Fluorescence anisotropy

Fluorescence anisotropy or fluorescence polarization is the phenomenon where the light emitted by a fluorophore has unequal intensities along different axes of polarization. Early pioneers in the field include Aleksander Jablonski, Gregorio Weber, and Andreas Albrecht. The principles of fluorescence polarization and some applications of the method are presented in Lakowicz's book. Fluorescence anisotropy or fluorescence polarization is the phenomenon where the light emitted by a fluorophore has unequal intensities along different axes of polarization. Early pioneers in the field include Aleksander Jablonski, Gregorio Weber, and Andreas Albrecht. The principles of fluorescence polarization and some applications of the method are presented in Lakowicz's book. The anisotropy (r) of a light source is defined as the ratio of the polarized component to the total intensity ( I T {displaystyle I_{T}} ): r = I z − I y I x + I y + I z {displaystyle r={frac {I_{z}-I_{y}}{I_{x}+I_{y}+I_{z}}}} When the excitation is polarized along the z-axis, emission from the fluorophore is symmetric around the z-axis(Figure). Hence statistically we have I x = I y {displaystyle I_{x}=I_{y}} . As I y = I ⊥ {displaystyle I_{y}=I_{perp }} , and I z = I ∥ {displaystyle I_{z}=I_{parallel }} , we have r = I ∥ − I ⊥ I ∥ + 2 I ⊥ = I ∥ − I ⊥ I T {displaystyle r={frac {I_{parallel }-I_{perp }}{I_{parallel }+2I_{perp }}}={frac {I_{parallel }-I_{perp }}{I_{T}}}} . 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 τ {displaystyle au } ), 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. The first concept to understand for anisotropy measurements is the concept of Brownian motion. Although water at room temperature contained in a glass to the eye may look very still, on the molecular level each water molecule has kinetic energy and thus there are a continuous number of collisions between water molecules. 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: ϕ r = η V k B T {displaystyle phi _{r}={{eta V} over {k{_{B}}T}}}