Jun Chu 1 , 2 , Young-Hee Oh 1 , 2 , 3 , Alex Sens 4 , Niloufar Ataie 4 , Hod Dana 5 , John J. Macklin 5 , Tal Laviv 6 , Erik S. Welf 7 , Kevin M. Dean 7 , Feijie Zhang 2 , 8 , Benjamin B. Kim 1 , Clement Tran Tang 4 , Michelle Hu 4 , Michelle A. Baird 9 , 10 , Michael W. Davidson 9 , 10 , Mark A. Kay 2 , 8 , Reto Fiolka 7 , Ryohei Yasuda 6 , Douglas S. Kim 5 , Ho-Leung Ng 4 , 11 , Michael Z. Lin 1 , 2 , 3
30 May 2016
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 due 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.