Using Bioluminescence Imaging in Behavioral Studies

By July 25, 2020 No Comments

Optics and imaging have revolutionized medical research in recent decades, leading to advancements in behavioral research techniques such as bioluminescence imaging (BLI).  BLI has grown tremendously across many fields including neuroscience, immunology, and oncology. BLI is an invaluable asset that has allowed researchers to visualize tumor growth, follow developmental processes, and track neuronal activity.[1]

In neuroscience, BLI has helped address important questions regarding neural circuitry, molecular interactions and anatomical substrates for behaviors. It is applied in a range of transgenic animals, including zebrafishes, fruit flies, and rodents.

In this article, we will focus on how behavioral scientists have used BLI to drive exciting neuroscience discoveries in rodent models.

What Is Bioluminescence Imaging?

BLI is an optic tool that allows the non-invasive study of biological processes in live cells and animals. This technique is made possible by using the natural light emission properties of organisms that naturally bioluminesce, including the:

  • Fireflies
  • Sea pansies
  • Jelly fish like Aequorea victoria
  • Bacteria like Vibrio fischeri and Photorhabdus

Luciferase and Luciferin

These organisms convert chemical energy into light, using a bioluminescent enzyme, a substrate and oxygen. Among them, the firefly luciferase and its substrate luciferin are the most popular.

Researchers can now use a variety of bioluminescent enzymes that span a broad spectrum of colors.

Table 1: Summary of major bioluminescent proteins used in neuroscience research.[2]

Genetic engineering technologies such as CRISPR/Cas9 have driven significant evolution in using bioluminescent proteins as genetically encoded reporters (proteins that can be seen from outside the cell). Take luciferase as an example, its genetic modifications have led to wide applications:

  • Luciferase proteins can be expressed in specific tissues and cellular processes using different promoters.
  • The use of trafficking or localization signal sequences can target luciferase proteins to different subcellular structures.
  • Luciferase proteins have been engineered to respond to intracellular molecules like Calcium and ATP. Thus, they can be used to quantify signalling and metabolic dynamics within cells.
  • Brighter and longer wavelength luciferases have been developed for in vivo

Thus, the development of bioluminescence imaging was highly driven by advancements in genetic engineering technologies.

How Is Bioluminescence Imaging Different from Fluorescence Imaging?

Both fluorescent imaging and BLI can be readily applied in vivo and in vitro to study biological processes. However, there are technical differences and relative strengths of each imaging modality, which can influence how one best addresses a particular research question.[2]

Like BLI, fluorescent molecules are versatile reporters for live cell imaging because they cover a wide spectrum of wavelengths.[3] However, fluorescent molecules require an exogenous light source to be excited. Light signals are only detected when fluorescent molecules return to their ground state from a higher-energy state. To generate stronger signals, one needs to increase the amount of excitation light, which would also increase risks of photobleaching and phototoxicity.

In contrast, bioluminescence is produced without any excitation light source. Because it generates light via an enzymatic reaction, the bioluminescent signal persists as long as the substrate is present. Therefore, bioluminescent signal is increased with the amount of substrate. In live animals, this is usually achieved by an injection of substrate.

Behavioral Research Study Comparing BLI and Fluorescence Imaging Findings

When a study compared both imaging tools in studying tumor-bearing mice, BLI showed superior sensitivity, deeper tissue penetration and wider dynamic range (ratio between the maximum and minimum measurable light intensities).[2]

In addition, luciferase-expressing tumors could be detected just 1 day after tumor cell inoculation, compared to 7 days for fluorescent tumors. As such, BLI tends to be the better choice for detecting weak signals, thereby winning precious time for disease intervention before its progression. On the other hand, fluorescence imaging does not require substrate injections, thereby minimizing animal handling. It also has excellent microscopic-level spatial resolution, making it a better choice for in vitro cell assays.

Why Is Bioluminescence Imaging Useful to Behavioral Scientists?

