Emission anisotropy polarization ratio

Characterization of the polarization state of fluorescence (polarization ratio, emission anisotropy)... 

In the expression of the polarization ratio, the denominator represents the fluorescence intensity in the direction of observation, whereas in the formula giving the emission anisotropy, the denominator represents the total fluorescence intensity. In a few situations (e.g. the study of radiative transfer) the polarization ratio is to be preferred, but in most cases, the use of emission anisotropy leads to simpler relations (see below). 

The components ly and Ih, vertically and horizontally polarized respectively, are such that Jz = Jy = Ix, Iy = IH (Figure 5.3). The total fluorescence intensity is then 2fy + Ih- The polarization ratio and the emission anisotropy are given by... 

In principle, pulsed excitation measurements can provide direct observation of time-resolved polarization decays and permit the single-exponential or multiexponential nature of the decay curves to be measured. In practice, however, accurate quantification of a multiexponential curve often requires that the emission decay be measured down to low intensity values, where obtaining a satisfactory signal -to-noise ratio can be a time-consuming process. In addition, the accuracy of rotational rate measurements close to a nanosecond or less are severely limited by tbe pulse width of the flash lamps. As a result, pulsed-excitation polarization measurements are not commonly used for short rotational periods or for careful measurements of rotational anisotropy. 

In Eq. (14.1), I is fluorescent intensity the subscript letters, V for vertical and H for horizontal, represent the polarization direction of the two polarizers on the excitation and emission light path, respectively and the ratio, ZHV/IhH) calibrates for the difference in the emission channel s sensitivity towards vertical and horizontal polarized components. Anisotropy, r, can be measured by either L-format or T-format. In the L-format, all four fluorescence intensities, Zw, h11, f iv. and ZHh> are measured using a single channel of a photodetector so that each intensity needs to be measured separately. If the fluorimeter has two emission channels then anisotropy can also be measured in a T-format, which allows fluorescence intensities pairs, Ivv//Vi i or If iv // ii i, to be measured simultaneously via the two emission channels. Thus, measurements in the T-format are faster than in the L-format. 

Anisotropy can be measured when a fluorescent molecule is excited with polarized light. The ratio of emission intensity in each polarization plane, parallel and perpendicular relative to the excitation polarization plane, gives a measure of anisotropy, more commonly and incorrectly referred to in HTS as fluorescence polarization (FP). This anisotropy is proportional to the Brownian rotational motion of the fluorophore. 

The use of polarized excitation for anisotropy measurements in coordination complexes is widespread and is useful in correlating the relative orientation of excitation and emission dipoles. For samples excited with vertically polarized light the anisotropy ratio is determined by measuring the intensity of emission of light polarized both parallel and perpendicular to the polarized excitation. In this case the anisotropy, r, is given by Equation (5). For a chromophore in a rigid matrix, where loss of polarization does not occur via molecular rotation, the value of r is related to the relative orientation of the absorption and emission dipoles, 0. Thus, the measurement... 

---