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Our group is developing a scanner that combines x-ray, single gamma, and optical imaging on the same rotating gantry. Two functional modalities (SPECT and optical) are included because they have different strengths and weaknesses in terms of spatial and temporal decay lengths in the context of in vivo imaging, and because of the recent advent of multiple reporter gene constructs. The effect of attenuation by biological tissue on the detected intensity of the emitted signal was measured for both gamma and optical imaging. Attenuation by biological tissue was quantified for both the bioluminescent emission of luciferace and for the emission light of the near infrared fluorophore cyanine 5.5, using a fixed excitation light intensity. Experiments were performed to test the feasibility of using either single gamma or x-ray imaging to make depth-dependent corrections to the measured optical signal. Our results suggest that significant improvements in quantitation of optical emission are possible using straightforward correction techniques based on information from other modalities. Development of an integrated scanner in which data from each modality are obtained with the animal in a common configuration will greatly simplify this process.