We cultured human U87 glioblastoma cells and incubated them with compound 15 to directly image the viability of the cells in the dark as well as after light irradiation. Because photosensitizers producing singlet oxygen induce oxidative cell death via apoptosis, we used the apoptotic marker Annexin V as an indicator of the toxicity caused by compound 15. Confocal fluorescence microscopy confirmed that U87 glioblastoma cells that had taken up compound 15 (as shown by intracellular red staining, Figure ) were apoptotic (blue cell membrane staining by Annexin V-Pacific Blue, Figure ) only after light irradiation but not in the dark. On the other hand, single treatments (i.e., compound 15 only or light irradiation only) did not cause cell death as confirmed by fluorescence microscopy and quantitative flow cytometry (Figure ). We also confirmed that compound 15 would compete with unlabeled D-glucose, which drastically reduced the uptake and phototoxicity of compound 15 (Figure ). Furthermore, as a control we used 2-NBDG, the non-photosensitive nitrobenzoxadiazole glucose analogue (Figure ) and confirmed that the selenium atom in the benzoselenadiazole core is essential to cause light-induced oxidative death (Figure ). Finally, we examined the toxicity of compound 15 in 3D cultures from U87-nlsCrimson glioblastoma cells, which express the fluorescent protein E2Crimson in their nuclei as a cell viability marker (λexc/em: 610/645 nm). U87-nlsCrimson cells were cultured in ultra-low adherence plates at 37 ºC, forming spheroids of around 250 µm in diameter after 3 days.