Fluorescence polarization anisotropy factors

Fluorescence polarization is the subject of Chapter 5. Factors affecting the polarization of fluorescence are described and it is shown how the measurement of emission anisotropy can provide information on fluidity and 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). 

This point is further exemplified in Fig. 8 where the relaxation times of several xanthene dye molecules rotation in -alcohols are plotted in the same hydrodynamic formalism. These data are from transient-induced dichorism and time-resolved fluorescence depolarization experiments. In the latter case, the anisotropy factor r(f), which describes the intensities of fluorescence /(/) parallel ( ) and perpendicular (J.) to the polarized exciting pulse, is related to the second Legendre polynomial, P, ... 

The polarization characteristics of monochromators have important consequences in the measurement of fluorescence anisotropy. Such measurements must be corrected for the varying efficiencies of each optical component this correction is expressed as the G-factor (Section 10.4). However, the extreme properties of the concave gratings (Figure 2.11) can cause difficulties in the measurement of fluorescence polarization. For example, assume that the polarization is to be measured at an excitation wavelength of 450 nm. The excitation intensities will be nearly equal with the excitation polarizers in each orientation, which makes it easier to compare the relative emission intensities. If the nission is unpdarized, the relative intensities of the parallel (U) and perpendicular (X) excit ion will be nearly equal. However, suppose the excic ion is at 340 nm, in which case the intensities the... 

In a practical experiment, a correction factor is introduced to take into account the sensitivity of the fluorescence spectrometer to anisotropy. The factor G = /hv//hh is used, where the subscript v and h correspond to the vertically polarized light (z direction) and the horizontally polarized light (x- and -axes), respectively, with the incident light polarized in the.v direction. Taking this correction factor into account, the value of the fluorescence anisotropy ratio is... 

Unlike the static linear dichroism, the anisotropy function r(t) exhibits nonzero values even in randomly oriented samples, because polarized excitation creates an anisotropic excited state population through the electric dipole orientational factor (x (if. However, polarized fluorescence experiments in oriented samples are potentially far more informative than in isotropically random samples (Section II,A). 

As a further com dicating factor, tryptophan dis days complex spectral properties due to the presence of two nearly isoenergetic excited states. and The electronic transitions display distinct absorption, emission, and anisotropy spectra and are differently sensitive to solvent polarity. The complexity of indole photc hysics has stimulated detailed studies of protein fluorescence but has also inhibited interpretation of the data. 

The time-resolved fluorescence anisotropy function, rif), is calculated using Equation 1 in which / (0 and IJJ) are the individual decays collected with the polarization analyzer set parallel and perpendicular to the vertically polarized excitation light. The G factor is included to account for any polarization bias of the detection system. The influence of this term was minimized by arranging the polarization analyzer to be the first element in the detection system and using a polarization pseudo-scrambler (Oriel 28115) immediately prior to the emission monochromator slit. 

Fluorescence anisotropy is the measure of emission depolarization of a fluorescent molecule excited by a polarized light [72]. Rotation of the molecule is commonly responsible for the emission depolarization. Several factors such as viscosity, temperature, structure of the surrounding solvent, and specific solute-solvent... 

Fluorescence anisotropy can also be calculated by the quotient between the fluorescence intensities obtained at the four different combinations of the excitation and emission polarizer positions, as shown by Equation (12.3), Iw and Ihh account for the fluorescence intensity obtained with both polarizers at the vertical and horizontal position, respectively. Ihv corresponds to the fluorescence emission obtained with excitation polarizer horizontally and emission polarizer vertically oriented. In contrast, lyn corresponds to the fluorescence emission obtained with excitation polarizer verticahy and emission polarizer horizontally oriented. The G factor (Equation 12.4) is a parameter that accounts for the sensitivity of the detection system for verticahy and horizontally polarized lights [3]. 

It is hence possible to calculate the G factor in a given spectral range by recording the fluorescence spectra of a sample which emits in that spectral window keeping the excitation polarizer in the horizontal position and the emission one first horizontal and then vertical. Indeed it is usually convenient to measure the G factor using the same sample under investigation. In this case it is necessary to record four different luminescence spectra exactly in the same experimental conditions changing the orientation of the two polarizer in all the possible combination. Two of the four spectra are used to calculate the G factor (Eq. 6.21) and the other two to calculate the anisotropy. In alternative it is possible to express the anisotropy as a function of the four spectra. 

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