Figure 4.

Two-photon excitation spectra. Superimposed excitation spectra obtained in BHK cells expressing constructs that incorporate either 2 active chromophores (GAkdY), an active and an inactivated chromophore (GAkdYmut and GmutAkdY), or a single active chromophore (GAk and dY). The dY spectrum was obtained from a construct expressing only the dark-YFP domain of GAkdY. Note that the GAkdY spectrum exhibited 2 discrete peaks at 920 and 960 nm. The latter was due to fluorescence emission by its dark-YFP moiety since both the GmutAkdY sensor, which contains the chromophore-inactivating glycine triplet (positions 65–67) in its GFP moiety, and dark-YFP alone showed a single fluorescence peak at 960 nm. Conversely, GAkdYmut and GAk exhibited almost identical spectra with a single peak at 920 nm.