We are used to seeing multicolored fluorescence images labeling either specific events or structures within cells. When compared to imaging with fluorescent methods, bioluminescence imaging methods provide the advantages of low background and subsequent higher signal to noise ratio—enhancing sensitivity. A key prerequisite for dual-imaging experiments is the ability to distinguish the signal from each event separately and clearly. However, compared to the large number of available fluorescent compounds (many spectrally distinct fluorescent proteins and dyes), there are not many different luciferases to choose from. This lack of variety has limited the capabilities of bioluminescence for imaging multiple molecular events in the same sample. Therefore, there is a need for new luciferases with substrates and emission spectra that are different from the beetle luciferases currently in widespread use.
A paper published in Molecular Imaging in October 2013 describes use of firefly and the new NanoLuc® Luciferase to image cell signaling events in cultured cells and in a mouse model system. The paper, authored by Stacer et al. of the University of Michigan, details a proof-of-concept experiment using firefly and NanoLuc luciferases to image two distinct events in the TGF-beta1 signaling pathway.
These data establish a dual molecular imaging reporter system to quantify activation and inhibition of two key components of TGF-beta signaling: receptor activity and transcription. The authors designed a firefly luciferase reporter for kinase activity of a TGF-beta1 receptor and a NanoLuc Luciferase-based reporter for TGF-beta-dependent transcription and were able to monitor the effect of TGF-beta or specific inhibitors on either receptor activation (firefly activity) or gene expression (NanoLuc activity). These measurements were possible because the two luciferases used had their own distinct emission spectra, substrates, and bioluminescence kinetics (firefly= flash type; NanoLuc= glow-type). The dual-assay was performed successfully in cells and in whole animals.
In other experiments, these authors investigated NanoLuc luciferase alone for imaging tumor growth and progression in mice, comparing results to those with another small luciferase used for imaging studies—Gaussia Luciferase. They also used tumor cells co-expressing both firefly and NanoLuc luciferases to compare the utility of both luciferases for monitoring superficial and deep-tissue tumor progression.
This is the first paper comparing the performance of NanoLuc luciferase with other luciferases for in vivo whole animal imaging, and demonstrating use of both firefly and NanoLuc luciferase in an in vivo, dual-imaging assay.
Here’s the Paper
Stacer AC, Nyati S, Moudgil P, Iyengar R, Luker KE, Rehemtulla A, & Luker GD (2013). NanoLuc reporter for dual luciferase imaging in living animals. Molecular imaging, 12 (7), 1-13 PMID: 24371848