Figure 15:

Single-color FRET-based methods.

Fluorescence quenching resonance energy transfer (FqRET)-based CaMKIIα biosensor green-Camuiα⁴²⁸. In the inactive state, the energy from excited EGFP is non-radiatively transferred to the dark acceptor REACh, which then dissipates that energy without emitting a photon. Upon activation, the conformational change moves REACh away from the EGFP, leading to increased EGFP emission. FqRET can also be quantified by fluorescence lifetime imaging (FLIM), where the lifetime, τ, is low in the high-FqRET state and vice versa. B) Homo-FRET-based NADP+ biosensor Apollo-NADP+³³⁸. Upon binding NADP+, G6PD dimerizes and thus allow FRET between the two FPs. Fluorescence polarization microscopy can be used where a polarized excitation source will only excite FP in the appropriate dipole orientation, resulting in the emitted photon also being polarized. Conversely, when FRET occurs between these two FPs, the emitted photons exhibit a mix of polarizations and thus decrease the polarized fluorescence signal.