Two-photon fluorescence imaging and specifically biosensing of norepinephrine on a 100-ms timescale

Norepinephrine (NE) is a key neurotransmitter in the central nervous system of organisms; however, specifically tracking the transient NE dynamics with high spatiotemporal resolution in living systems remains a great challenge. Herein, we develop a small molecular fluorescent probe that can precisely anchor on neuronal cytomembranes and specifically respond to NE on a 100-ms timescale. A unique dual acceleration mechanism of molecular-folding and water-bridging is disclosed, which boosts the reaction kinetics by ˃10⁵ and ˃10³ times, respectively. Benefiting from its excellent spatiotemporal resolution, the probe is applied to monitor NE dynamics at the single-neuron level, thereby, successfully snapshotting the fast fluctuation of NE levels at neuronal cytomembranes within 2 s. Moreover, two-photon fluorescence imaging of acute brain tissue slices reveals a close correlation between downregulated NE levels and Alzheimer’s disease pathology as well as antioxidant therapy.