1：Experimental Design and Method Selection:

The probe Mito-NSH was designed based on a novel fluorophore (Mito-NOH) with a large Stokes shift, using thiolysis of dinitrophenyl ether for H2S detection. Methods included synthesis, spectroscopic analysis, selectivity tests, cytotoxicity assays, and fluorescence imaging in cells.

2：Sample Selection and Data Sources:

HeLa cells were used for bioimaging experiments, sourced from XiangYa School of Medicine, Central South University. Chemical reagents were procured from Adamas.

3：List of Experimental Equipment and Materials:

Equipment included BRUKER 400 MHz and 125 MHz NMR spectrometers, LCMS-IT-TOF mass spectrometer, Leici PHS-3C pH meter, Shimadzu UV-2550 spectrophotometer, Hitachi F-4600 fluorometer, Leica TCS-SP5 II confocal microscope, PerkinElmer Operetta CLS High-Content Analysis System, and HPLC system with YMC ODS column. Materials included DMSO, NaHS, PBS buffer, and various biological analytes.

4：Experimental Procedures and Operational Workflow:

Synthesis of compounds via chemical reactions, purification by chromatography. Absorption and fluorescence spectra were recorded in PBS buffer. Titration with NaHS for concentration dependence. Selectivity tests with various ions and molecules. Cytotoxicity assays using MTT method. Cell culture and imaging with probe incubation and H2S treatment.

5：Data Analysis Methods:

Fluorescence intensity measurements, quantum yield calculation using Rhodamine B as standard, statistical analysis for detection limit (3σ/κ), and image analysis with software like Image Pro Plus 6.0.