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

Giorgia Zambito,Thomas A Kirkland,Laura Mezzanotte,Lance P Encell,Mary P Hall,Yanto Ridwan,Natasa Gaspar,Fabio F Stellari,Monika G Wood,Ce Shi,Clemens Löwik

doi:10.1016/j.isci.2020.101986

Giorgia Zambito, Thomas A Kirkland + Show 9 more

Open Access

https://doi.org/10.1016/j.isci.2020.101986

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Abstract

SummaryFor 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.

Highlights

Bioluminescence imaging (BLI) has become a highly adopted technique for preclinical and non-invasive study of biological events in vivo (Kaskova et al, 2016; Mezzanotte et al, 2017)
SUMMARY 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
We describe the development of a new click beetle luciferase mutant, CBG2, with a redshifted color emission

Summary

Introduction

Bioluminescence imaging (BLI) has become a highly adopted technique for preclinical and non-invasive study of biological events in vivo (Kaskova et al, 2016; Mezzanotte et al, 2017). Red-shifted luciferase mutants improve the sensitivity of BLI and allow tracking of single cells over time in deep tissue (Branchini et al, 2010; Iwano et al, 2018). It is still challenging to visualize multiple biological processes over time in deep tissue because current BLI offerings are limited. In many of the systems currently used, sequential administration of multiple substrates is required, making interpretation of data challenging (Maguire et al, 2013; Taylor et al, 2018). The signal for CBG99 was attenuated in deep tissue, resulting in acquisition of predominantly the red contribution (Mezzanotte et al, 2011)

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