Emission Anisotropy Spectra

From the practical point of view it is not necessary to determine the value of both 5v and 5h since only their ratio (conventionally referred to as G factor) appears in the expression of the FA. 

Once G is defined and determined it becomes possible to express r as a function of the two experimental spectra lyy and /vh- Of course G is a wavelength dependent factor which is characteristic of the instruments and once it has been determined for a sample it can be used to correct the FA of other samples. 

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As seen from (12) and Fig. 6, the peaks in the excitation anisotropy spectrum indicate a small angle between the absorption and emission transition dipoles suggesting allowed 1PA transitions while valleys indicate large angles between these two dipoles, suggesting a forbidden 1PA transition. Due to selection rules for symmetrical cyanine-like dyes, the valleys in the anisotropy spectrum could indicate an allowed 2PA transition as demonstrated in Fig. 6. Thus, an excitation anisotropy spectrum can serve as a useful guide to suggest the positions of the final states in the 2PA spectra. 

Fig. 4 Excitation and emission spectra of complex 16 in CHCI3, CH3CN, CH3OH and buffer at room temperature (A,ex = 400 nm). The solid line shows the excitation anisotropy spectrum in 100% glycerol at - 60 °C with the emission wavelength tuned to 550 nm [51]...

Quenching resolved emission anisotropy experiments could be performed at emission wavelengths in the blue (< 330 nm) and red (> 330 nm) portions of the spectrum to yield a more consistent data surface. However, this could be possible if at each edge of the fluorescence spectrum the emission occurs mainly from the buried or the surface Trp residues. Unfortunately, this is not the case since for example at 315 nm, the fractional contribution to the total fluorescence of the surface Trp residue is 42%. 

Dependencies of luminescence bands (both fluorescence and phosphorescence), anisotropy of emission, and its lifetime on a frequency of excitation, when fluorescence is excited at the red edge of absorption spectrum. Panel a of Fig. 5 shows the fluorescence spectra at different excitations for the solutes with the 0-0 transitions close to vI vn, and vra frequencies. Spectral location of all shown fluorescence bands is different and stable in time of experiment and during lifetime of fluorescence (panel b)... 

Figure B9.3.1 shows the parallelism between the increase in emission spectrum displacement and fluorescence anisotropy observed for the red-edge of most vibronic bands and especially for the 0-0 one. It can be interpreted in terms of inhomogeous spectral broadening due to solvation heterogeneity. The decrease in energy transfer that is observed upon red-edge excitation is evidence that energy hopping is not chaotic but directed toward lower energy chromophores, as in photosynthetic antennae.

Fig. 2c. It can be seen that at 530 nm, the fluorescence decays mono-exponentially with the fluorescence lifetime of 3.24 ns. The rise of the emission seen below 50 ps in the corresponding FlUp data is obviously not resolved here. In contrast, the TCSP data at 450 nm is described by a triple-exponential decay whose dominant component has a correlation time well below the time resolution. This component is obviously equivalent to the fluorescence decay observed in the FlUp experiment. A minor contribution has a correlation time of about 3.2 ns and reflects again the fluorescence lifetime that was also detected at 530 nm. The most characteristic component at 450 nm however has a time constant of about 300 ps. It is important to emphasize that this 300 ps decay does not have a rising counterpart when emission near the maximum of the stationary fluorescence spectrum is recorded. In other words, the above mentioned mirror image correspondence of the fluorescence dynamics between 450 nm and 530 nm holds only on time scales shorter than 20 ps. Finally, in contrast to picosecond time scales, the anisotropy deduced from the TCSPC data displays a pronounced decay. This decay is reminiscent of the rotational diffusion of the entire protein indicating that the optical chromophore is rigidly embedded in the core of the 6-barrel protein.

Since electronic transitions differ from one excitation wavelength to another, the value of P would change with excitation wavelength. Emission generally occurs from the lowest excited state Si Vo, and so one can measure anisotropy or polarization along the absorption spectrum at a fixed emission wavelength. We obtain a spectrum called the excitation polarization spectrum or simply the polarization spectrum (Figure 11.2). 

The simplest steady-state measurements of fluorescence properties such as the fluorescence emission spectrum or the steady-state anisotropy can be carried out in a standard fluo-rometer with excitation from a lamp source and a monochromator. Common lamp sources in commercial fluorometers include xenon lamps for excitation from the UV to the near-IR (250 nm to llOOnm). 

A conclusion can be drawn from the above results is that the dynamics and the structure of the microenvironment of the Trp residues and the tertiary structure of the protein have an important effect on the fluorescence intensity, the position of the maximum, the center of gravity of the spectrum and finally on the dependence of the anisotropy on the emission and excitation wavelengths. 

When a protein contains two classes of intrinsic fluorophore, one at the surface of the protein and the second embedded in the protein matrix, fluorescence intensity quenching with cesium or iodide allows obtaining the spectra of these two classes. A selective quenching implies that addition of quencher induces a decrease in the fluorescence observables (intensity, anisotropy and lifetime) of the accessible class. At high quencher concentration the remaining observables measured will reflect essentially those of the embedded fluorophore residues. In this case, one can determine the fraction of fluorescence intensity that is accessible (fa). Knowing fa along the emission spectrum will allow us to draw the spectrum of each class of fluorophore (Lehrer, 1971). 

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