1：Experimental Design and Method Selection:

The probe ZPAC was designed based on a FRET platform using coumarin as the donor and 4-(4-(dimethylamino)styryl)-1-methylpyridin-1-ium as the acceptor, with piperazine as the linker. The experimental methods included synthesis, spectroscopic characterization (UV-Vis, fluorescence, NMR, HRMS, IR), and biological imaging.

2：Sample Selection and Data Sources:

Probe ZPAC was synthesized from commercial materials. Hypochlorous acid and other reactive species (ROS, RNS, metal ions, biological thiols) were prepared from corresponding salts. RAW

3：7 cells were used for bioimaging. List of Experimental Equipment and Materials:

2 Equipment included Bruker Avance (300 MHz) for NMR, Q-TOF6510 mass spectrometer for HRMS, VERTEX 70 ET-IR Bruker Optics for IR, Hitachi U-4100 spectrophotometer for UV-Vis, Perkin-Elmer LS-55 luminescence spectrophotometer for fluorescence, PHS-3C pH meter, and LSM 700 laser confocal microscope for cell imaging. Materials included silica gel 60F254 for TLC, ethanol, PBS with CTAB, and various chemicals for synthesis and species preparation.

4：Experimental Procedures and Operational Workflow:

Synthesis involved multi-step reactions with purification by column chromatography. Spectroscopic measurements were conducted in PBS with CTAB at pH

5：4, with fluorescence parameters set to excitation wavelength 410 nm, slit 10/6, width Cell culture involved incubating RAW 7 cells in DMEM-H with FBS, followed by treatment with LPS and probe ZPAC, and imaging under λex = 405 nm. Data Analysis Methods:

Fluorescence intensity ratios (I472/I600 for in vitro, Iblue/Ired for cells) were used for quantitative analysis. Statistical analysis included linear regression for detection limits and co-localization coefficients for subcellular localization.