Predicted FRET efficiencies fluorescent protein pairs.

The Overlap Integral (J) of the spectra for each pair was calculated using the freeware program a|e—UV-Vis-IR Spectral Software (http://www.fluortools.com/software/ae), which uses the normalised emission spectrum of the donor and the normalised excitation spectrum of the acceptor corrected for the published Extinction Coefficient (as detailed in Table 1) to determine the value of J in nm4/M.cm. The sources of each spectral dataset are detailed in the Supporting Information. The Forster Radius (r0) for the pair was then calculated according to the equation detailed in the Materials and Methods section. Once r0 was established an expected FRET Efficiciency at a given separation distance (r) was easily calculated according to the relationship E = r06/(r06+r6).