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

Nat Biotechnol. 2016 Jul;34(7):760-7. doi: 10.1038/nbt.3550. Epub 2016 May 30.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, N.I.H., Extramural

MeSH terms

  • Fluorescent Dyes / chemical synthesis
  • Fluorescent Dyes / pharmacokinetics
  • Lighting / methods
  • Luminescent Measurements / methods*
  • Luminescent Proteins / chemical synthesis*
  • Luminescent Proteins / pharmacokinetics*
  • Microscopy, Fluorescence, Multiphoton / methods*
  • Molecular Imaging / methods*
  • Staining and Labeling

Substances

  • Fluorescent Dyes
  • Luminescent Proteins