February 15, 2010 (Vol. 30, No. 4)

Simple Steps for Successful Bioluminescence Animal Imaging

Luciferin is the basic substrate of bioluminescent assays. Based on a simple reaction where ATP fuels luciferase catalysis of firefly (beetle) luciferin to produce light, bioluminescence has broad applications. Its employment as a biomass indicator allows for the detection of microorganisms in consumer products, manufacturing plant surfaces, and has even been used to determine if life exists on the moon and Mars.

Bioluminescence is being used in in vitro and in vivo monitoring of biological processes including gene expression and protein-protein interactions. Luciferin is used, for example, in reporter gene assays to study gene regulation and function where expression of the luciferin-tagged reporter is a marker to indicate successful uptake of the gene of interest in recombinant DNA techniques. As a detection reagent, the luciferin-luciferase reaction is utilized in pyrosequencing to accomplish fast detection of bases in today’s high-throughput DNA sequencing systems.

More recently, bioluminescence imaging has emerged as a powerful technique for the direct study of different cell populations in live small animals such as mice. In this technique, cells (e.g., cancer cells, stem cells, T cells) are designed to express luciferase and illuminate. Noninvasive visualization in live animals using a sensitive charge-couple device camera thus makes it possible for real-time observation of disease progression and regression at different times over the course of therapeutic treatment. Employed to understand the molecular basis of pathologies such as neurodegenerative disorders, cardiovascular disorders, obesity, and cancer, this technique is of immense value for clinical, diagnostic, and drug development.

Scientists at Regis Technologies have identified common weaknesses in luciferase assays. Problems often occur before the assay begins. Firefly luciferin is a sensitive molecule, and even well-planned experiments can give erroneous results if the luciferin is mishandled and damaged before use. In this article, we describe the optimal salt, purity, handling, and storage of luciferin to ensure successful and consistent results in animal imaging and other assays.

Purchasing Considerations

Researchers often wonder which form of luciferin to use. The potassium and sodium salts of luciferin are usually used interchangeably. However, the molecule’s dissolution is an important factor to consider for some assays. The sodium salt is more soluble in water (>100 mg/mL) than the potassium salt (55 mg/mL). The free-acid form, while still available, is not often used due to its difficulty of dissolution.

Because a few impurities can impact experiments in different ways, the initial purity of the reagent may be an important consideration.

L-luciferin, an enantiomer of D-luciferin (Figure 1), is a known impurity in synthetic luciferin. While shown to function in luciferase reactions, a significant shift in peak light emission is likely to occur depending on the data-collection parameters used. L-luciferin is easily detected and quantified by chiral HPLC and should be reported on the manufacturer’s certificate of analysis. Most luciferin should contain less than 0.5% L-luciferin, although higher limits can likely be tolerated as long as the L-luciferin levels remain constant throughout the experiment.

Dehydroluciferin (Figure 1) is an impurity that acts as an inhibitor of luciferase, suppressing the flash height and total integration. A sample doped with 1% dehydroluciferin shows less than half the flash height and less than 25% of the total integration over 15 minutes. Dehydroluciferin can form during the synthesis or storage of luciferin. Selecting a manufacturer that supplies low dehydroluciferin levels will help to ensure reliable and repeatable results.

Many common trace impurities from the synthesis of luciferin can inhibit other enzymes in complex systems and may cause experimental variability due to batch-to-batch or manufacturer-to-manufacturer differences.

Figure 1. Structural comparison of D-luciferin and impurities


Firefly luciferin is sensitive to light, oxygen, and moisture and must be protected. Light and oxygen can catalyze the oxidation of luciferin to dehydroluciferin. Left in an amber bottle under nitrogen at room temperature, luciferin will begin to decompose to dehydroluciferin after a month (Figure 2), but is stable in an unopened bottle for at least two years in a freezer. Dissolved in water with sufficient oxygen, luciferin will convert to dehydroluciferin in only a few days.

Luciferin should be purchased and stored in the smallest quantity possible to prevent decomposition to dehydroluciferin from multiple freeze-thaw-open cycles. If purchased in bulk, dividing the luciferin reagent into single-use amber vials will help ensure stability. Long-term storage as a frozen solution is not recommended for sensitive applications such as whole-animal studies. To help slow decomposition in frozen solutions, sparge the solution with nitrogen or argon prior to freezing.

Figure 2. Luciferin decomposition to dehydroluciferin versus time at 25ºC and -20ºC


Whenever opened, the luciferin bottle must be allowed to come fully to room temperature and then purged with nitrogen or argon before being resealed. If luciferin solutions are to be used within a few hours, few precautions are needed. Solutions that will be used over a day should be sparged with nitrogen or argon. Solutions should not be used over several days, as dehydroluciferin formation will occur. 

Scott B. Mohler ([email protected]) is a product manager at Regis Technologies. Website: www.registech.com.

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