The development or impairment of a behavior cannot be fully understood just by investigating a single time point but requires longitudinal investigations. Thus, researchers need a reporting method that is safe for live animals and can be performed at multiple time points. BLI meets these requirements and is therefore useful for studying cognitive behaviours associated with brain development and degeneration.

Since BLI does not require an excitation light source, it poses no risk of phototoxicity. Currently available BLI techniques allow detection of signals through the intact skull in free-behaving rodents without adverse impacts. Thus, researchers can monitor cellular processes associated with their behaviors of interest over time. For researchers who routinely use brain slice culture models, high resolution BLI can also be used for hours without phototoxicity.

Next, we will look at some examples of in vitro and in vivo BLI applications in studying different neuroscience questions involving behavioral elements.

Examples of Bioluminescence Imaging Applications in Behavioral Neuroscience

In vivo and in vitro studies often go hand-in-hand in biomedical research as they put together different pieces of a puzzle. In vitro BLI imaging can probe more detailed cellular activities whilst in vivo BLI can report where and when a process occurs in live, free-moving animals.

In Vitro Slice Culture Imaging

Linking Tau and Cognitive Decline in Alzheimer’s Disease (AD)

Hippocampal accumulation of hyperphosphorylation of tau is one of the prominent features of AD. Kandimalla et al. first used behavioral tests to demonstrate behavioral impairments in tau mice.[4]

However, the functional relevance of hyperphosphorylated tau in AD was poorly understood. The authors suspected that disrupted mitochondrial function may be one way that tau impairs cognitive functions in AD. To this end, they used BLI to report mitochondrial ATP levels and found a reduction associated with hippocampal tau accumulation.

This particular in vitro BLI assay was based on the reaction of ATP with firefly luciferase and luciferin. A luminometer measured bioluminescence from the reaction, which is linear to the ATP concentration. This way, the ATP levels could be compared between tau mice and wildtype mice.

Using both behavioral and BLI assays, the researchers gained support for the notion that hippocampal accumulation of tau is associated with both ATP and cognitive impairments.

As shown in Table 2 below, tau mice had compromised behavioral findings in spatial navigation and motor coordination. These results were accompanied by altered mitochondrial function as indicated by BLI findings.

This prompts future research to establish a causal relationship between the two.

Behavioral testDescriptionResultsImplicationsParallel BLI Findings
Rotarod testA test for motor coordination required for spatial navigation. The rodent is required to move forward in a rotating horizontal rodReduced latency to fall on an accelerating rotarod in tau miceImpaired motor learning and coordinationMitochondrial ATP levels, as reported by bioluminescence,  are significantly lower in tau mice relative to wildtype mice.
Morris water maze testA spatial learning task that exploits rodents’ motivation to escape to hidden platforms when forced to swimLonger latency time and lower motor ability in tau miceImpaired spatial learning and cognitive function

Table 2: Behavioral tests and outcomes in Kandimalla et al. (2018).[4]

Identifying Brain Regions with Disrupted Circadian Rhythms in Depression

Landgraf et al. hypothesized that depression-like behaviors are associated with abnormal circadian clocks in mood-regulating brain regions.[5] To test it, one needs a reporter for circadian clock activity as well as a behavioral paradigm that induces depression.

Therefore, the researchers cloned a luciferase gene after a circadian clock gene sequence to produce a bioluminescent fusion protein. In transgenic mice carrying this transgene, researchers could induce depression-like behaviors and then quantify bioluminescence as a readout of clock gene expression.

To induce depression-like behaviors, the researchers used a learned helplessness protocol. Learned helplessness is when the animal “gives up” on escaping a stressful situation, leading to increased feelings of depression. Mice were given increasing intensities of electric shocks in a closed shuttle box for two days. On the third day, the chamber door is kept open and mice were tested for their ability to escape the chamber under shocks.

If learned helplessness is acquired, the mice would show an increased number of escape failures and a longer escape latency time relative to control mice that were not exposed to shocks on previous days. The mouse brains were then collected to produce slice cultures containing different brain regions. Using BLI imaging, an absence of circadian clock activity in the nucleus accumbens and periaqueductal grey was found.

