1：Experimental Design and Method Selection:

The study involved synthesizing a new styryl dye (probe 2) and characterizing its optical properties, followed by application in live cell imaging to assess mitochondrial selectivity and biocompatibility. Theoretical models include intramolecular charge transfer (ICT) for explaining photophysical properties.

2：Sample Selection and Data Sources:

Chemical synthesis used reagents from Acros Organics. Cell studies used COS-7 (normal) and A549 (cancer) cell lines cultured in DMEM and RPMI media, respectively, with reagents from Sigma-Aldrich and Fisher Scientific.

3：List of Experimental Equipment and Materials:

Instruments included Hewlett Packard-8453 diode array spectrophotometer for UV-vis studies, HORIBA Fluoromax-4 spectrofluorometer for fluorescence studies, Zeiss LSM 710 confocal microscope with 63x oil objectives for imaging, Spectramax 5e microplate reader for viability assays. Materials included commercial dyes (MitoTracker Green FM, LysoTracker Green DND-26), CellTiter-Glo Luminescent cell viability assay kit, and various chemicals for synthesis.

4：Experimental Procedures and Operational Workflow:

Synthesis of probe 2 via reaction of dialdehyde with 4-methylpyridinium salt in ethanol, purification by precipitation. Optical characterization in solvents like DCM, DMSO, ethanol, water. Cell staining with 500 nM probe 2 for 30 minutes at 37°C, imaging without washing for wash-free assessment. Co-localization studies with MitoTracker Green and LysoTracker Green. Cell viability testing using CellTiter-Glo assay.

5：Data Analysis Methods:

Fluorescence quantum yields calculated using Rhodamine 6G as standard. Co-localization analyzed with Zeiss LSM software and ImageJ for Mander's overlap coefficient. Cell viability LC50 calculated using dose-response formula in Origin8 software.