Red-shifted click beetle luciferase mutant expands the multicolor bioluminescent palette for deep tissue imaging

For in vivo multicolor bioluminescence applications, red and near-infrared signals are desirable over shorter wavelength signals because they are not as susceptible to light attenuation by blood and tissue. Herein, we describe the development of a new click beetle luciferase mutant, CBG2, with a red-shifted color emission. When paired with NH2-NpLH2 luciferin, CBG2 (λ = 660 nm) and CBR2 (λ = 730 nm) luciferases can be used for simultaneous dual-color bioluminescence imaging in deep tissue. Using a spectral unmixing algorithm tool it is possible to distinguish each spectral contribution. Ultimately, this enzyme pair can expand the near-infrared bioluminescent toolbox to enable rapid visualization of multiple biological processes in deep tissue using a single substrate. [Display omitted] •CBG2 is a new click beetle mutant derived from CBG99 luciferase•It has a far red-shifted bioluminescent emission with NH2-NpLH2 luciferin (λ = 660 nm)•CBG2 with CBR2 luciferase and NH2-NpLH2 allows fast acquisition of data in deep tissue Optical Imaging; Biological Services; Biophysics