1：Experimental Design and Method Selection:

The study involved characterizing two-photon absorption cross-sections (σ2PA) using an automated z-scan setup, assessing cellular uptake via confocal laser scanning microscopy (LSM), and quantifying phototoxicity using the PrestoBlue metabolic assay in a 3D hydrogel-based osteosarcoma cell culture. The design included testing three TPE-PS (P2CK, Eosin Y, TPP) at optimal wavelengths determined by z-scan.

2：Sample Selection and Data Sources:

MG63 osteosarcoma cells and adipose-derived stem cells (ASC/TERT) were used. Cells were encapsulated in gelatin-norbornene (GelNB) hydrogels. Photosensitizers were dissolved in PBS or DMSO at specified concentrations.

3：List of Experimental Equipment and Materials:

Equipment included a PerkinElmer Lambda 750 spectrometer for UV-VIS spectra, a home-built z-scan setup with a MaiTai DeepSee femtosecond laser, LSM 700 confocal microscope, BioTek Synergy H1 plate reader, and various objectives (e.g., ZEISS 10×/0.3 NA, 2.5×/0.085 NA). Materials included GelNB hydrogel, dithiothreitol (DTT), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (Li-TPO), and photosensitizers (P2CK, Eosin Y, TPP).

4：3 NA, 5×/085 NA). Materials included GelNB hydrogel, dithiothreitol (DTT), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (Li-TPO), and photosensitizers (P2CK, Eosin Y, TPP). Experimental Procedures and Operational Workflow:

4. Experimental Procedures and Operational Workflow: Z-scan measurements were performed at multiple wavelengths and powers. Cells were encapsulated in hydrogels, incubated with PS, irradiated with two-photon laser at specific wavelengths and intensities, and assessed for viability using PrestoBlue assay. Cellular uptake was imaged with LSM after PS incubation. Co-culture experiments involved irradiating spheroids and monitoring damage localization.

5：Data Analysis Methods:

σ2PA was calculated from z-scan data. Cell viability was quantified using PrestoBlue fluorescence. Statistical analysis used one-way ANOVA with Bonferroni post-test.