1：Experimental Design and Method Selection:

The study involved synthesizing Iso-PG based on a previously described strategy, followed by photophysical characterization using steady-state and time-resolved fluorescence measurements to investigate ground-state equilibria, excited-state proton transfer (ESPT) reactions, and applicability in fluorescence lifetime imaging microscopy (FLIM). Theoretical models for acid-base equilibria and ESPT kinetics were employed.

2：Sample Selection and Data Sources:

Aqueous solutions of Iso-PG were prepared at various pH values (3.48 to 9.73) and acetate buffer concentrations (0 to 0.7 M). Artificial serum was used for FLIM studies. Data were acquired from absorption and fluorescence spectra.

3：48 to 73) and acetate buffer concentrations (0 to 7 M). Artificial serum was used for FLIM studies. Data were acquired from absorption and fluorescence spectra. List of Experimental Equipment and Materials:

3. List of Experimental Equipment and Materials: Equipment included spectrophotometers for absorption and fluorescence measurements, time-resolved fluorescence instrumentation for lifetime studies, and FLIM setup. Materials included Iso-PG dye, buffers, and synthetic serum components.

4：Experimental Procedures and Operational Workflow:

Synthesis of Iso-PG was performed via organolithium addition and hydrolysis. Absorption spectra were recorded as a function of pH. Steady-state fluorescence was measured at different pH values. Time-resolved fluorescence decays were collected at various excitation and emission wavelengths, pH values, and buffer concentrations. FLIM images were obtained in artificial serum with varying acetate concentrations.

5：Data Analysis Methods:

Global fitting of absorbance data to acid-base equilibrium equations determined pKa and molar absorption coefficients. Fluorescence data were fitted to kinetic models for ESPT reactions to extract rate constants. FLIM data were analyzed for average lifetime changes.