. In the absence of any background fluorescence interference to the time-dependent anisotropy decay profile, the internal rotational relaxation time should correlate with the strength of binding with the polar material in the polar core. However, spectral interference from the aromatic moieties of sulfonates is substantial, so that the values of internal rotational relaxation time can only be used for qualitative comparison. 

The measurements of the fluorescence dynamics were made by a femtosecond fluorescence up-conversion apparatus similar to that described elsewhere [2], The fivhm of the instrumental response was 110 fs. The polarization axis of the pump pulse was set at 54.7° with respect to the probe to suppress the anisotropy effects. 

Sample preparation was given elsewhere [2]. Femtosecond fluorescence upconversion and picosecond time-correlated single-photon-counting set-ups were employed for the measurement of the fluorescence transients. The system response (FWHM) of the femtosecond fluorescence up-conversion and time-correlated single-photon-counting setups are 280 fs and 16 ps, respectively [3] The measured transients were fitted to multiexponential functions convoluted with the system response function. After deconvolution the time resolution was 100 fs. In the upconversion experiments, excitation was at 350 nm, the transients were measured from 420 nm upto 680 nm. Experiments were performed under magic angle conditions (to remove the fluorescence intensity effects of rotational motions of the probed molecules), as well as under polarization conditions in order to obtain the time evolution of the fluorescence anisotropy. 

The most natural method of detecting the optical polarization produced in the excited state consists of using the polarization characteristics of molecular fluorescence from this state in some transition b — c which is convenient for observation see Fig. 1.2. The same radiation, in the cycle a —> b c may also provide information on the initial state a if the process of absorption is non-linear. A more direct way of probing the anisotropy of angular momenta distribution in the ground state consists of monitoring the absorption from this level by means of a test beam. 

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