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Keywords:

  • fluorescence;
  • FRET (fluorescence resonant energy transfer);
  • kinases;
  • microscopy
Thumbnail image of graphical abstract

Donor, acceptor, and tuner: FRET-based ratiometric live-cell imaging captures emissions of two fluorophores within the constraints of a FRET pair. A method for engineering FRET-circuit-based biosensors has been developed by introducing an intermediate fluorophore, which facilitates energy transfer and allows tuning of the circuit efficiency. A protein kinase A biosensor was thus generated with a larger dynamic range than its precursors.