Absolute measurement of fast and slow neuronal signals with fluorescence lifetime photometry at high temporal resolution.

Dynamic signaling by extracellular and intracellular molecules impacts downstream pathways in a cell-type-specific manner. Fluorescent reporters of such signals are typically optimized to detect fast, relative changes in concentration of target molecules. They are less well suited to detect slowly changing signals and rarely provide absolute measurements. Here, we developed fluorescence lifetime photometry at high temporal resolution (FLIPR), which utilizes frequency-domain analog processing to measure the absolute fluorescence lifetime of genetically encoded sensors at high speed but with long-term stability and picosecond precision. We applied FLIPR to investigate dopamine signaling in functionally distinct striatal subregions. We observed higher tonic dopamine levels in the tail of the striatum compared with the nucleus accumbens core and differential and dynamic responses in phasic and tonic dopamine to appetitive and aversive stimuli. Thus, FLIPR reports fast and slow timescale neuronal signaling in absolute units, revealing previously unappreciated spatial and temporal variation even in well-studied signaling systems.