Fluorescence-Lifetime-Modulating Sensor for Dopamine Sensing

Measuring the precise dynamics of specific neuromodulators in neurons is essential for understanding how information is transmitted and processed in the brain. Although intensiometric neural activity sensors such as GCaMP, the Light and GRAB family are widely used, simultaneously monitoring multiple neurochemicals in live neurons with a high signal-to-background ratio remains challenging. Unlike intensity-based sensors, which are prone to artifacts from fluctuating excitation power and rapid photobleaching, fluorescence lifetime-based sensors provide more stable readouts, as demonstrated by the GRAB_ACh3.0 sensor. Here we introduce a novel genetically encodable fluorescence-lifetime-based dopamine sensor, dHaloLife, which integrates a G protein-coupled dopamine receptor with a HaloTag protein. The HaloTag domain can be labeled with cell-permeable rhodamine derivatives such as the Janelia Fluor (JF) dyes. Dopamine binding induces a conformational change in the receptor which alters the local environment of the HaloTag-conjugated fluorophore, thereby modulating its fluorescence lifetime. In live-cell experiments, our dopamine sensor dHaloLife exhibited high specificity for dopamine, showing no response to acetylcholine, serotonin, or GABA stimulation. This establishes dHaloLife as a proof of concept for lifetime-based neuromodulator sensors. Looking ahead, we envision the development of additional genetically encoded lifetime sensors with distinct fluorescence lifetime signatures and dynamic response ranges upon ligand binding, enabling multiplexed, real-time monitoring of multiple neurochemicals in live neurons and in vivo.