Overview of interaction and photophysical model. (A) In the underlying interaction, a donor-tagged protein binds an acceptor-tagged protein with dissociation constant K
. (B) During a FRET experiment, photophysical events occur such as FRET, which occurs between donors and acceptors in complex with efficiency E
, and the excitation of and emissions from donors and acceptors, where the factor relating fluorescence observed in channel i to the concentration of species S is . We include all possible spectral overlap effects, allowing the donor and acceptor to be excited and emit photons in all spectral channels.