The challenge of detecting and locating bioluminescent light emissions from within living subjects has been met by several commercial suppliers of in vivo imaging equipment (). A basic imaging system consists of a light-tight imaging chamber into which the subject is placed and a high quantum efficiency charged coupled device (CCD) camera, usually super cooled to less than −80 °C to reduce thermal noise, that collects emitted light. The camera typically first takes a photographic image of the subject followed by a bioluminescent image. When superimposed, regions of bioluminescence become mapped to the subject’s anatomy for pinpoint identification of source emissions. Acquisition times can range from a few seconds to several minutes depending on signal strength. Software displays the image in a pseudo colored format and provides the tools needed to quantify, adjust, calibrate, and background correct the resulting image. Integrated gas anesthesia systems, heated stages, and isolation chambers are typically available to accommodate animal handling.
Commercial manufacturers of in vivo imaging systems.
The technology incorporated into in vivo imaging systems is rapidly advancing to meet user needs in a greater diversity of application backgrounds. CCD cameras are being replaced by more sensitive intensified CCD (ICCD) and electron multiplying CCD (EMCCD) cameras that can manage acquisition times of millisecond durations. These fast processing times along with powerful software now permit real-time tracking of conscious, moving subjects (see, for example, the IVIS Kinetic system from Caliper Life Sciences). Anesthesia can have dramatic, unknown, and interfering effects on animals, and the ability to image in its absence is a major step forward in in vivo imaging technology. However, these newer imaging systems still remain far too expensive for the typical researcher and to date most imaging is still performed on anesthetized animals. Imaging systems are additionally becoming better integrated with existing medical technologies for multi-parameter analyses. For example, electrocardiogram (ECG), X-ray, or computed tomography (CT) procedures can operate in parallel with imaging acquisition. The ability of software to overlay and map these data to the bioluminescent image offers unique opportunities to visualize physiological status and kinetics.
The major drawback of in vivo
imaging systems is its limited depth penetration under whole animal imaging conditions. In most cases, using a CCD camera to image luminescent or fluorescent signals at depths beyond a few centimeters produces inconsistent results. Without major advances in imaging sensitivity, either with the camera systems, the internal signal, or almost certainly both in tandem, in vivo
imaging applications may become limited solely to small animals and the translational leap to humans will never occur. Rather than relying on a camera to visualize the signal externally, it may be feasible and potentially more practical to monitor the signal internally using implantable sensors. Although not yet a viable technology, proof-of-concept microluminometer integrated circuits of only a few square millimeters in size have been developed and validated for bioluminescent signal acquisition [15
]. These so-called bioluminescent bioreporter integrated circuits, or BBICs, were specifically designed for capturing the 490 nm bioluminescent light signal emitted by the bacterial Lux proteins, and accommodated on-chip transmitters for wireless data transmission. Effectively interfacing the microluminometers with the luciferase reporter systems, maintaining reporter viability, and implanting the chips would remain challenging, as would the regulatory and safety constraints associated with any human implantation experimental approaches.