1：Experimental Design and Method Selection:

The probe DCM-OH was designed based on dicyanomethylene-4H-pyrans (DCM dyes) for detecting ONOO?. The synthesis involved multi-step organic reactions, and the detection mechanism was verified through ESI-MS, NMR, and HPLC analysis. Fluorescence and UV-vis spectroscopy were used to characterize the probe's response to ONOO?. Cell imaging and in vivo imaging were performed to assess biological applications.

2：Sample Selection and Data Sources:

Chemical reagents were purchased from commercial suppliers. HepG2 and RAW264.7 cells were cultured in DMEM medium. Kunming mice were used for in vivo studies. ONOO? was generated using SIN-1 as a donor, and other ROS/RNS analytes were used for selectivity tests.

3：7 cells were cultured in DMEM medium. Kunming mice were used for in vivo studies. ONOO? was generated using SIN-1 as a donor, and other ROS/RNS analytes were used for selectivity tests. List of Experimental Equipment and Materials:

3. List of Experimental Equipment and Materials: Equipment included Bruker AV-400 NMR spectrometer, Agilent Q-TOF mass spectrometer, SHIMADZU UV-3600 spectrophotometer, Hitachi F-7000 Fluorescence Spectrophotometer, Leica SP8 confocal laser scanning microscope, and PerkinElmer IVIS in vivo imaging system. Materials included silica gel for chromatography, various chemicals for synthesis, DMEM medium, fetal bovine serum, penicillin-streptomycin, LPS, IFN-γ, SIN-1, AG, TEMPO, and MTT assay reagents.

4：Experimental Procedures and Operational Workflow:

Synthesis of DCM-OH involved steps from compound 1 to final product with purification by column chromatography. Spectral measurements were conducted in PBS buffer. Cell imaging involved pretreatment with probes and stimulants/inhibitors, followed by confocal microscopy. In vivo imaging involved LPS-induced inflammation in mice, followed by probe injection and imaging with IVIS.

5：Data Analysis Methods:

Fluorescence intensity data were analyzed for detection limit calculation (3σ/k). Statistical analysis included linear regression for concentration-response curves. Cell viability was assessed using MTT assay. Imaging data were processed for fluorescence intensity comparisons.