Spectral FRET functionality

One way to reduce the number of independent variables in the FRET-adjusted spectral equation is to use samples with a fixed donor-to-acceptor ratio. Under these conditions, the values of d and a are no longer independent, but rather the concentration of d is now a function of a and vice-versa. This approach is typical for the situation of FRET-based biosensor constructs. These sensors normally are designed to have a donor fluorophore attached to an acceptor by a domain whose structure is altered either as a result of a biological activity (such as proteolysis or phosphorylation), or by its interaction with a specific ligand with which it has high affinity. In general, FRET based biosensors have a stoichiometry of one... 

Figure 2 FRET characteristics, (a) The FRET efficiency as a function of R/Rq is shown f = 1/(1 + (R/Rq) ). Proper selection of FRET pairs so that distances of interest lie near Rg where the FRET efficiency-distance slope is greatest will give the most sensitivity, (b) The spectral overlap requirement for FRET between the donor emission and the acceptor fluorescence spectrum is shown for the organic cyanine Cy3-Cy5 donor-acceptor pair.

---