What is Bioluminescence?
Bioluminescence is the production and emission of light by living organisms, a form of chemiluminescence. This remarkable phenomenon occurs widely among various species. Marine vertebrates and invertebrates both exhibit this trait. It’s also found in some fungi and microorganisms. This includes certain bioluminescent bacteria. Terrestrial arthropods, such as fireflies, display this as well. In some animals, the light they emit is bacteriogenic. This light is produced by symbiotic bacteria. Examples include those from the genus Vibrio. In other creatures, the light is autogenic. This means it’s produced by the animals themselves.
The principal chemical reaction in bioluminescence involves a light-emitting molecule (luciferin) and an enzyme (luciferase). The luciferase enzyme catalyzes the oxidation of the luciferin, which produces light. In some species, the luciferase requires other cofactors. These include calcium or magnesium ions. Sometimes, the energy-carrying molecule adenosine triphosphate (ATP) is also needed. In evolution, luciferins show little variation. In contrast, luciferases vary widely between different species. This variation is evidence that bioluminescence has arisen over 40 times in evolutionary history.
The History of Bioluminescence
Bioluminescence is an ancient phenomenon. Both Aristotle and Pliny the Elder mentioned that damp wood sometimes gives off a glow. Many centuries later, Robert Boyle showed that oxygen was involved in the process, in both wood and glowworms. It was not until the late nineteenth century that bioluminescence was properly investigated. The phenomenon is widely distributed among animal groups, especially in marine environments. On land, it occurs in fungi, bacteria, and some groups of invertebrates, including insects.
Bioluminescence in nature
Animals use bioluminescence in various ways. These include counterillumination camouflage and mimicry of other animals. They might use it to lure prey. They also signal to other individuals of the same species, often to attract mates. Researchers are exploring the use of bioluminescent systems for street and decorative lighting. Additionally, a bioluminescent plant has been created.
Bioluminescence has found extensive applications in various fields of research. It is routinely used for gene assays, the detection of protein–protein interactions, high-throughput screening in drug discovery, hygiene control, and the analysis of pollution in ecosystems. In addition, bioluminescence plays a crucial role in in vivo imaging in small mammals. Beyond sensing and imaging, bioluminescence has been used in biomedicine for the photo-uncaging of small molecules, photodynamic therapy, and the control of neurons. There has also been an increase in blue-sky research such as the engineering of various light-emitting plants
The Many Uses of Luciferase in the biomedical space
Luciferase is a powerful tool in biotechnology. This enzyme is used to track various molecules within cells. These molecules would otherwise be invisible. It serves as a biomarker or reporter. The ability to engineer the gene for luciferase into the system is significant. Scientists can then track results using light. This process lets them observe cellular changes in real time. Therefore, luciferase is invaluable for diagnostic purposes. It also greatly advances scientific research.
It is routinely used for gene assays, the detection of protein–protein interactions, high-throughput screening in drug discovery, hygiene control, and the analysis of pollution in ecosystems. In addition, bioluminescence plays a crucial role in in vivo imaging in small mammals. Beyond sensing and imaging, bioluminescence has been used in biomedicine for the photo-uncaging of small molecules, photodynamic therapy, and the control of neurons.
Firefly Luciferase: Nature’s Flashlight
The most well-known luciferase comes from fireflies. These insects create unique light patterns. They do this by bringing air into their abdomens. In the presence of luciferin and luciferase, this results in light flashes. The luciferase of fireflies has been extensively studied. It’s used in various applications. One use is as a reporter gene in molecular biology. Another is for detecting ATP in hygiene monitoring systems. This enzyme catalyzes the oxidation of luciferin. In the presence of ATP and magnesium, light is produced. This happens in a two-step reaction. It ultimately leads to the creation of light.
Renilla Luciferase: A Glow from the Sea
Renilla luciferase is a bioluminescent enzyme derived from the marine organism Renilla reniformis, commonly known as the sea pansy. This enzyme catalyzes the oxidation of its substrate, coelenterazine, resulting in the emission of light.
The enzyme is often used in dual-reporter assays alongside firefly luciferase. In these assays, the firefly luciferase is typically used to measure experimental promoter activity, while the Renilla luciferase serves as a control to normalize the results for variations in cell viability and transfection efficiency. This normalization is essential for accurate and reliable experimental data. Because Renilla luciferase uses a different substrate than firefly luciferase, the two enzymes can be assayed separately in the same sample, providing a powerful tool for understanding complex biological processes.
