1：Experimental Design and Method Selection:

Utilized a one-step condensation reaction to synthesize ESIPT-active AIEgens from 2-(hydrazonomethyl)phenol and benzaldehyde derivatives. Theoretical calculations (TD-DFT at B3LYP/6-31G* level) were employed to understand photophysical mechanisms.

2：Sample Selection and Data Sources:

HeLa cells were used for biological imaging and cytotoxicity studies. Commercial probes (e.g., Nile red, LysoTracker Deep Red) were used for co-localization assays.

3：List of Experimental Equipment and Materials:

NMR spectrometer (Varian VNMRS 400), UV-Vis spectrometer (Shimadzu UV-2600), fluorescence spectrometer (Perkin-Elmer LS 55), quantum yield analyzer (Hamamatsu C11347-11), lifetime instrument (Edinburgh FLS 980), confocal microscope (LSM710), plate reader (Perkin-Elmer Victor3TM). Chemicals purchased from Energy Chemical, Derthon, Meryer, Bide Pharmatech, Sigma-Aldrich.

4：Experimental Procedures and Operational Workflow:

Synthesis involved stirring reactants in ethanol, filtration, and purification. Cell imaging involved incubating cells with probes, rinsing, and imaging. ROS generation analysis used DCFH-DA as an indicator with white LED irradiation. Cytotoxicity was assessed via MTT assay.

5：Data Analysis Methods:

NMR, HRMS, single crystal X-ray diffraction for characterization. Fluorescence spectra, quantum yields, lifetimes, and statistical analysis (e.g., Pearson correlation coefficient for co-localization) were performed.