Neuromodulators such as acetylcholine are chemicals made in the brain that can transform animal behavior. Neuromodulator dynamics are essential for their functions. Optical sensors have transformed the study of neuromodulators because they capture their dynamics with high spatial and temporal resolution. However, fluorescence intensity-based sensors are restricted to measure acute changes within one animal over a short period because intensity varies with sensor expression level and excitation light fluctuation. In contrast, fluorescence lifetime is impervious to sensor expression level or excitation light power, allowing comparison between individuals and across long periods. Here, we discover fluorescence lifetime response in multiple intensity-based neuromodulator sensors, and show that fluorescence lifetime predicts animal behavior states accurately despite varying excitation power or changes in sensor expression level across weeks and animals.
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