What should I consider when selecting donor/acceptor FRET pairs?

There are several factors one should consider when selecting donor/acceptor FRET pairs. 

1. One factor is that the emission spectrum of the donor fluorophore should have overlap with the excitation spectrum of the acceptor fluorophore. This ensures efficient energy transfer between the two fluorophores. Thus, one should choose fluorophores with significant spectral overlap to maximize FRET efficiency.  
2. Another factor is that the acceptor fluorophore should have a high absorption coefficient in order to efficiently capture energy transferred from the donor. Acceptor fluorophores with high absorption properties, such as red fluorescent protein (RFP) variants or Cy5, are preferred for FRET experiments because they efficiently absorb photons emitted by the donor, maximizing energy transfer between the donor and acceptor fluorophores. 
3. A third factor is quantum yield, which refers to the efficiency with which a fluorophore converts absorbed photons into emitted photons. In the context of FRET, selecting a donor fluorophore with a high quantum yield is crucial because it ensures that a large proportion of absorbed photons are converted into fluorescence emission. This means that more energy is available for transfer to the acceptor fluorophore. Donors with high quantum yields, such as green fluorescent protein (GFP) variants or cyan fluorescent protein (CFP), are commonly chosen for FRET experiments because they provide strong fluorescence signals, contributing to efficient FRET measurements.