In summary, this study established an association between absent circadian rhythms in specific brain regions and depression-like behaviors. Other researchers can use a similar approach to find brain regions or biological processes associated with behaviors like depression. With sophisticated gene editing techniques and a myriad of behavioral assays, researchers can probe correlates of many behaviors.

In Vivo Imaging

Using Bioluminescent Xenograft to Track Depression-promoted Cancer Invasion

Cheng et al. hypothesized that focal adhesion kinase (FAK) mediates depression-promoted prostate cancer. [6] To test it, one needs to establish that:

  1. Depression can be effectively induced.
  2. Prostate cancer invasion is exacerbated in depressed mice as compared to the non-depressed mice.
  3. FAK is responsible for depression-promoted cancer invasion.

To validate the first point, appropriate behavioral tests need to show depression-like behaviors in the subject mice. To validate the second and third points, an in vivo visualization tool like BLI is needed to track tumor invasion.

Therefore, the researchers first exposed mice to unpredictable mild stress and showed depression-like behaviors using three behavioral tests.

Behavioral testDescriptionResults
Sucrose Preference TestAnimals are presented with either plain water or a sucrose drink. Reduced sucrose preference is correlated with depression.Reduced sucrose consumption after stress exposure
Forced Swimming TestAlso known as the “Behavioral despair test”. Rodents are placed in a confined space filled with water. Their escape immobility time is measured as a readout of depression.Increased immobility time after stress exposure
Tail Suspension TestAn animal is suspended from a box from the top, unable to touch any surfaces. The immobile period in which the animal gives up trying to escape is indicative of depression-like behavior.Increased immobility time after stress exposure

Table 3: Behavioral tests for depression-like behaviors and outcomes in Cheng et al. (2018).[6]

The researchers then created bioluminescent tumor cell cultures by infecting tumor cells with lentiviruses carrying a luciferase gene. Next, bioluminescent tumor cells were xenografted (a tissue graft from a human patient) onto stressed and non-stressed mice to compare tumor invasion in vivo.

It was found that the depressed mice had significantly higher bioluminescence intensity compared to the non-depressed mice. This indicates that tumor invasion was indeed more severe when the mice had been previously stressed to develop depression. Lastly, FAK knockdown rescued this depression-promoted tumor invasion, supporting the hypothesis.

This study by Cheng et al. is a perfect example of how behaviors like depression can have a physiological impact such as promoting cancer. Using behavioral assays, BLI and molecular manipulations, researchers can make mechanistic breakthroughs that have important clinical implications.

Studying the Therapeutic Potential and Biodistribution of Interleukin-2 (IL-2) in AD

The importance of neuroinflammation in neurodegenerative disorders is becoming increasingly clear. For example, AD patients have decreased levels of an inflammation regulator, IL-2, in their hippocampi.[7] As such, Alves et al. investigated whether IL-2 administration can have positive therapeutic effects in an AD mouse model.

The researchers administered recombinant adeno-associated virus carrying the luciferase and IL-2 gene. They found a number of favourable cellular outcomes, as a result of IL-2 administration, including decreased amyloid plaque and improved synaptic plasticity. Using a Morris Water Maze task, the authors also demonstrated that these cellular improvements were associated with reduced memory deficits.

Due to the bioluminescent properties of IL-2-transduced cells, the authors could analyse the biodistribution of IL-2 in living mice. Luciferase bioluminescence was found only in peripheral organs but not in the brain. This indicates that an increase in brain IL-2 was due to peripheral passage instead of local production by brain cells.

In summary, this study not only demonstrated IL-2’s therapeutic potential up to the behavioral level, but also clarified its biodistribution patterns. These results suggest its promising to deliver IL-2 through peripheral administration routes in future clinical trials targeting AD.

