A highly sensitive genetically encoded red cAMP sensor for multiplex imaging in vivo.

Circularly permuted green fluorescent protein (cpGFP)-based high-performance cAMP sensors have enabled real-time monitoring of cAMP dynamics with high spatiotemporal resolution in living animals. However, their utility is hampered by significant spectral overlap with other green/yellow fluorescent indicators and blue/cyan light-activated optogenetic actuators, limiting their compatibility in multiplexed imaging applications. While existing red cAMP sensors offer great spectral separation, they often suffer from a limited dynamic range ( < 1.5-fold in HEK293T cells), low cellular brightness, aggregation, or significant blue-light-induced photoactivation. Here, we report R-Flamp1, a red cAMP sensor with a large dynamic range ( > 10-fold in HEK293T cells), enhanced cellular brightness, appropriate cAMP affinity (Kd ~1.9 μM), subsecond response kinetics, and minimal photoactivation under blue or cyan light exposure. Using R-Flamp1, we visualized region-specific cAMP dynamics, and when paired with green indicators, revealed differential activation patterns between cAMP and neuromodulators or calcium using two-photon imaging and fiber photometry during various behaviors. These findings provide valuable insights into the role of cAMP signaling in complex behaviors.