Bioluminescence images of U2OS cell lines expressing beetle luciferases (CBG99, CBR, Luc2) or NanoLuc at 25 and 37°C. Cells were incubated in DMEM containing 10% FBS with 5% CO2. The substrate was 1 mM beetle luciferin for beetle luciferase or 12.5 μM furimazine for NanoLuc. Images (A, B, D, E) were captured using an LV200 microscope with UPlanFLN 40× Oil objective lens (NA = 1.30, I = 0.026) and DP70 colour CCD camera. Exposure time was 2 min for beetle luciferase or 10 sec for NanoLuc cell line. The arrowheads denote 1, 2 and 3 cells expressing CBG99, CBR and Luc2 luciferase, respectively. Images (C, F) were captured using an IX70 microscope with UPlanFLN 40× Oil objective lens (NA = 1.30, I = 0.001) and DP70 colour CCD camera. Exposure time was 10 min for beetle luciferase or 1 min for NanoLuc cell line. All the images were displayed with no level adjustment. Scale bars, 100 μm (A, B, D, E) and 20μm (C, D).