NanoLuc Luciferase: The Shrimp’s Shine
The NanoLuc luciferase, derived from the deep-sea shrimp Oplophorus gracilirostris, is another powerful tool for research scientists. This form of luciferase has recently been used to push the boundaries of bioluminescent imaging, particularly in neuroscience. A breakthrough came when researchers found a way to optimize the substrate for NanoLuc to penetrate the blood-brain barrier, a previously insurmountable obstacle for bioluminescent imaging of brain tissues. This development allowed real-time tracking of brain activity in living animals, providing a powerful tool for neuroscience research.
Illuminating Research: The Extensive Applications of Luciferase
Bioluminescent Reporter: Luciferase is often used as a reporter gene to help researchers track and visualize gene expression and cellular events. By linking the luciferase gene to a gene of interest, the bioluminescence (light production) can serve as a “report” of the target gene’s activity.
Non-Invasive Studies: Bioluminescence imaging, which involves luciferase, is a powerful tool for non-invasive studies of the effects of treatments on cells and tissues. It enables repeated observations over time, allowing the study of cellular changes for hours or days. The luminescent signal is strong and can be used in live animals without interfering with normal cellular processes, providing a great advantage over other methods like fluorescence imaging.
As research continues to evolve and advance, it is certain that luciferase will remain an indispensable player, illuminating new frontiers in our quest for knowledge and understanding. Whether it is firefly, Renilla, or NanoLuc luciferase, each contributes in unique ways to our collective scientific journey, sparking curiosity and igniting innovation for a brighter future.
One of the world’s leading manufacturers in the field of products utilizing the power of luminescence for understanding cell biology and drug mechanisms of action is Promega, which offers a variety of kits to study the different luciferases, a few examples are:
The Nano-Glo® Luciferase Assay System, which provides a new bright luminescent substrate, furimazine, developed for NanoLuc® Luciferase. This product has led to a 100-fold improvement in signal strength from the original wild type NanoLuc® Luciferase.
Steady-Glo® Luciferase Assay System: This system is designed to provide a stable luminescent signal when used with firefly luciferase. It is particularly useful for high-throughput applications as the luminescent signal remains stable for several hours.
Bio-Glo™ Luciferase Assay System: This system uses a proprietary formulation of luciferase and substrate to provide a bioluminescent reporter assay that is sensitive, robust, and easy to use. It is ideal for measuring firefly luciferase activity in high-throughput screening application using Promega’s cell based biologics assay featuring genetically engineered cell lines that respond with luminescence to various types of complex stimulations.
The EnduRen™ Live Cell Substrate, which is a novel, patented substrate for Renilla luciferase offering unparalleled sensitivity, and has the ability to generate light in live cells over long periods of time.
Dual-Luciferase® Reporter Assay System: This system provides a way to perform two reporter assays in the same sample. It uses firefly and Renilla luciferase enzymes to monitor the activity of two different promoters. The firefly luciferase reporter is used to measure the experimental promoter activity, while the Renilla luciferase reporter is used as a control.
And finally, Promega also offers the GloMax® line of plate readers: these are microplate readers with superior sensitivity to luminescence some also feature the ability to capture fluorescence, and absorbance.
Comparing Bioluminescence to Biofluorescence
Fluorescence and luminescence are both forms of photoluminescence, which is the emission of light from a substance after it absorbs photons. However, they differ in their mechanisms and, consequently, in their intensity and sensitivity.
While bioluminescence involves the emission of light through a chemical reaction, biofluorescence is a different process where organisms absorb low wavelength or dim light, then emit high wavelength light that makes them glow against a dark background. The light emitted is a completely different color from what’s absorbed — usually green, red, or orange. Unlike bioluminescence, biofluorescence does not involve a chemical reaction nor does it generate light from the organism’s own power source. Instead, it relies on the absorption and re-emission of light from an external source.
In general, the intensity and sensitivity of both fluorescence and luminescence depend on the specific substances involved, the conditions (like temperature and pressure), and the equipment used to detect the light. Both methods can be very sensitive and are used in a variety of scientific and industrial applications, including medical diagnostics, forensic analysis, and materials science.
In general the sensitivity of luminescence-based bioassays is usually about 10-100 folds greater and the linear range is more dynamic compared to fluorescence based assays.
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ONE-Glo™ Luciferase Assay System – E6110 | E6120 | E6130
Enhanced Efficiency substrate for firefly luciferase for High- and Ultrahigh-Throughput Reporter Assays.
Dual-Glo® Luciferase Assay System – E2920 | E2940 | E2980
Simultaneous Detection of Firefly and Renilla Luciferase Activities in One Sample
The inclusion of Renilla luciferase as an internal control enhances result accuracy.
Each reporter exhibits an extended functional duration of up to 2 hours.
