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Optical molecular imaging in small animals harnesses the power of highly specific and biocompatible contrast agents for drug development and disease research1-7. However, the widespread adoption of in vivo optical imaging has been inhibited by its inability to clearly resolve and identify targeted internal organs. Optical tomography8-11 and combined X-ray and micro-computed tomography (micro-CT)12 approaches developed to address this problem are generally expensive, complex or incapable of true anatomical co-registration. Here, we present a remarkably simple all-optical method that can generate co-registered anatomical maps of a mouse's internal organs, while also acquiring in vivo molecular imaging data. The technique uses a time series of images acquired after injection of an inert dye. Differences in the dye's in vivo biodistribution dynamics allow precise delineation and identification of major organs. Such co-registered anatomical maps permit longitudinal organ identification irrespective of repositioning or weight gain, thereby promising greatly improved accuracy and versatility for studies of orthotopic disease, diagnostics and therapies.

All-optical anatomical co-registration for molecular imaging of small animals using dynamic contrast