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

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 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.

Imaging Adenosine Triphosphate (ATP)

“…These BRET sensors have been developed to improve brightness and red-shifted, luciferase-based imaging (Chu et al , 2016). Saito et al developed an ATP sensor, Nano-lantern (ATP1) in which a split Renilla luciferase (Rluc8) is modified with the ε subunit of the F 0 F 1 -ATP synthase and Venus (Saito et al , 2012).…”

Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts

“…A key determinant of the activity of both natural and engineered microbes in vivo is their location within the host organism 8,9 . However, existing methods for imaging cellular location and function, primarily based on optical reporter genes, have limited deep tissue performance due to light scattering or require radioactive tracers 10–12 . Here we introduce acoustic reporter genes – genetic constructs that allow bacterial gene expression to be visualized in vivo using ultrasound, a widely available, inexpensive technique with deep tissue penetration and high spatial resolution 13–15 .…”

Engineering BRET-Sensor Proteins

“…In analogy to the BRET/FRET-sensor approach described in this chapter, the Nanolanterns could be used as luminescent donors to construct BRET/FRET-sensors that allow both bioluminescent and fluorescent ratiometric detection. Another elegant approach was recently reported by Lin and coworkers, who fused NanoLuc to CyOFP1, a bright orange-red fluorescent protein that is excitable by cyan light (Chu et al, 2016). The high spectral overlap between NanoLuc emission and CyOFP excitation ensures efficient BRET when NanoLuc is tightly coupled to a CyOFP1 domain.…”