Monitoring Peripheral Circadian Rhythms in Mouse Embryos and Pups

Canaple et al. were interested in the temporal and spatial characteristics of peripheral circadian rhythms in developing mice.[8] To study it noninvasively, a bioluminescent model was generated using a luciferase reporter gene driven by a clock gene promoter. When the bioluminescent signal changes in intensity and temporal patterns, the researchers could learn about the dynamics of peripheral clocks.

Bioluminescence was monitored in utero through to postnatal stages. It appeared since an early embryonic stage but only acquired a pattern during late gestation. Interestingly, the maternal eating behavior is crucial for controlling the patterns of bioluminescence in pup peripheral clocks.

Maternal food restriction during gestation and parturition both led to a phase delay of the circadian profile in the pups. These results indicate that offspring rely on maternal signals to adjust their biological clocks, possibly because they confer a survival advantage.

From this study, one could appreciate that BLI can be used longitudinally spanning different developmental stages. Further, behaviors can be modified in the mother to study transgenerational physiological impacts.

Single-cell Imaging (AkaBLI) to Identify Neural Ensemble for Real-time Spatial Learning

BLI imaging using the traditional luciferin substrate is not ideal for deep brain structures due to poor delivery across the blood brain barrier. Thus, Iwano et al. developed an improved AkaBLI system that overcame this limitation.[1] The improved system can penetrate most tissues, including subcortical brain structures such as the striatum.

Apart from improved penetration, the AkaBLI system also has superior sensitivity that enables researchers to monitor single neurons in real time. As a proof of principle, the authors non-invasively monitored the hippocampal cells that respond to novel environments in mice. Previously, researchers could only use post-mortem histology to identify neurons involved in learning within a limited time window before sacrifice. Using the AkaBLI system, a luciferase gene Venus-Akaluc was expressed under the c-Fos promoter, a marker for activity-dependent learning.

When mice were exposed to a novel environment, c-Fos-dependent expression of Venus-Akaluc lit up in a small population of activated neurons. The researchers concluded that this activation is the neural ensemble for spatial learning in the hippocampus.

Therefore, the AkaBLI system allowed the researchers to monitor single cells responsible for spatial learning in a novel environment. Its ability to penetrate brain tissues, coupled with high spatial and temporal resolution, makes it a powerful improvement. In the era of single cell research, AkaBLI is likely to be widely used in upcoming research for identifying neural ensembles underlying behaviors.

Advantages and Disadvantages of Bioluminescence Imaging

Like every research tool, BLI has its advantages and limitations. While bioluminescence offers a means to report the biological process of interest, researchers also need to be aware of its shortcomings.


The advantages of BLI include:

  • Non-invasive: Genetically encoded reporters that are expressed in vivo, allowing for longitudinal studies in free-behaving animals.
  • Non-phototoxic: BLI does not require an exogenous excitation light for signal detection, thereby avoiding photobleaching and phototoxicity.
  • Easily crosses the blood-brain barrier: BLI substrates can cross the blood-brain barrier, allowing detection of signals through the intact skull.
  • Higher sensitivity: BLI is highly sensitive due to negligible nonspecific background signal, in contrast to the auto-fluorescence problem in fluorescence imaging.
  • Versatile: Suitable for imaging light-sensitive cells (eg. retinal neurons) since no excitation light is required
  • Can study molecular interactions: Bioluminescent proteins can be fused to different proteins or expressed in different tissues/cells to probe diverse cellular and molecular interactions.


The disadvantages of BLI are made up of the following:

  • Signal quantification: Presence of the bioluminescent transgene, and the distribution of substrate molecules may vary among different tissues, affecting signal quantification.
  • Use of foreign proteins: Expression of foreign proteins such as luciferase may affect the biological process being studied. For example, bioluminescent tumors seem to grow slower than non-bioluminescent tumors.[9]
  • Confounds: Signal quantification can be arbitrary and variable as it depends on the luminometer, substrate, enzyme and cofactors.
  • Enzymatic stability: Endogenous and exogenous factors may affect enzymatic stability. For example, oxidative stress in apoptotic cells can rapidly decrease the stability of firefly luciferase.[10]

To overcome the limitations of BLI, significant improvements have been made to reduce the variabilities of the enzyme, the substrate and the luminometers.[11] Today, researchers have access to BLI kits with high sensitivity, stability, solubility and biodistribution profiles.


