What factors affect the degree of spectral overlap?

The extent to which the emission spectrum of the donor fluorophore overlaps with the absorption spectrum of the acceptor fluorophore directly affects the efficiency of FRET. Greater spectral overlap enhances the probability of energy transfer. The efficiency of energy transfer relies on several factors, including how much the emission spectrum of the donor overlaps with the absorption spectrum of the acceptor, how the donor and acceptor molecules are oriented relative to each other, the brightness of the donor and the distance between them.