(A) Strategy of fluorescent imaging of the activated SARM1. (B) Designing based on pyridine and styryl derivatives with a donor-π-acceptor framework. (C) Structure of PC6. (D) The kinetics of the fluorescence increase at the maximal absorbance wavelengths catalyzed by SARM1-dN, in the presence of 100 μM NMN, 100 μM NAD, and 50 μM PCs. (E) Time-dependent changes of the emission spectra at the isosbestic point (350 nm). (F) HPLC analysis of PC6 reaction. Red line: in the presence of PC6, NMN, and NAD; Gray line: without PC6. Insert: MS analysis and structure of PAD6. (G) Absorbance and fluorescence spectra of 25 μM PC6/PAD6. (H) Emission spectra with dose of NMN (10, 20, 40, 60 μM) in the presence of NAD, PC6, and SARM1-dN. Inset: the initial rates plotted to NMN concentrations. (I) Emission spectra with doses of SARM1-dN in the presence of NMN, NAD, and PC6; Inset: the initial rate plotted to SARM1 concentrations. (J) The reaction rates of 10 μM PC6, in the presence of 100 μM NMN and 100 μM NAD, compared with NAD analogss (100 μM) catalyzed by SARM1. (K) The reaction rates of 10 μM PC6 catalyzed by SARM1, NADase, and CD38. PC = pyridine conjugate, NMN = nicotinamide mononucleotide.