Principle
In fluorescence, a molecule absorbs a photon and gets excited to a higher energy state. After a short delay (the average represented as the fluorescence lifetime ), it comes down to a lower state by losing some of the energy as heat and emitting the rest of the energy as another photon. The excitation and de-excitation involve the redistribution of electrons about the molecule. Hence, excitation by a photon can occur only if the electric field of the light is oriented in a particular axis about the molecule. Also, the emitted photon will have a specific polarization with respect to the molecule.
When polarized light is applied to a group of randomly oriented fluorophores, most of the excited molecules will be those oriented within a particular range of angles to the applied polarization. If they do not move, the emitted light will also be polarized within a particular range angles to the applied light. This intrinsic anisotropy (denoted r0) is usually measured by embedding the fluorophore in a frozen polyol.
When the fluorophores can freely change their orientation before re-emitting the photons, the degree of polarization of the emitted light will be reduced. The degree of decorrelation in the polarization of the incident and emitted light depends on how quickly the fluorophore orientation gets scrambled ( the rotational lifetime ) compared to the fluorescence lifetime . The scrambling of orientations can occur by the whole molecule tumbling or by the rotation of only the fluorescent part. The rate of tumbling is related to the measure anisotropy by the relationship:
Where r is the observed anisotropy, r0 is the intrinsic anisotropy of the molecule, is the fluorescence lifetime and is the rotational time constant.
This analysis is valid only if the fluorophores are relatively far apart. If they are very close to another, they can exchange energy by FRET and because the emission can occur from one of many independently moving (or oriented) molecules this results in a lower than expected anisotropy or a greater decorrelation. This type of homotransfer Förster resonance energy transfer is called energy migration FRET or emFRET
Read more about this topic: Fluorescence Anisotropy
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