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.Optical Imaging; Biological Services; Biophysics

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Department of Radiology

Zambito, G. (Giorgia), Hall, M. P., Wood, M. G., Gaspar, N. (Natasa), Ridwan, Y., Stellari, F.F. (Fabio F.), … Mezzanotte, L. (2021). Red-shifted click beetle luciferase mutant expands the multicolor bioluminescent palette for deep tissue imaging. iScience, 24(1). doi:10.1016/j.isci.2020.101986