The efficiency of energy transfer depends upon

1. the distance between D and A
2. the relative orientation of the transition dipoles of D and A
3. the extent of the overlap between D’s emission spectrum and A’s absorption spectrum

Efficiency of energy transfer

                                           where,

r is the distance between D and A R0 (also called the Förster distance) is the distance (r) between D and A at which the efficiency of energy transfer is 50%, and is characteristic of a D-A FRET pair.

 

                                                   

Resonance energy transfer can be used to determine the distances between D and A, and is therefore also termed as molecular ruler.

Fluorescence anisotropy: The radiation emitted by a sample following excitation with polarized light can be polarized. Polarization is measured in terms of anisotropy. Zero anisotropy implies isotropic/non-polarized radiation while non-zero anisotropy implies some degree of polarization.
Figure 6.5 shows how fluorescence anisotropic measurements are made.

Figure 6.5 A schematic diagram showing the measurement of fluorescence anisotropy