When choosing FRET pairs, care must be taken to choose a complementary pair of fluorophores that overcome the inherent issues that affect FRET efficiency. The QY (quantum yield) of the donor, EC (extinction coefficient) of the acceptor, spectral overlap, and inter-fluorophore distance and orientation are the key elements that affect the efficiency of FRET pairs.

When choosing FRET pairs, choose the highest quantum yield donor, the highest absorbing acceptor, and fluorophores having substantial overlap in their spectral profiles. This combination will help maximize the FRET signal.