Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Jul 7;2(3):100662.
doi: 10.1016/j.xpro.2021.100662. eCollection 2021 Sep 17.

Near-infrared bioluminescence imaging of two cell populations in living mice

Giorgia Zambito  1   2 , Laura Mezzanotte  1   2
Affiliations

Near-infrared bioluminescence imaging of two cell populations in living mice

Giorgia Zambito et al. STAR Protoc. .

Abstract

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).

Keywords: Biotechnology and bioengineering; Model Organisms; Molecular/Chemical Probes.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Chemical structures of D-LH2 luciferin and its analog NH2-NpLH2
Figure 2
Figure 2
Illustration of the spectral unmixing for co-localized bioluminescent signals in vivo 1) Create and save the specific libraries for pure (100% cell ratio) HEK-CBG2 and HEK-CBR2 in the database. 2) Use the relevant libraries to unmix the emission outputs when HEK-CBG2 and HEK-CBR2 are co-injected i.v. in the same mouse. 3) Use the spectral unmixing algorithm to separate and quantify the unmixed images. Normalized emission curves can be plotted highlighting the luciferase emission peaks. Scale bar: Radiance (ph/s/cm2/sr).
Figure 3
Figure 3
Step-by-step spectral unmixing (A) Save the specific luciferase spectrum to create a pure (100% cell ratio) HEK-CBG2 or HEK-CBR2 library. (B) Perform spectral unmixing by loading opportune pure libraries. The algorithm will be able to distinguish each luciferase contribution even if the two luciferases are colocalized in the same area. Scale bar: radiance (ph/s/cm2/sr). A detailed guide on spectral unmixing is reported as technical note on Perkin Elmer website.
See this image and copyright information in PMC

References

    1. Aswendt M., Vogel S., Schäfer C., Jathoul A., Pule M., Hoehn M. Quantitative in vivo dual-color bioluminescence imaging in the mouse brain. Neurophotonics. 2019;6:1–11. - PMC - PubMed
    1. Hall M.P., Woodroofe C.C., Wood M.G., Que I., van’t Root M., Ridwan Y., Shi C., Kirkland T.A., Encell L.P., Wood Click beetle luciferase mutant and near infrared naphthyl-luciferins for improved bioluminescence imaging. Nat. Commun. 2018;9:132. - PMC - PubMed
    1. Knol-Blankevoort V.T., Mezzanotte L., Rabelink M.J., Löwik C.W., Kaijzel E.L. Development of a multicolor bioluminescence imaging platform to simultaneously investigate transcription factor NF-κB signaling and apoptosis. Methods Mol. Biol. 2016;1461:255–270. - PubMed
    1. Mezzanotte L., Aswendt M., Tennstaedt A., Hoeben R., Hoehn M., Löwik C. Evaluating reporter genes of different luciferases for optimized in vivo bioluminescence imaging of transplanted neural stem cells in the brain. Contrast Media Mol. Imaging. 2013;8:505–513. - PubMed
    1. Stowe C.L., Burley T.A., Allan H., Vinci M., Kramer-Marek G., Ciobota D.M., Parkinson G.N., Southworth T.L., Agliardi G., Hotblack A. Near-infrared dual bioluminescence imaging in mouse models of cancer using infraluciferin. Elife. 2019;8:e45801. - PMC - PubMed

Publication types