The sample is excited with the linearly polarized light and emission is recorded at 90°. A polarizer is placed before the detector that allows intensity measurement of the light polarized parallel (I _ll) and perpendicular to the direction of excitation radiation. The anisotropy (r) is given by

Molecular tumbling before emission changes the orientation of the transition dipole moment, resulting in the loss of polarization (Figure 6.6). As rotational diffusion of the molecules depends on their sizes, fluorescence anisotropy can be used to measure the diffusion coefficient and therefore the sizes of the molecules.

Figure 6.6 Depolarization of radiation as a result of molecular tumbling.

We shall, in the next lecture, discuss the biological fluorophores and the applications of fluorescence in understanding the biomolecules.