A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo

Jun Chu, Younghee Oh, Alex Sens, Niloufar Ataie, Hod Dana, John J. Macklin, Tal Laviv, Erik S. Welf, Kevin M. Dean, Feijie Zhang, Benjamin B. Kim, Clement Tran Tang, Michelle Hu, Michelle A. Baird, Michael W. Davidson, Mark A. Kay, Reto Fiolka, Ryohei Yasuda, Douglas S. Kim, Ho Leung NgMichael Z. Lin

Research output: Contribution to journalArticle

86 Scopus citations

Abstract

Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals owing to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright, engineered, orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins.

Original languageEnglish (US)
Pages (from-to)760-767
Number of pages8
JournalNature biotechnology
Volume34
Issue number7
DOIs
StatePublished - Jul 1 2016

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology
  • Molecular Medicine
  • Biomedical Engineering

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    Chu, J., Oh, Y., Sens, A., Ataie, N., Dana, H., Macklin, J. J., Laviv, T., Welf, E. S., Dean, K. M., Zhang, F., Kim, B. B., Tang, C. T., Hu, M., Baird, M. A., Davidson, M. W., Kay, M. A., Fiolka, R., Yasuda, R., Kim, D. S., ... Lin, M. Z. (2016). A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo. Nature biotechnology, 34(7), 760-767. https://doi.org/10.1038/nbt.3550