1：Experimental Design and Method Selection:

The study designed cyanine-based fluorescent probes (probe 1 and probe 2) using a reaction-based approach involving thiolation of dinitrophenol ether to release a fluorescent dye upon H2S exposure. Theoretical models include photo-induced electron transfer (PET) for fluorescence quenching and activation.

2：Sample Selection and Data Sources:

Probes were synthesized from commercial reagents. Cell lines (e.g., Hela, HepG2) from ATCC and Chinese Academy of Sciences were used for in vitro experiments. Kunming mice from Sino-British SIPPR/BK Lab. Animal Ltd. were used for in vivo imaging.

3：List of Experimental Equipment and Materials:

Equipment includes NMR spectrometers (Bruker AVANCE 400 MHz and 100 MHz), UV-vis spectrophotometer (Hitachi U-2900), fluorescence spectrophotometer (Hitachi F-2500), LC-MS systems (Agilent Technologies 6530 Accurate-Mass Q-TOF LC-MS, Waters systems), confocal microscope (LSM710, Carl Zeiss), and in vivo imaging system with CCD camera. Materials include various chemicals (e.g., NaHS, amino acids, trackers like Lyso-Tracker Green), solvents, and cell culture media.

4：Experimental Procedures and Operational Workflow:

Synthesis of probes via reactions in acetic anhydride with sodium acetate, purification by HPLC. Photophysical characterization in PBS buffer. Selectivity tests with various analytes. Cell culture and imaging with probe incubation and H2S treatment. In vivo imaging in mice with intraperitoneal injections of probe and NaHS, followed by fluorescence measurement over time.

5：Data Analysis Methods:

Fluorescence intensities were measured and analyzed for enhancement, detection limits, and colocalization coefficients. Statistical analysis included calculation of IC50 for toxicity and overlap coefficients for subcellular localization.