1：Experimental Design and Method Selection:

The study involved synthesizing asymmetric diarylethenes (DAEs) with push-pull substituents, characterizing their photophysical properties, and evaluating their performance in photoswitching and microscopy. Methods included synthesis via iodine-lithium exchange and Pd-catalyzed reactions, photophysical measurements (absorption, emission, quantum yields), fatigue resistance testing by alternating UV and visible light irradiation, and microscopy (confocal and RESOLFT).

2：Sample Selection and Data Sources:

Samples were synthesized DAEs (e.g., 1-MeO,N, 1-MeO,COMe, 1-CN,N, 1-CN,COMe, 1-H,CH) in solvents like acetonitrile, methanol, and water. Bioconjugates with BSA and antibodies were prepared for microscopy studies.

3：List of Experimental Equipment and Materials:

Equipment included HPLC for analysis, spectrometers for absorption and emission measurements, lasers (365 nm, 470 nm, 355 nm, 488 nm) for irradiation, a Leica SP5 confocal microscope, and a modified 1C RESOLFT QUAD Scanning microscope. Materials included solvents, reagents for synthesis (e.g., N-hydroxysuccinimide, EDCI), and biological samples (BSA, antibodies, Vero cells).

4：Experimental Procedures and Operational Workflow:

Synthesis followed specific routes (Schemes 1 and 2). Photophysical properties were measured by irradiating solutions to photostationary states and analyzing with HPLC and spectroscopy. Fatigue resistance was tested by cyclic irradiation and monitoring absorption/emission. Microscopy involved immunostaining cells, switching dyes with lasers, and imaging.

5：2). Photophysical properties were measured by irradiating solutions to photostationary states and analyzing with HPLC and spectroscopy. Fatigue resistance was tested by cyclic irradiation and monitoring absorption/emission. Microscopy involved immunostaining cells, switching dyes with lasers, and imaging. Data Analysis Methods:

5. Data Analysis Methods: Data were analyzed using HPLC, spectroscopy for quantum yields and lifetimes, exponential fitting for kinetics, and Lorentzian fitting for microscopy resolution (FWHM).