bioluminescence imaging

Live-Cell Imaging: It’s Time to See What Else Your Luminescence Assays Can Tell You

Posted on by Kelly Grooms
luminescent cells behind a molecular structure

Luminescent live-cell assays are powerful tools for cellular biology research. They offer both qualitative and quantitative insights into processes such as gene expression, cell viability, metabolic activity, protein and small molecule interactions, and targeted protein degradation. But what if you could go beyond the numbers and actually see what is happening in your cells? With luminescent imaging, you have the opportunity to uncover more dynamic data by visualizing what happens with your cells in real time.

Why Luminescent Imaging?

Bioluminescent reporters such as NanoLuc® Luciferase reporters are well-suited for use in bioluminescent imaging studies. The extreme brightness means that exposure times can be reduced, compared to the time required for other luminescent reporter proteins. Its small size also makes it less likely to perturb the normal biology or functionality.

Another benefit of bioluminescence for imaging is the inherent stability and sustainability of the bioluminescent signal, which does not require external excitation like fluorescent tags.  This allows direct visualization of protein dynamics in living cells without the need for repeated sample excitation. The lack of external excitation also reduces the risk of phototoxicity and photobleaching, common issues that can adversely affect cell viability and signal integrity over time.

Applications Across Cellular Research

Luminescent imaging complements traditional luminescence assays by adding spatial and temporal dimensions. With luminescent live-cell imaging, researchers can visualize NanoLuc® Luciferase assays to gain a deeper understanding of the real-time cellular processes occurring in each experiment. Applications include:

  • Determining which cells provide signal
  • Analyzing mixed cell populations
  • Identifying rare events
  • Monitoring protein:protein interactions
  • Identifying protein localization and translocation
  • Tracking protein degradation and stability over time
  • Visualizing ligand:protein interactions (target engagement)

Luminescent Imaging in Action

Targeted Protein Degradation

Selectively targeting proteins for removal from the cell—instead of inhibiting protein activity—is a newer approach with therapeutic potential. In this method, the protein is targeted for degradation using the cell’s natural ubiquitin proteasome system (UPS). The degradation process is initiated by compounds such as molecular glues and proteolysis targeting chimeras (PROTACs) linking the target protein to an E3 ligase. Once this linkage occurs, the cell’s UPS does the rest.

Luminescent substrates with increased signal stability, such as the Nano-Glo® Extended Live Cell Substrate, enables researchers to image targeted protein degradation in their cells in real time. In the example shown below, Nano-Glo® Vivazine™ Live Cell Substrate was used to image degradation of the GSPT1 protein by the CC-885 degrader over 5 hours.

gif showing luminescent signal disappearing with protein degradation

Targeted protein degradation over time. HEK293 cells expressing endogenous HiBiT-tagged GSPT1 and stably expressing LgBiT were treated with CC-885 degrader or DMSO control treatment. Assayed with Nano-Glo® Vivazine™ Live Cell Substrate and imaged over 5 hours using GloMax® Galaxy Bioluminescence Imager.

Combining Luminescent and Fluorescent Imaging to Detect Protein:Small Molecule Interactions

Using bioluminescence resonance energy transfer (BRET)-based assays such as NanoBRET® assays allows you to detect protein:protein interactions by measuring energy transfer from a bioluminescent protein donor to a fluorescent protein acceptor. These assays can be used to monitor changes in protein interactions over time, making them a useful tool for small-molecule screening.

The schematic below illustrates how the NanoBRET® NanoGlo® Detection Systems can be used to visualize target engagement. The cells on the left are expressing a NanoLuc® fusion protein, resulting in a luminescent signal. Adding a fluorescent small tracer (center) results in energy transfer and a fluorescent signal (right). Using an imaging platform that has luminescence and fluorescence imaging capabilities will let you see this energy transfer in action.

schematic showing cells detected by luminescent and fluorescent imaging
Detecting protein:small molecule interactions with NanoBRET® NanoGlo® Detection Systems.  HCT116 cells expressing a PRMT5–NanoLuc® fusion were supplemented with a fluorescent small molecule tracer (center panel). Before tracer addition, luminescent signal indicates energy is present on the donor protein (left; 3-minute exposures for 15 minutes). Binding of fluorescent tracer results in energy transfer and fluorescent signal (right; 3-minute exposures for 60 minutes). Images were captured on the GloMax® Galaxy Bioluminescence Imager.

