1：Experimental Design and Method Selection:

The study involved designing and synthesizing two pH ?uorescent probes based on indole derivatives and the Tr?ger's Base framework via Knoevenagel reaction. Theoretical calculations using density functional theory (DFT) with basis set 6-31G(d) in Gaussian 09 were employed to understand electronic properties.

2：Sample Selection and Data Sources:

Probes 1 and 2 were synthesized and characterized. Stock solutions (10 μM) were prepared in DMSO/H2O (4/1, v/v). pH values were adjusted using hydrochloric acid and sodium hydroxide. Metal ions and small bioactive molecules were tested for selectivity.

3：List of Experimental Equipment and Materials:

Equipment included Bruker AVANCE III MR spectrometer for NMR, Agilent 6210 ESI/TOF mass spectrometer for HRMS, Edinburgh FLS920 TCSPC ?uorescence spectrophotometer for PL spectra, Cary 60 UV-Vis spectrophotometer for absorption spectra, Edinburgh Instruments FLS 980-STM for fluorescence lifetimes, Hamamatsu UV-NIR absolute PL quantum yield spectrometer C13534 for quantum yields, Milli-Q water purification system for deionized water, and PHSJ-3F pH meter for pH measurements. Materials included reagents from Adamas-beta, solvents like DMSO, and synthesized compounds.

4：Experimental Procedures and Operational Workflow:

Synthesis involved reacting TB2 with indole derivatives under nitrogen atmosphere at 85°C for 24 hours, followed by purification via column chromatography. Spectroscopic experiments were conducted at room temperature with 10 μM concentration, using specific excitation wavelengths (370 nm for 1, 380 nm for 2) and slit settings. pH titration, reversibility, photostability, and selectivity tests were performed.

5：Data Analysis Methods:

Data analysis included NMR and HRMS for characterization, UV-vis and fluorescence spectra for photophysical properties, linear regression for pH response (e.g., R2 = 0.9920 for probe 1), and DFT calculations for orbital distributions.