We report a pH-insensitive and photostable ultramarine fluorescent protein, Sirius, with an emission peak at 424 nm, the shortest emission wavelength among fluorescent proteins reported to date. The pH-insensitivity of Sirius allowed prolonged visualization of biological events in an acidic environment. Two fluorescence resonance energy transfer (FRET) pairs, Sirius-mseCFP and Sapphire-DsRed, allowed dual-FRET imaging with single-wavelength excitation, enabling detection of Ca2+ concentration and caspase-3 activation in the same apoptotic cells.
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We thank R.E. Campbell (University of Alberta) and A. Miyawaki (RIKEN) for providing the cDNA encoding EBFP2 and SapRC2, respectively. This work was partly supported by grants from Scientific Research on Advanced Medical Technology of the Ministry of Labor, Health and Welfare of Japan and the Japanese Ministry of Education, Science and Technology.
Supplementary Figures 1–7, Supplementary Table 1, Supplementary Notes 1–2 and Supplementary Methods (PDF 2041 kb)
Imaging the phagocytosis of bacteria by Dictyostelium discoidium. Tracking of bacteria expressing EGFP (green color in left panel) and Sirius (cyan color in right panel) phagocytosed by a Dictyostelium cell. (MOV 3573 kb)
Dual functional imaging using two FRET-based indicators. Dual FRET ratio imaging of HeLa cells co-expressing two FRET indicators, SC-SCAT3 (left) and SapRC2 (right). (MOV 2915 kb)
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Tomosugi, W., Matsuda, T., Tani, T. et al. An ultramarine fluorescent protein with increased photostability and pH insensitivity. Nat Methods 6, 351–353 (2009). https://doi.org/10.1038/nmeth.1317
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