Abstract
Measurements of fluorescence anisotropy are a powerful tool in biochemical research and medical testing. Upon excitation with polarized light the emission from many samples is also polarized. The extent of polarization of the emission is described in terms of the anisotropy (r). Samples exhibiting nonzero anisotropies are said to display polarized emission. The origin of anisotropy is the existence of transition moments for absorption and emission that lie along specific directions within the fluorophore structure. In homogeneous solution the ground-state fluo-rophores are all randomly oriented. When exposed to polarized light, those fluorophores that have their absorption transition moments oriented along the electric vector of the incident light are preferentially excited. Hence the excited-state population is partially oriented. A significant fraction of the excited molecules have their transition moments oriented along the electric vector of the polarized exciting light.
The emission can become depolarized by a number of processes, the relative importance of which depends upon the sample under investigation. All chromophores have transition moments that occur along a specific direction in the molecular axis. Rotational diffusion changes the direction of the transition moments and is one common cause of depolarization. Anisotropy measurements reveal the average angular displacement of the fluorophore that occurs between absorption and subsequent emission of a photon. This angular displacement is dependent upon the rate and extent of rotational diffusion during the lifetime of the excited state. The rate of rotational diffusion depends on the viscosity of the solvent and the size and shape of the rotating molecule. The rotational rate of fluorophores in solution is dependent upon the viscous drag imposed by the solvent. A change in solvent viscosity will result in a change in fluorescence anisotropy. For small fluorophores in low-viscosity solutions the rate of rotational diffusion is typically faster than the rate of emission. Under these conditions the emission is depolarized and the anisotropy close to zero.
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(2006). Fluorescence Anisotropy. In: Lakowicz, J.R. (eds) Principles of Fluorescence Spectroscopy. Springer, Boston, MA. https://doi.org/10.1007/978-0-387-46312-4_10
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