Fluorescence anisotropy solvent effects

One of the most popular applications of molecular rotors is the quantitative determination of solvent viscosity (for some examples, see references [18, 23-27] and Sect. 5). Viscosity refers to a bulk property, but molecular rotors change their behavior under the influence of the solvent on the molecular scale. Most commonly, the diffusivity of a fluorophore is related to bulk viscosity through the Debye-Stokes-Einstein relationship where the diffusion constant D is inversely proportional to bulk viscosity rj. Established techniques such as fluorescent recovery after photobleaching (FRAP) and fluorescence anisotropy build on the diffusivity of a fluorophore. However, the relationship between diffusivity on a molecular scale and bulk viscosity is always an approximation, because it does not consider molecular-scale effects such as size differences between fluorophore and solvent, electrostatic interactions, hydrogen bond formation, or a possible anisotropy of the environment. Nonetheless, approaches exist to resolve this conflict between bulk viscosity and apparent microviscosity at the molecular scale. Forster and Hoffmann examined some triphenylamine dyes with TICT characteristics. These dyes are characterized by radiationless relaxation from the TICT state. Forster and Hoffmann found a power-law relationship between quantum yield and solvent viscosity both analytically and experimentally [28]. For a quantitative derivation of the power-law relationship, Forster and Hoffmann define the solvent s microfriction k by applying the Debye-Stokes-Einstein diffusion model (2)... 

A typical time evolution of fluorescence anisotropy is a monotonously decreasing function. However, the sum of several exponentials with both positive and negative prefactors derived on the basis of a rigid rotor model does not preclude increasing or even a non-monotonous time evolution. The non-monotonous time evolution has been observed for perylene excited to S2 quite far in the blue region with respect to the emission [11], It starts, as predicted for the perpendicular orientation of dipole moments, at ro = -0.2, but increases rapidly to a slightly positive transient value and then decreases more slowly to = 0. The non-monotonous r t) decay can be rationalized by the solvent effect on the rotation of the flat disc-like perylene around three different axes. 

Such is the newness of appreciation of near-IR fluorescence techniques that there is a dearth of examples in the literature of implementations of many of the classic fluorescence methods in the IR. Anisotropy is one striking example of this. However, in a comprehensive study of the anisotropy decay of dyes, including oxazine fluorescence at 720 nm, in mixed isotropic solvents Dutt et al.( T7 TS) have investigated the effects of viscosity on molecular rotation. 

Further structural information has been derived from the recent use of optical spectroscopy as a probe of structure at the L-L interface the two general approaches pursued are (i) total internal reflectance (TIR) fluorescence spectroscopy of adsorbed solute species and (ii) nonlinear spectroscopic methods to generate interface-sensitive spectra of the solvent molecules in the interfacial region. The former approach uses the polarization of the emitted signal, from an s-polarized excitation, to determine the effective dimensionality of the interface. The anisotropy of the fluorescent response from an interface that is flat on the length-scale of the adsorbed probe ( lnm) is [33]... 

The case of probes undergoing isotropic rotations in a homogeneous isotropic medium will be examined first. Rotations are isotropic when the probe has a spherical shape, but it is difficult to find such probes because most fluorescent molecules are aromatic and thus more or less planar. Nevertheless, when a probe interacts with the solvent molecules through hydrogen bonds, experiments have shown that in some cases the observed rotational behaviour can approach that of a sphere. In the case of rod-like probes whose direction of absorption and emission transition moments coincide with the long molecular axis (e.g. diphenylhexatriene Figure 8.4), the rotations can be considered as isotropic because any rotation about this long axis has no effect on the emission anisotropy. 

The use of 1-dimethylamino-5-naphthyl sulphonate ("dansyl") as a label was first introduced in 1952 for the determination of the rotational diffusion of protein molecules from the anisotropy of the dansyl fluorescence (1). Somewhat later, (2) it was found that the emission intensity of fluorophores of this type is strongly increased when they are adsorbed from water solution on proteins This increase in quantum yield of emission (accompanied by a blue-shift of the emission spectrum) could be correlated with a decreasing polarity of the medium (3). The effect can be explained as follows If the dipolar interaction of the excited chromophore with the solvent is smaller than this interaction in the ground state, the emitted quantum will be increased at the same time, the probability of nonradiative deactivation will be reduced, increasing the quantum yield of fluorescence (4). If the fluorescent label can be attached to a well-defined site on a protein, its emission characteristics will "report" about the polarity of this site (5) and the use of... 

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