Fig. 4. Cytotoxicity of UCNPs-DI in the orthogonal excitation.

a MCF7 cells were incubated with different concentrations of UCNPs-DI for 4 h, then cells were irradiated with a 980 nm PW laser (10 ns, 0.5 W/cm²) or CW laser (0.5 W/cm²) for 3 min. The relative viabilities of MCF7 cells were determined by the standard MTT assay. b Relative viabilities of cells pretreated UCNPs-DI (200 μg/mL, 4 h) recorded under different laser power density. c Intracellular ¹O₂ generation detected in SOSG-stained MCF7 cells with different treatments. Scale bar, 50 μm. d Confocal fluorescence microscope images of calcein AM and PI co-stained MCF7 cells after various treatments indicated. Green and red colors represented live and dead cells, respectively. Scale bar, 100 μm. e Flow cytometric analysis of MCF7 cells death after different treatments. In c–e, the nanoagent was used to treat the cells for 4 h at the concentration of 200 μg/mL and the 980 nm PW (10 ns) or CW laser was employed to irradiate the cells for 3 min at the power density of 0.5 W/cm². Mean values and error bars are defined as mean and SD, respectively. All data in a and b are presented as mean ± SD (n  =  3). Statistical differences p values were calculated by Student’s two-sided t-test (^nsP > 0.05).