FIGURE 1.

FRET principles and FRET biosensors. (A) Excitation (Ex.) and Emission (Em.) wavelength of ECFP and YPet, as a FRET donor and receptor, respectively. The emission wavelength of ECFP overlaps with the excitation wavelength of YPet, enables the energy transfer between donor and receptor when their distance is less than 10 nm. (B) The terminology used to describe and quantify the performance of FRET biosensors. The figure was modified based on previous publication (Komatsu et al., 2011). (C) The cartoon illustrates the FRET change of a Src kinase biosensor at the presence of Src kinase or phosphatase. (D) Time course quantification of the Src FRET biosensor and its loss of function mutants in response to EGF stimulation in HeLa cells. Y662F and Y664F, single loss-of-function mutations in the substrate of the biosensor; DM, double mutation (Y662F and Y664F); R175V, loss-of-function mutation in SH2 domain. (E) FRET responses of a cell with clear directional wave propagation away from the site of mechanical stimulation. In these images, a pulling force was applied via laser tweezers on a bead coated with fibronectin on the cell membrane. (C–E) were obtained from a previous publication (Wang et al., 2005).