Bioluminescence is appealing as an approach for in vivo
optical imaging in mammalian tissues because these tissues have low intrinsic bioluminescence; therefore, images can be generated with remarkably high signal-to-noise ratios. A variety of different bioluminescent systems have been identified in nature, each requiring a specific enzyme and substrate. Although the most commonly used bioluminescent reporter for research purposes has been luciferase from the North American firefly (Photinus pyralis
; FLuc), useful luciferases have also been cloned from jellyfish (Aequorea
), sea pansy (Renilla
; RLuc), corals (Tenilla
), click beetle (Pyrophorus plagiophthalamus
), and several bacterial species (Vibrio fischeri
, V. harveyi
Firefly luciferase was cloned in 1985 (23
). Three years later, an assay to measure luciferase in mammalian cell lysates was developed (24
) that enabled the luciferase gene to become a useful tool for in vivo
studies of gene regulation. Luciferase is an excellent marker for gene expression because of its lack of post-translational modifications and an in vivo
of approximately 3 h (25
). Firefly luciferase produces photons in a reaction that requires ATP, magnesium, and a benzothiazoyl–thiazole luciferin (26
). Light emission from the firefly luciferase-catalyzed luciferin reaction is broad-band (530–640 nm) and peaks at 562 nm (27
). This emission spectrum, coupled with the optical properties of biological tissue, allows light (especially with spectral content above 600 nm) to penetrate through several centimeters of tissue. Therefore, it is possible to detect light emitted from internal organs in mice that express luciferase as a reporter gene.
The sensitivity of detecting internal light sources is dependent on several factors, including the level of luciferase expression, the depth of labeled cells within the body (the distance that the photons must travel through tissue), and the sensitivity of the detection system (26
). Key advances in detector technology have led to substantial improvement in sensitivity and image quality. Photons are detected by specialized charge coupled device (CCD) cameras that convert photons into electrons after striking silicon wafers. CCD cameras spatially encode the intensity of incident photons into electrical charge patterns to generate an image. For BLI, the noise of the systems is reduced by super-cooling the CCD camera and mounting the camera in a light-tight box. These cameras are run by a computer for image acquisition and analysis. Second-generation cameras that are much smaller and can be accommodated on bench tops make the technology feasible and practical for day-to-day experimentation.
Although BLI has been used successfully in a variety of applications to obtain semiquantitative information regarding biological processes in vivo
, several issues must be considered when applying this technology. Simple quantification of light emission may not provide a true representation of the biological effect studied. The luciferase reaction is a complex interaction of a variety of molecules, including ATP, oxygen, and luciferin (substrate). If ATP, oxygen, or exogenously administered luciferin is not abundantly present, light emission may not be a true representation of luciferase activity (26
). Another issue with BLI is the limited and wavelength-dependent transmission of light through animal tissues. As a general rule, there is an approximate 10-fold loss of photon intensity for each centimeter of tissue depth. Also, images are surface weighted, meaning that light sources closer to the surface of the animal appear brighter compared with deeper sources because of tissue attenuation properties (28
). In addition, dynamic changes may occur in geometry (e.g., growing tumor, scar tissue) and/or the optical properties of tissues that affect light scatter or absorption and detected bioluminescence. Thus, although BLI provides a unique and powerful methodology, quantitative analysis must be approached with caution, and validation for each specific application is necessary.