Zhong, C.; Arai, S.; Okada, Y.

Single fluorescent protein (FP)-based FLIM (fluorescence lifetime imaging) biosensors are potent tools for quantitatively imaging intracellular processes with high spatial and temporal resolution. They only require a single wavelength for detection, which facilitates multi-color imaging. However, the development of single FP-based FLIM biosensors has been limited by the absence of a general design framework and the complexity of the screening process. In this study, we engineered FLIM biosensors capable of detecting ATP (adenosine triphosphate), cAMP (cyclic adenosine monophosphate), citrate, and glucose by inserting each sensing domain into the mTurquoise2 fluorescent protein between Tyr-145 and Phe-146 using peptide linkers. Through efficient linker screening, we successfully developed FLIM biosensors exhibiting an effective dynamic range from 0.5 to 1.0 ns upon analyte binding. This demonstrates that the qmTQ2-ATP-0 backbone is a universal platform for developing mTQ2-based biosensors. As a proof-of-concept, we demonstrated the capabilities of these FLIM biosensors in monitoring the intracellular dynamics of ATP and cAMP alongside dual-color imaging. Therefore, our work presents an accessible methodology for establishing a single FP-based FLIM biosensor platform for quantitative imaging.