Thirst-quenching cocktail enables noninvasive bioluminescence imaging

Gwynne D. Koch

Bioluminescence imaging is commonly used for visualizing the regulation of gene expression in live whole animals to monitor rates of tumor growth, infection, development and circadian rhythms. Genetically engineered mice express the gene for the enzyme luciferase, which initiates a chemical reaction that emits light when exposed to the substrate luciferin. Typically, luciferin is injected into animals before they are anesthetized for imaging. However, stress caused by handling the animals and tissue damage resulting from injection can affect gene expression and change the phase of an animal’s circadian rhythms -- the roughly 24-hour cycle in the physiological processes of living organisms.

According to Daniel J. Hiler, a graduate assistant of Michael E. Geusz at Bowling Green State University in Ohio, biologists studying these rhythms want to minimize any unnecessary effects on the animal to prevent changes in the system as a whole. Therefore, he and a team of researchers evaluated an alternative method of administering luciferin.

The scientists provided the substrate to the mice in their drinking water or in apple juice. Fortunately, the luciferin-laced liquids did not appear to be distasteful to the animals.

Scientists have shown that administering the light-emitting substrate luciferin in drinking water can effectively cause bioluminescence in mice engineered to express the firefly luciferase gene. The pseudo-color image (right) overlaid on a bright-field reference image (left) shows the pattern of bioluminescence after oral administration of luciferin. The maximum intensity for the bioluminescence pseudo-color is 2030 analog-to-digital units.

About one hour after luciferin was provided, luminescence images were captured using a 50-mm f/1.2 Nikon lens attached directly through an F-mount to a liquid-nitrogen-cooled CCD camera from Photometrics (a division of Roper Scientific Inc.) of Tucson, Ariz. The mice were kept in the dark or in red light during substrate administration and imaging to prevent exposure to light that could cause excitation and possible degradation of the substrate. The fast camera lens and the high quantum efficiency of the back-thinned, back-illuminated 512 × 512-pixel sensor with 24-μm-wide pixels were important features for image collection.

The researchers used exposures of 20 to 60 s and 4 × 4 binning to increase the amount of light detected by each pixel. Images were collected with V++ software, also from Photometrics. To bring out dimmer body areas, the scientists further processed the images using ImageJ, a free software program available from the National Institutes of Health.

The team found that luciferin is effective in producing luminescence when it is delivered orally and that the signal is comparable to the signal resulting from injection.

Oral administration of luciferin has several advantages. Because it is noninvasive, it minimizes stress to the animal. Slight variations in the distribution pattern of luciferin that is administered orally may enable visualization of body areas that injection does not. Furthermore, continuous oral intake may provide sustained levels of luciferin throughout tissues -- which is not possible with injection -- providing greater flexibility in scheduling imaging sessions.

According to Hiler, although luciferin costs have declined in recent years, the dosage of the substrate in the animal’s drinking water was expensive relative to the labor and effects of using injection techniques. The scientists found that an effective oral dose of luciferin for detecting expression was 1 to 5 millimoles in 1 to 2 ml of water. Further studies evaluating the dose-response curve may determine whether reduced amounts of luciferin would be effective.

Photochemical and Photobiological Sciences, November 2006, pp. 1082-1085.