Epifluorescence analysis of Ultramarine and ShadowR fusion proteins. (a) Tandem constructs of mEGFP (green), mRuby2 (red), and mScarlet (red) with Ultramarine or ShadowR were expressed in HeLa cells and imaged under an epifluorescence microscope. All cells in the image field were identified by Hoechst 33342 staining (blue). Scale bar, 100 µm. (b–d) The fluorescence intensities of mEGFP (b), mRuby2 (c), and mScarlet (d) in the image field were measured and divided by the cell number determined by Hoechst staining and counting. Ten images per condition were used for the analysis. Each image contains 500–1000 cells, and the data are presented as mean ± SEM. Asterisks denote statistical significance (t test, *P < 0.05, **P < 0.01, ***P < 0.001, N.S. = not significant).