Fluorescence lifetime enables high-resolution analysis of neuromodulator dynamics across time and animals

Optical sensors have transformed the field of neuromodulation because neuromodulator dynamics are essential for their function. Despite their high spatial and temporal resolution, these fluorescence intensity-based sensors are sensitive to sensor expression level and excitation light fluctuation, thus preventing analysis of neuromodulators across time or animals. Here, we screened neuromodulator sensors and discovered that multiple sensors showed response in fluorescence lifetime, a property independent of sensor expression or excitation light power. The acetylcholine sensor GRAB-ACh3.0 showed the largest lifetime change. Fluorescence lifetime of GRAB-ACh3.0 responds to transient ACh release, is dose sensitive, and is insensitive to excitation laser power. In mice across sleep/wake and running/resting states, fluorescence lifetime, in contrast to intensity, predicts behavior states accurately despite change in sensor expression level across weeks and animals. Thus, fluorescence lifetime of neuromodulator sensors enables comparison of neuromodulator dynamics at high resolution across different animals, brain regions, disease models, and chronic time scales.