If a fluorescent molecule is excited with polarized light then the emission will also be polarized. The extent of polarization of the emission is usually described in terms of anisotropy (r). As molecules are tumbling in solution, the emitted light is then depolarized. The depolarization of the fluorescent molecule is dependent on the size and shape of the rotating molecule and also the viscosity of the solution. The smaller the molecule, the more rapidly it rotates and the more the light is depolarized and hence the lower the anisotropy. If a larger molecule interacts with the fluorescent molecule the rotation of the complex will be slower than of the unbound molecules and result in an increase in the fluorescence anisotropy. Molecular interactions can be analyzed using this approach where the smaller molecule is fluorescently labeled and the concentration of the larger binding partner is varied. A cartoon depicting the concept of fluorescence anisotropy is shown below.
In PCL we often use fluorescence anisotropy as one tool in our tool box to characterize a molecular interaction. For example we may use it to define appropriate binding conditions for a more detailed SPR study. Or after making some initial fluorescence anisotropy measurements we validate the interaction with the fluorescently labeled molecule by SPR.
Examples of how PCL uses fluorescence anisotropy to study molecular interactions: