1：Experimental Design and Method Selection:

The study involved designing and synthesizing a new fluorogenic molecule TPA-BTTDO with aggregation-induced emission (AIE) and twisted intramolecular charge transfer (TICT) properties. Nanoparticles (NPs) were fabricated by encapsulating TPA-BTTDO within a DSPE-PEG2000 matrix using nanoprecipitation to enhance water dispersibility and biocompatibility for bioimaging applications.

2：Sample Selection and Data Sources:

HeLa cells, NIH 3T3 cells, and ktr-3 cells were used as biological samples. Commercial probes like Lysotracker DND 99, BODIPY, and Mitotracker were employed for colocalization studies.

3：List of Experimental Equipment and Materials:

Instruments included a Bruker AV 500 spectrometer for NMR, GCT premier CAB048 mass spectrometer for HRMS, Shimadzu UV-2600 spectrophotometer for UV-vis, Horiba Fluoromax-4 spectrofluorometer for PL, Hamamatsu C11347 Quantaurus_QY for quantum yields, Zeiss LSM 710 confocal microscope for imaging, Malvern ZSE for DLS, Qsonica Sonicators for nanoparticle preparation, Invitrogen Countess II for cell counting, JEOL JEM-1400 Plus TEM for morphology, and Thermo Fisher microplate reader for MTT assays. Materials included TPA-BTTDO, DSPE-PEG2000, solvents (THF, etc.), cell culture reagents (DMEM, FBS), and inhibitors (e.g., dynasore).

4：Experimental Procedures and Operational Workflow:

Synthesis of TPA-BTTDO via Pd-catalyzed coupling, preparation of NPs by mixing TPA-BTTDO and DSPE-PEG2000 in THF, adding water under ultrasound, removing THF with N2, and dialysis. Cell viability was assessed using MTT assay. Cells were incubated with NPs, co-stained with commercial probes, and imaged using CLSM. Internalization kinetics, endocytosis inhibition assays (using low temperature and inhibitors), and time-dependent imaging were performed. Lambda-scanning was used for emission analysis.

5：Data Analysis Methods:

Colocalization was quantified using overlap and Pearson correlation coefficients. Statistical analysis of cell viability and photostability was conducted. Spectra were analyzed for emission shifts and quantum yields.