1：Experimental Design and Method Selection:

The study involved designing and synthesizing a novel fluorescent probe DCM-ML based on dicyanomethylene-4H-pyran with a morpholine group for lysosome targeting. Photophysical properties were characterized using spectroscopy, and biological imaging was performed using confocal microscopy (one- and two-photon excitation).

2：Sample Selection and Data Sources:

Cell lines used include HeLa, MCF-7, and A549. Commercial dyes like Lysotracker DND-99 and Mitotracker MTDR were used for colocalization studies. Stimulants such as chloroquine and dexamethasone were applied to induce pH changes and apoptosis.

3：Commercial dyes like Lysotracker DND-99 and Mitotracker MTDR were used for colocalization studies. Stimulants such as chloroquine and dexamethasone were applied to induce pH changes and apoptosis. List of Experimental Equipment and Materials:

3. List of Experimental Equipment and Materials: Equipment includes confocal microscope, spectrophotometer, and MTT assay kit. Materials include DCM-ML, cell culture reagents, glycerol for viscosity control, and buffers for pH titration.

4：Experimental Procedures and Operational Workflow:

Synthesis of DCM-ML, measurement of fluorescence in solutions of varying viscosity and pH, cytotoxicity assessment via MTT assay, cell staining and imaging, colocalization with commercial trackers, time-lapse imaging under stimulant treatment, and photostability tests.

5：Data Analysis Methods:

Data analysis involved calculating fluorescence intensity, quantum yield, Pearson's coefficient for colocalization, and statistical analysis of cytotoxicity. DFT and TD-DFT calculations were performed using Gaussian 09 software for mechanistic insights.