Fluorescence anisotropy
Early pioneers in the field include Aleksander Jablonski, Gregorio Weber,[1] and Andreas Albrecht.
[2] The principles of fluorescence polarization and some applications of the method are presented in Lakowicz's book.
[3] The anisotropy (r) of a light source is defined as the ratio of the polarized component to the total intensity (
Hence, excitation by a photon can occur only if the electric field of the light is oriented in a particular axis about the molecule.
A nanoparticle (yellow dot in the figure) suspended in solution will undergo a random walk due to the summation of these underlying collisions.
The rotational correlation time (Φr), the time it takes for the molecule to rotate 1 radian, is dependent on the viscosity (η), temperature (T), Boltzmann constant (kB) and volume (V) of the nanoparticle:[5] The second concept is photoselection by use of a polarized light.
Taking the idealistic simplest case a subset of dye molecules suspended in solution that have a mono-exponential fluorescence lifetime
The fluorescence sum and difference can be constructed by addition of the intensities and subtraction of the fluorescence intensities respectively: Dividing the difference by the sum gives the anisotropy decay: The grating factor G is an instrumental preference of the emission optics for the horizontal orientation to the vertical orientation.
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
The scrambling of orientations can occur by the whole molecule tumbling or by the rotation of only the fluorescent part.
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.
Again using the idealistic simplest case a subset of dye molecules suspended in solution that have a mono-exponential fluorescence lifetime
measured with the unpolarized emission set-up and the second decay time will be due to the loss of fluorescence as Brownian motion results in dye molecules moving from an initial vertical polarized configuration to an unpolarized configuration.
but the second one will have a negative pre-exponential factor resulting from the introduction of excited molecules that were initially vertically polarized and became depolarized via Brownian motion.
The fluorescence sum and difference can be constructed by addition of the decays and subtraction of the fluorescence decays respectively: Dividing the difference by the sum gives the anisotropy decay: In the simplest case for only one species of spherical dye: Fluorescence anisotropy can be used to measure the binding constants and kinetics of reactions that cause a change in the rotational time of the molecules.
If the fluorophore is a small molecule, the rate at which it tumbles can decrease significantly when it is bound to a large protein.
If the fluorophore is attached to the larger protein in a binding pair, the difference in polarization between bound and unbound states will be smaller (because the unbound protein will already be fairly stable and tumble slowly to begin with) and the measurement will be less accurate.
[8] Fluorescence anisotropy is also applied to microscopy, with use of polarizers in the path of the illuminating light and also before the camera.
This technique has also been used to detect the binding of molecules to their partners in signaling cascades in response to certain cues.
The phenomenon of emFRET and the associated decrease in anisotropy when close interactions occur between fluorophores has been used to study the aggregation of proteins in response to signaling.
