Multicolor bioluminescence imaging using near-infrared emitting luciferases is an attractive application to detect two cell populations within one animal model. Herein, we describe how to distinguish dual-color bioluminescent signals co-localized in the same compartment. We tested CBG2 click beetle (λ = 660 nm) and CBR2 click beetle (λ = 730 nm) luciferases paired with NH2-NpLH2 luciferin. Following a spectral unmixing algorithm, single spectral contributions can be resolved and quantified, enabling the visualization of multiple cell types in deep tissue by injection of a single substrate. For complete details on the use and execution of this protocol, please refer to Zambito et al. (2020).
Bibliographical noteFunding Information:
We acknowledge the funding for this work provided by the European Commission under the H2020 -MSCA-RISE award grant number 777682 (CANCER). The graphical abstract and figures were partially created with BioRender.com .
© 2021 The Author(s)