In conclusion, BLI is a very useful tool for the thriving field of neuroscience. Its safe in vivo application is the most exciting advantage as it bridges the gap between behavioral and cellular level information. Its versatile applications as reporters for both protein localization and transcriptional activity allow researchers to answer a myriad of behavioral questions. The inherent advantages of BLI, namely low background noise and no phototoxicity, have been coupled with clever genetic engineering. Having said that, optimization and caution for variability are still required when researchers adopt BLI in behavioral research.


  1. Iwano, S. et al. Single-cell bioluminescence imaging of deep tissue in freely moving animals. Science 359, 935-939, doi:10.1126/science.aaq1067 (2018).
  2. Choy, G. et al., 2003. Comparison of noninvasive fluorescent and bioluminescent small animal optical imaging. BioTechniques, 35(5), pp.1022–1030.
  3. Tung, J. K., Berglund, K., Gutekunst, C. A., Hochgeschwender, U. & Gross, R. E. Bioluminescence imaging in live cells and animals. Neurophotonics 3, 025001, doi:10.1117/1.NPh.3.2.025001 (2016).
  4. Kandimalla, R., Manczak, M., Yin, X., Wang, R. & Reddy, P. H. Hippocampal phosphorylated tau induced cognitive decline, dendritic spine loss and mitochondrial abnormalities in a mouse model of Alzheimer’s disease. Hum Mol Genet 27, 30-40, doi:10.1093/hmg/ddx381 (2018).
  5. Landgraf, D., Long, J. E. & Welsh, D. K. Depression-like behaviour in mice is associated with disrupted circadian rhythms in nucleus accumbens and periaqueductal grey. Eur J Neurosci 43, 1309-1320, doi:10.1111/ejn.13085 (2016).
  6. Cheng, Y. et al. Depression promotes prostate cancer invasion and metastasis via a sympathetic-cAMP-FAK signaling pathway. Oncogene 37, 2953-2966, doi:10.1038/s41388-018-0177-4 (2018).
  7. Alves, S. et al. Interleukin-2 improves amyloid pathology, synaptic failure and memory in Alzheimer’s disease mice. Brain 140, 826-842, doi:10.1093/brain/aww330 (2017).
  8. Canaple, L., Grechez-Cassiau, A., Delaunay, F., Dkhissi-Benyahya, O. & Samarut, J. Maternal eating behavior is a major synchronizer of fetal and postnatal peripheral clocks in mice. Cell Mol Life Sci 75, 3991-4005, doi:10.1007/s00018-018-2845-5 (2018).
  9. Baklaushev, V. P. et al. Luciferase Expression Allows Bioluminescence Imaging But Imposes Limitations on the Orthotopic Mouse (4T1) Model of Breast Cancer. Sci Rep 7, 7715, doi:10.1038/s41598-017-07851-z (2017).
  10. Czupryna, J. & Tsourkas, A. Firefly luciferase and RLuc8 exhibit differential sensitivity to oxidative stress in apoptotic cells. PLoS One 6, e20073, doi:10.1371/journal.pone.0020073 (2011).
  11. Badr, C. E. Bioluminescence imaging: basics and practical limitations. Methods Mol Biol 1098, 1-18, doi:10.1007/978-1-62703-718-1_1 (2014).
Author Details
Yunan has a Bachelor of Biomedical Science from the University of Queensland, and a passion for embryo development and neuroscience. She is currently writing about science as a freelancer in sunny Brisbane. Learn more and connect with her –
Yunan has a Bachelor of Biomedical Science from the University of Queensland, and a passion for embryo development and neuroscience. She is currently writing about science as a freelancer in sunny Brisbane. Learn more and connect with her –