Immunotherapy (IMT) is a cancer treatment that harnesses activated T cells to induce a targeted immune response against the cancer. Preclinical IMT research has been limited because there are no effective methods to longitudinally image T cell biodistribution in mouse models, including ovarian cancer. This is an urgent unmet need because the only viable methods to monitor activated T cells, the immune marker most associated with antitumor response and prognosis, immunohistochemistry and FACs, are both terminal and ex vivo. Fluorescence imaging (FLi) offers many advantages for monitoring T cell migration, including the relatively long photostability of fluorescent ligands, ease of use, and its low cost. However, FLi does not provide 3D spatial maps of fluorescent reporters due to diffuse light propagation in animal tissue. In addition, fluorescence intensities on the tissue surface also depend on the animal?s size, pose, and shape and, hence, limit quantification and reproducibility. Last, there is no anatomical reference that also provides a template for automated organ delineation along with T cell biodistribution analysis. Therefore, InVivo Analytics seeks funding to develop InVivoFLUOR, an automated data analysis tool for 3D fluorescence tomography (FLt) of mouse models. InVivoFLUOR is comprised of: a Body Conforming Animal Mold (BCAM) for multi-source transillumination FLt and spatial registration of the animal?s geometry and pose; an Organ Probability Map (OPM) for providing an organ template; and a cloud-based FLt algorithm. We will demonstrate its feasibility on an IMT example for determining the spatial biodistribution of fluorescence-labeled T cells and, in combination with bioluminescence imaging (BLi), will compare localization of ovarian cancer cells. In Aim 1 we will confirm the ability to quantitatively determine fluorescent targets inside a small animal. The spatial distribution of known fluorescent targets will be reconstructed and compared to ex vivo data. In Aim 2 we will confirm the ability to determine the in vivo T cell biodistribution at tumor sites. The fluorescence-labeled T cell distribution will be calculated and coregistered to the anatomy based on the OPM and to disseminated ovarian tumors. The ability to instantaneously quantify the T cell distribution in the same animal longitudinally, as opposed to sacrificing a different animal at every time point for T cell counting via FACS or histology, neither of which can identify sites where the activated T cells may be ?hiding?, has an impact on the development of and outcome of new IMTs with high accuracy. InVivoFLUOR will enable cross-platform data comparison and analysis, eliminate operator-dependent variability, increase data reproducibility, and will facilitate the translation of new therapeutics. The successful completion of the proposed project will help to commercialize InVivoFLUOR and will find immediate application in the pharmaceutical industry for rapid development of novel IMTs.