Emission polarization

In the early 1990s, a new spin polarization mechanism was posPilated by Paul and co-workers to explain how polarization can be developed m transient radicals in the presence of excited triplet state molecules (Blattler et al [43], Blattler and Paul [44], Goudsmit et al [45]). While the earliest examples of the radical-triplet pair mechanism (RTPM) mvolved emissive polarizations similar in appearance to triplet mechanism polarizations, cases have since been discovered m which absorptive and multiplet polarizations are also generated by RTPM. 

Pulsed method. Using a pulsed or modulated excitation light source instead of constant illumination allows investigation of the time dependence of emission polarization. In the case of pulsed excitation, the measured quantity is the time decay of fluorescent emission polarized parallel and perpendicular to the excitation plane of polarization. Emitted light polarized parallel to the excitation plane decays faster than the excited state lifetime because the molecule is rotating its emission dipole away from the polarization plane of measurement. Emitted light polarized perpendicular to the excitation plane decays more slowly because the emission dipole moment is rotating towards the plane of measurement. 

Figure 4.9 illustrates time-gated imaging of rotational correlation time. Briefly, excitation by linearly polarized radiation will excite fluorophores with dipole components parallel to the excitation polarization axis and so the fluorescence emission will be anisotropically polarized immediately after excitation, with more emission polarized parallel than perpendicular to the polarization axis (r0). Subsequently, however, collisions with solvent molecules will tend to randomize the fluorophore orientations and the emission anistropy will decrease with time (r(t)). The characteristic timescale over which the fluorescence anisotropy decreases can be described (in the simplest case of a spherical molecule) by an exponential decay with a time constant, 6, which is the rotational correlation time and is approximately proportional to the local solvent viscosity and to the size of the fluorophore. Provided that... 

Appendix) how a signal proportional to the total fluorescence intensity can be measured by using excitation and/or emission polarizers at appropriate angles. 

As previously outlined, the emission correction factors must be recorded with an emission polarizer in a defined orientation (preferably at the magic angle), and this orientation must be kept unchanged for recording emission or excitation spectra irrespective of whether or not the fluorescence is polarized. 

Distortion of the fluorescence response measured by the detection system (monochromator + detector) arises when the emitted fluorescence is partially polarized. As explained in the Appendix, a response proportional to the total fluorescence intensity can be observed by using two polarizers an excitation polarizer in the vertical position, and an emission polarizer set at the magic angle (54.7°) with respect to the vertical, or vice versa (see the configurations in Figure 6.3). 

Effect of polarity on fluorescence emission. Polarity probes... 

Steady state emission polarization measurements reflect the time over which the excited species remains rigidly bound to DNA, yielding some idea of the movement and orientation of the luminophore in the DNA microenvironment. 

Figure 15 Polarized fluorescence microscopy images of a 2.5-p,m-long Ox +-loaded zeolite L crystal after excitation at 545-580 nm (cutoff 605 nm). The arrows indicate the transmission direction of the emission polarizer.

The emitted light is detected along y through a polarizer oriented either along z (Fz) or along x (Fx). In fluorescence polarization studies with continuous excitation (steady-state experiments), the emission anisotropy r and the emission polarization p are defined in eqs 8a and 8b. 

Excitation-wavelength-dependent emission polarization studies indicate the presence of an overlapping xy polarized transition in the bluer part of the 290-315-nm range, as indicated in Fig. 5. The combination of static absorption, time-resolved emission, and emission quantum yield measurements suggests that the emitting state has the same polarization (z axis, linear), but is not the same state as that giving rise to the 362-nm absorption peak. These assignments for the 3.5-nm particles are summarized in Fig. 5. 

B) FRET efficiency as a function of Mg2+ ion concentration for the SB and BC vectors. The data have been fitted to a two-state ion binding model. Fluorescence emission spectra were recorded at 4 °C using an SLM-Aminco 8100 fluorimeter with modernized Phoenix electronics (ISS Inc., Champaign, IL, USA). Spectra were corrected for xenon lamp fluctuations and instrumental variations, and polarization artifacts were avoided by crossing excitation and emission polarizers at 54.7°. 

In the case of a water/DCE interface [(c) and (d)], on the other hand, a fitting of the data by a single-exponential function cannot be attained by setting an emission polarizer at 45°, as confirmed by deviations of Re and Cr from the optimum values (c). When the fluorescence decay profile is measured by setting an emission polarizer at 54.7° (d), fluorescence anisotropy can be reasonably fitted by a single-exponential function including the time response in the initial stage of excitation (see also and... 

The angle of the emission polarizer in respect to that of the excitation laser beam. 

The angle of an emission polarizer, by which the fluorescence decay is best fitted by a single-exponential function. 

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