Conventional optical imaging instruments are often limited to acquiring still images and only offer quantification of signal which reveals the expression of a molecule at fixed moment in time. Yet biological phenomena are always dynamic, and so a single observation at a fixed time point more often than not results in an incomplete view of the overall process, which results in misinterpretation of the available data. Such 'Snap-shot”-like observation results in the loss of information on fast moving dynamic phenomena. Conventional non invasive optical in vivo molecular imaging modalities also have the following limitations:

• Animals are required to be stationary which necessitates the use of anesthesia. Anesthesia is a time consuming step and reduces the throughput of an imaging system.
• Anesthesia can introduce bias by disturbing the physiology and affecting signal expression, so affecting directly the pathology of an animal model. (cf Anesthesia and other considerations for in vivo imaging of small animals, Hildebrandt et al, ILAR 2008).
• Imaging an animal in motion is not possible, so monitoring calcium activity in the brain during sleep and wake cycles or observing a bioluminescent signal in a contracting muscle non invasively in vivo is not possible.

The In Actio module makes it possible to image non anesthetized, freely moving animals and opens previously unimagined new avenues for research . The In Actio module simultaneously records both bioluminescence signal and a bright field video of the animal under infra red illumination for co-registration; thereby extending the the imaging capabilities of the Photon Imager from recording dynamic signal from an anesthetized animal, to recording dynamic signal from a free moving animal. The In Actio module offers the following advantages in circumstances where there is no need for multiple views of the animal, as provided by the 3D Module:

• Improved physiological relevance: observe physiological or pathological phenomena free from any perturbation by anesthesia.
• Reduce the time for animal preparation: Anesthesia is no longer required and animal stress is reduced.
• Higher throughput: Without anesthesia the experimental procedure is made faster and several animals can be imaged simultaneously.

• Molecular approaches to behavioral studies. Ex: calcium flux during sleep and wake cycles
• Functional physiological studies, i.e. calcium imaging during muscular contraction
• Biodistribution of therapeutic molecules at the subcutaneous tumor site in awake animals

Some of these applications are thoroughly described in the publication of Rogers et al: Non-invasive in vivo imaging of calcium signaling in mice, PLoS ONE, 2(10): e974 (2007) (link)