1：Experimental Design and Method Selection:

The study employs a hydroxyl protection and deprotection strategy using an ESIPT-based fluorophore (HBTN) linked to a levulinyl group to create a probe (HBTN-Se) that responds selectively to sodium selenite, enabling ratiometric fluorescence imaging.

2：Sample Selection and Data Sources:

HepG2 (cancer) and L-02 (normal) cells were cultured in DMEM with FBS, penicillin, and streptomycin. Sodium selenite and other analytes were prepared as stock solutions.

3：List of Experimental Equipment and Materials:

Instruments include Bruker Ascend 400M and 700M NMR spectrometers, Bruker Apex IV FTMS for MS, Beijing Purkinje TU-1901 UV-visible spectrophotometer, Hitachi F-7000 fluorescence spectrophotometer, OLYPUS inverted fluorescence microscope, and Thermo Scientific Multiskan FC microplate reader. Materials include HBTN-Se, Na2SeO3, various analytes, ethanol, PBS buffer, DMEM, FBS, penicillin, streptomycin, MTT, DMSO, and reagents for synthesis.

4：Experimental Procedures and Operational Workflow:

Synthesis of HBTN-Se via reaction of HBTN with levulinic acid. Optical studies involved adding analytes to HBTN-Se solution and measuring spectra after 5 min. Cell imaging involved treating cells with Na2SeO3 and HBTN-Se, followed by fluorescence microscopy. Cytotoxicity was assessed using MTT assay.

5：Data Analysis Methods:

Fluorescence ratios (I475nm/I410nm) were calculated for quantitative detection. Statistical analysis included mean ± standard deviation from triplicate experiments. NMR and MS were used for mechanism confirmation.