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

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

Author information

1
Department of Bioengineering, Stanford University, Stanford, California, USA.
2
Department of Pediatrics, Stanford University, Stanford, California, USA.
3
Department of Neurobiology, Stanford University, Stanford, California, USA.
4
Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii, USA.
5
Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, Virginia, USA.
6
Max Planck Florida Institute, Jupiter, Florida, USA.
7
Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
8
Department of Genetics, Stanford University, Stanford, California, USA.
9
Department of Biological Science, Florida State University, Tallahassee, Florida, USA.
10
National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida, USA.
11
University of Hawaii Cancer Center, Honolulu, Hawaii, USA.

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.

PMID:
27240196
PMCID:
PMC4942401
DOI:
10.1038/nbt.3550
[Indexed for MEDLINE]
Free PMC Article

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