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Developing a novel tool capable of real-time monitoring and accurate quantification of NO is critical to understanding its role in physiological and pathological processes. Herein, a two-photon ratiometric fluorescent probe (NOP) was developed for real-time imaging and quantification of NO based on fluorescence resonance energy transfer-photoinduced electron transfer (FRET-PET). In this developed probe, coumarin (CM) and naphthalimide with o -phenylenediamine (NPM) were rationally designed as a fluorescent donor and acceptor, respectively, to enable a ratiometric fluorescence response to NO. The developed NO probe demonstrated good detection linearity with the concentration of NO in the range of 0.100-200 μM, with a detection limit of 19.5 ± 1.00 nM. Considering the advantages of high selectivity, good accuracy and rapid dynamic response (<15 s), the developed NO probe was successfully applied for real-time imaging and accurate quantification of NO in neural stem cells (NSCs) and different regions of mouse brain tissue with a penetration depth of 350 μm. Using this powerful tool, it was found that NO regulated the activation and differentiation of quiescent NSCs (qNSCs). In addition, NO-induced differentiation of qNSCs into neurons was found to be dose-dependent: 50.0 μM NO caused about 50.0% of qNSCs to differentiate into neurons. Moreover, different regions of the mouse brain were observed to be closely related to the concentration of NO, and the concentration of NO in the DG region was found to be lower than that in the S1BF, CA1, LD and CPu of the Alzheimer's disease (AD) mouse brain. The symptoms of AD mice were significantly improved through the treatment with NO-activated NSCs in the DG region., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)