Expert Insights: A Look Forward at Multiplexing for in vivo Bioluminescence Imaging

Bioluminescent in vivo imaging tools

NanoLuc, NLuc

With advancements made over the past few decades, the future of in vivo bioluminescence imaging (BLI) continues to gain momentum. In vivo BLI provides a non-invasive way to image endogenous biological processes in whole animals. This provides an easier method to assess relevant systems and functions. Unlike fluorescent imaging, BLI relies on a combination of enzymes and substrates to produce light, greatly reducing background signal (Refaat et al., 2022). Traditional fluorescent tags are also quite large and may interfere with normal biological function. In vivo BLI research has been around for quite some time, primarily utilizing Firefly luciferase (Luc2/luciferin). A recent advancement was the creation of the small and bright NanoLuc® luciferase (NLuc). Promega offers an wide portfolio of NLuc products that provide ways to study genes, protein dynamics, and protein:protein interactions. To fully grasp the power of these tools, I interviewed several key investigators to determine their perspectives on the future of in vivo BLI. I was specifically interested in their thoughts on NLuc multiplexing potential with Firefly (FLuc), and future research areas. These two investigators are Dr. Thomas Kirkland, Sr. Scientific Investigator at Promega, and Dr. Laura Mezzanotte, Associate Professor at Erasmus MC.

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Illuminating the Brain with a New Bioluminescence Imaging Substrate

Bioluminescence imaging is a powerful tool for non-invasive studies of the effect of treatments on cells and tissues. The luminescent signal is strong, and can be used in vivo, enabling repeated observations over time, allowing longitudinal study of cellular changes for hours or days. Bioluminescence imaging can be used in live animals over varying periods of time, without interfering with normal cellular processes.

Fluorescence imaging is also used in cellular studies. Although it can provide a stronger signal than luminescence, fluorescence requires light for excitation, and thus its in vivo use is limited at a tissue or cell depth greater than 1mm.

NanoLuc® Luciferase. Small, bright and now useful in brain bioluminescence imaging.

In addition, autofluorescence can be an issue with fluorescence imaging, as cellular components and surrounding proteins and cells can fluoresce when exposed to light. Autofluorescence can result in high background signals, making it difficult to distinguish true fluorescence from background.

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Bioluminescent Sharks Set the Sea Aglow

Many deep sea creatures are bioluminescent. However, before documenting the luminescence of the kitefin shark, Dalatias licha, there has never been a nearly six-foot long luminous vertebrate creature. In a recent study, Mallefet and colleagues examined three species of sharks: Dalatias licha, Etmopterous lucifer, and Emopterus granulosus and documented their luminescence for the first time. These bioluminescent sharks are the largest bioluminescent creatures known.

Researchers studied three species of bioluminescent sharks near the Chatham Islands, New Zealand
Coastline of one of the Chatham Islands, New Zealand
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CRISPR/Cas9 HiBiT Knock-In: A Scalable Approach for Studying Endogenous Protein Dynamics

Studying protein function in live cells is limited by the tools available to analyze the expression and interactions of those proteins. Although mass spectrometry and antibody-based protein detection are valuable technologies for protein analysis, both methods have drawbacks that limit the range of targets and contexts in which proteins can be investigated.

Mass spectrometry is often poor at detecting low-abundance proteins. Antibody-based techniques require high quality, specific antibodies, which can be difficult to impossible to acquire. Both methods require cell lysis, preventing real-time analysis and limiting the physiological relevance, and both methods can be limiting for higher-throughput analysis. While plasmid-based overexpression of tagged target proteins simplifies detection and can allow for real time analysis, protein levels don’t typically resemble endogenous levels. Overexpression also has the potential to create experimental artifacts or limit the dynamic range of an observed response.

In 2018, Promega R&D scientists published a paper in ACS Chemical Biology demonstrating the use of CRISPR/Cas9 to integrate the 11 amino acid, bioluminescent HiBiT tag directly into the genome to serve as an easily measured reporter for endogenous proteins. This provides a highly quantitative method for investigating cellular protein dynamics that sidesteps the need for cloning and other drawbacks to conventional methods, including the ability to measure changing protein dynamics in real-time. (For more details about CRISPR/Cas9 knock-in tagging and other applications, read this blog.)

While their findings showed that this method provides efficient and specific tagging of endogenous proteins, the research was limited to just five different proteins within a single signaling pathway in two cell lines. This left unanswered questions about whether this approach was scalable, had broader applications and how accurately the natural biology of the cells was represented.

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Making BRET the Bright Choice for In vivo Imaging: Use of NanoLuc® Luciferase with Fluorescent Protein Acceptors


Live animal in vivo imaging is a common and useful tool for research, but current tools could be better. Two recent papers discuss adaptations of BRET technology combining the brightness of fluorescence with the low background of a bioluminescence reaction to create enhanced in vivo imaging capabilities.

The key is to image photons at wavelengths above 600nm, as lower wavelengths are absorbed by heme-containing proteins (Chu, J., et al., 2016 ). Fluorescent protein use in vivo is limited because the proteins must be excited by an external light source, which generates autofluorescence and has limited penetration due to absorption by tissues. Bioluminescence imaging continues to be a solution, especially firefly luciferase (612nm emission at 37°C), but its use typically requires long image acquisition times. Other luciferases, like NanoLuc, Renilla, and Gaussia, etc. either do not produce enough light or the wavelengths are readily absorbed by tissues, limiting their use to near-surface imaging.

The two papers discussed here illustrate how researchers have combined NanoLuc® luciferase with a fluorescent protein to harness bioluminescent resonance energy transfer (BRET) for brighter in vivo imaging reporters.

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