A1r hd multiphoton confocal laser scanning microscope

HeLa cells (cancerous cervical tumor cell line) should be incubated in a 35 mm confocal culture dish at 37 °C for 12 h firstly. After cells adhered onto the surface of the dish and reached a density of 5 × 10⁴ cells per dish, they could be treated with 2 mL DMEM, including Ru-Tb NPs (30 μg mL⁻¹) for 6 h. The supernatant cell culture medium should be removed and the cells were washed by PBS three times before microscopic measurements. Unlike the adhered cells, the 3D multi-cellular tumor spheroids (MCTs) should be cultured for 7 days in a normal dish whose bottom was carpeted with agarose. Then the MCTs were treated with the same DMEM consisting Ru-Tb NPs for 12 h and transferred to the confocal dish before imaging. In all the experiments, luminescence and differential interference contrast (DIC) images were recorded with a Nikon A1R HD multiphoton confocal laser scanning microscope. Emission wavelengths were collected at 585–625 nm (red channel, for Ru-Poly) as the cells were excited at 488 nm, while the emissions at 525–565 nm (green channel, for Tb-Poly) were monitored under mercury lamp excitation. The same two emission channels were observed under two-photon excitation at 720 nm, while scanning Z-stack of MCTs.

Hela (human cervical cancer) were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and cultured in an atmosphere containing 5% CO₂ at 37 °C for intracellular fluorescence imaging. The cells were cultured in 6 cm culture dishes with 4 mL of H-DMEM medium containing 10% (v/v) FBS. During the culture of cells, a fresh medium was replaced every two days. Then the Hela cells were seeded in confocal culture dishes with a density of 1 × 10⁵ cells. 24 h later, the original culture medium was substituted with 2 mL new culture medium containing 200 μL of NPs, followed by 12 h incubation to load these NPs into cells. Then, the cells were washed three times with PBS (pH 7.4) before microscopy viewing. Intracellular fluorescent imaging was conducted on a Nikon A1R HD multiphoton confocal laser scanning microscope. The NPs were respectively excited by 638 nm, 405 nm, 405 nm, 405 nm, 638 nm, and 561 nm with emission collected at 665–705 nm, 500–550 nm, 581–654 nm, 581–654 nm, 765–900 nm, and 581–654 nm (ZnPc, C6, PpIX, NAPP, ICG, RhB). In all the experiments, fluorescent images were collected with a 40× immersion objective.