Bringing the Power of Luminescent Imaging to Your Lab

glomax galaxy imager and computer screen

Having the right tools is critical to unlocking the full potential of bioluminescence imaging. The GloMax® Galaxy Bioluminescence Imager is uniquely positioned to offer researchers the power of imaging in an accessible, benchtop instrument. The Galaxy is a fully equipped microscope that can visualize output from NanoLuc® Technologies and offers luminescence, fluorescence and brightfield imaging capabilities. By offering a user-friendly platform for live-cell luminescent imaging, the GloMax® Galaxy empowers researchers to enrich their understanding of functional and dynamic cellular events across a cell population.

Conclusion

Luminescent imaging can enrich what we learn from live-cell assays and offers an unprecedented view into the dynamics of cellular processes. From monitoring drug responses to visualizing protein interactions, this technology delivers insights that go beyond the capabilities of traditional assays.

Whether you’re studying cancer biology, drug development or cellular signaling, luminescent imaging can help you uncover what’s hidden in your data and see your research in a whole new light.

Additional Resources

GloMax® Galaxy Luminescent Imager, NanoBRET® Nano-Glo® Detection Systems and Nano-Glo® Vivazine live Cell Substrate are for Research Use Only. Not for Use in Diagnostic Procedures.

Visualize Protein:Protein Interactions with Bioluminescence Imaging

Posted on by Simon Moe

If you’re familiar with bioluminescence, you’ve probably used it in plate-based experiments to track various biological processes. You understand it provides distinct advantages over traditional fluorescence assays, particularly when it comes to sensitivity. However, there’s always that one nagging question: how representative is the signal on a cell-to-cell level?

Traditional approaches to decipher cell-to-cell signal rely on complex, time-intensive measures that only approximated the findings acquired through bioluminescence. That’s where the GloMax® Galaxy Bioluminescence Imager comes in. This new tool will enhance your ability to visualize proteins using NanoLuc® technology, going beyond simple numeric outputs to reveal what’s happening in individual cells.

NanoLuc® technology is well-known for its ability to assist in detecting subtle protein interactions in complex biological systems. This bright luminescent enzyme enables a much broader linear range than fluorescence, improving detection of small changes in protein activity, such as proteins interacting. Microplate readers measuring NanoLuc® assays rely on signal generated from many cells. This results in an approximation of what is occurring biologically. Truly validating those luminescent readings within a cell population has been challenging—until now. The GloMax® Galaxy allows you to perform bioluminescence imaging, moving beyond the numbers, offering the power to visualize protein interactions directly.

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Silencing the Immunogenicity of AAV Vectors 

Posted on by Jordan Nutting

Recombinant adeno-associated viral (AAV) vectors are an appealing delivery strategy for in vivo gene therapy but face a formidable challenge: avoiding detection by an ever-watchful immune system (1,2). Efforts to compensate for the immune response to these virus particles have included immunosuppressive drugs and engineering the AAV vector to be especially potent to minimize its effective dosage. These methods, however, come with their own challenges and do not directly solve for the propensity of AAV vectors to induce immune responses.  

A recent study introduced a new approach to reduce the inherent immunogenicity of AAV vectors (2). Researchers strategically swapped out amino acids in the AAV capsid to remove the specific sequences recognized by T-cells that elicit the most pronounced immune response. As a result, they significantly reduced T-cell mediated immunogenicity and toxicity of the AAV vector without compromising its performance.  

Read on to get more of the study details, which include the use of NanoLuc® luciferase and Nano-Glo® Fluorofurimazine In Vivo Substrate for in vivo bioluminescent imaging of the AAV variants’ distribution and transduction efficiency in mice. 

A teal colored ribbon model of a AAV virus capsid floats against a black background.
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Expert Insights: A Look Forward at Multiplexing for in vivo Bioluminescence Imaging

Posted on by Simon Moe

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

Posted on by Kari Kenefick

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

Posted on by Riley Bell

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

Posted on by Darcia Schweitzer

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

Posted on by Kyle Hooper
13305818-cr-da-nanoluc-application_ligund

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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