The concept of bioluminescence to study biochemistry has been around for many years, for example, as the basis for quantifying ATP in snap frozen histological specimens or tissue extracts (
1,
2). However,
in vivo application has been spearheaded by Contag
et al. (
3) and promoted by Xenogen (now Caliper Lifesciences). In less than a decade, BLI has become a routine modality for use in cancer biology, particularly suited for assessing tumor burden and metastatic spread. In vivo BLI has been reviewed many times (
3–
6) and readers are directed to these papers and other chapters of this book for further insight.
In its most popular format, the bioluminescent reaction requires luciferase enzyme derived from the American firefly (Photinus pyralis) and D-luciferin substrate. Luciferase is generated by cells following transfection. It is important to select clones with high stable expression, usually based on lentiviral transfection, which tends to be more stable than plasmid transfection. It is important to recognize that clones isolated for high expression may not behave identically to parallel lines or the parental system (e.g., differential growth rates). Thus, tumor models can be highly effective in terms of assessing tumor development and response to therapy, but they may not perfectly replicate parental cell lines.
Pharmacokinetics of the luciferin substrate are important. Remarkably, luciferin appears to readily permeate every tissue including crossing the blood-brain and -placental barriers (
4). However, the kinetics of light-emission can differ with tumor location, and thus, it is critical to establish reproducibility of light-emission curves prior to embarking on large scale studies. The most popular route of administration of luciferin is IP (intra peritoneal) (
7), but while this is apparently facile, we find a significant failure rate (
8), where no light-emission is observed following substrate administration, yet repeat one hour later gives expected bioluminescence. We attribute this to poor injection, possibly into the intestines. Intravenous (IV) administration can give much higher light-emission (
9), but more transiently so that any variation in the timing of image capture and/or integration time can generate poorer reproducibility (
8). Intravenous injection is also technically more challenging. Direct intra tumor (IT) injection generates the most intense bioluminescence, but is obviously invasive and only feasible for easily accessible tumors (
7,
10). We favor subcutaneous (SC) administration of luciferin in the back neck region. The technique is facile with overwhelming success in observing expected signal and the kinetics provide intense light over several minutes (
8,
11).
Light detection is strongest from subcutaneous tumor sites although in this case caliper measurements may be just as effective and cheaper for simple tumor volume assessment. However, BLI is particularly effective for low tumor burdens, and indeed, sub-palpable volumes can be detected and quantified. For large tumors, self absorption and scatter of light can bias apparent relative tumor volume. Planar BLI appears to accurately reflect the volume of small tumors, but becomes less linear for larger tumors, although continuing to increase monotonically (
12,
13). Light is subject to significant absorption and scattering from deep tumors, and thus, equivalent tumors located at depth are expected to provide much less detectable light. Thus, for longitudinal studies it is crucial to view an animal from the same direction on successive occasions to ensure a reproducible solid viewing angle and consistent absorption by any intervening tissues. Nude mice are preferred, though light may also be detected from white or black mice with hair: some investigators prefer to shave the animals or apply depilating agents.
Bioluminescent imaging systems can be constructed quite easily and cheaply based on several recipes in the literature, primarily from the amateur astronomy field, where there is a similar need to detect weak signals against a low background based on long-term signal integration (
14). To date, our BLI service uses a home built system, which has been described elsewhere (
7,
8,
15). The primary protocol below describes the procedures with this system (Cyclops). However, the instrument is technically complex requiring a BLI technician and engineering support. Sophisticated commercial systems are available, which are user friendly (Caliper Xenogen
1 and Berthtold
2), and we have recently acquired both IVIS
® Lumina and Spectrum systems for use by multiple research teams. These provide both bioluminescence and fluorescence imaging including depth resolved capabilities for the Spectrum.
D-luciferin can cost $100 per 100 mg, but bulk purchases should allow better than $400 per gram, which is important for high-throughput screening.
Although BLI is simple, several properties require consideration. The light-emission can by characterized by parameters including area under the curve (AUC), maximum signal intensity, time to maximum intensity or light integration over a specified period. We routinely use a dose of 450 mg/kg administered subcutaneously into an anaesthetized nude mouse with imaging for a period of five minutes starting ten minutes after luciferin administration. Weak signals may require longer integration to achieve useful signal to noise, but many investigators prefer a constant acquisition method even though small tumors than provide essentially zero signal.
The publisher's final edited version of this article is available at 
