Fluorescence quenching, resonance energy transfer and anisotropy

Fluorescence spectroscopy comprises of experiments exploiting various different phenomena related to it. Discussion of all these experiments is beyond the scope of this course, but we shall have a quick look at a few important phenomena related to fluorescence.


Fluorescence quenching: A decrease in fluorescence intensity is referred to as quenching. A molecule that quenches the fluorescence of a fluorophore is called a quencher. A quencher can be either a collisional quencher or a static quencher. A collisional quencher brings about decrease in fluorescence intensity by de-exciting the excited fluorophore through collisions. Addition of another non-radiative process to the system leads to lower quantum yield. A static quencher forms a non-fluorescent complex with the fluorophore. It effectively leads to a decrease in the concentration of the fluorophore thereby decreasing the fluorescence emission intensity.


Resonance energy transfer: Resonance energy transfer (RET), also known as fluorescence resonance energy transfer (FRET) is an excited state phenomeneon wherein energy is transferred from a donor molecule (D) to an acceptor molecule (A). The prerequisite for the energy transfer is that there should be an overlap between the emission spectrum of the D and the absorption spectrum of the A (Figure 6.4).

 

Figure 6.4 Diagrammatic representation of spectral overlap between donor’s emission and acceptor’s absorption spectrum.