PROJECT SUMMARY/ABSTRACT Over the past 10 years, our group has made multi-disciplinary efforts to bridge the technological gap between radiation methods for human treatment and pre-clinical research. We employed the Bioengineering Research Partnership mechanism to construct an advanced small animal radiation research platform (SARRP). The SARRP is equipped with cone-beam CT (CBCT) to guide focal irradiation and was commercialized by Xstrahl in 2010. The system was transformative for pre-clinical radiation research as 66 machines are now in use world-wide by > 300 investigators. Recognizing that CBCT imaging is inadequate for localizing tumor models growing in a low image contrast environment, we developed bioluminescence tomography (BLT) on board the SARRP to complement CBCT guidance. The effort was supported by the previous academic-industrial partnership (AIP) mechanism. The BLT utility is the ?first? to localize the center of mass (CoM) of an optical target for irradiation. Although the success of this partnership led to the commercialization of the BLT system by Xstrahl in 2016, the adoption of the BLT platform by users is lackluster. It is clear that investigators highly desire the ability to define the 3D shape of the target volume on the SARRP to fully complement modern human treatment. It becomes imperative that we need to advance BLT guidance to a new level of 3D target shape delineation beyond CoM and to allow quantitative assessment of target response to radiation. On-board PET imaging has been introduced to enable biology-guided RT in humans. Small animal radiation research would also be greatly enhanced with capabilities of biologic and functional targeting and assessment beyond anatomy. Fluorescence tomography (FT) would allow in-depth biological and mechanistic interrogation of key information about the tumor and its microenvironment response to RT. It complements bioluminescence imaging to study tumor and normal tissue response, when the use of engineered bioluminescent models is not available. The research potentials of FT are particularly significant when considering combinational molecular therapeutic strategies with radiation, such as immunotherapy. Fluorescence imaging also has an added advantage to be amenable to clinical translation as a surrogate for PET imaging. Integrated CBCT/BLT/FT capabilities that complements the SARRP would provide a powerful platform for pre-clinical radiation research. Xstrahl and the Johns Hopkins team will continue the partnership to develop and commercialize these new capabilities. Our aims are (1). Design and construct a new advanced BLT/FT system to guide irradiation on- board the SARRP and for off-line imaging studies, (2). Develop quantitative BLT/FT for target volume determination and treatment assessment, (3). Assess and validate the optical system performance with phantom and in vivo model. The successful dissemination of new advanced molecular optical imaging capabilities significantly enhances the conduct of radiation research for the SARRP users.