UNLABELLEDSpatial and temporal coregistration of nuclear and optical images can enable the fusion of the information from these complementary molecular imaging modalities. A critical challenge is in integrating the optical and nuclear imaging hardware. Flexible fiber-based fluorescence-mediated tomography (FMT) systems provide a viable solution. The various bore sizes of small-animal nuclear imaging systems can potentially accommodate the FMT fiber imaging arrays. In addition, FMT imaging facilitates coregistration of the nuclear and optical contrasts in time. Herein, we combine a fiber-based FMT system with a preclinical SPECT/CT platform. Feasibility of in vivo imaging is demonstrated by tracking a monomolecular multimodal imaging agent (MOMIA) during transport from the forepaw to the axillary lymph node region of a rat.METHODSThe fiber-based, video-rate FMT imaging system is composed of 12 sources (785- and 830-nm laser diodes) and 13 detectors. To maintain high temporal sampling, the system simultaneously acquires ratio-metric data at each detector. A 3-dimensional finite element model derived from CT projections provides anatomically based light propagation modeling. Injection of a MOMIA intradermally into the forepaw of rats provided spatially and temporally coregistered nuclear and optical contrasts. FMT data were acquired concurrently with SPECT and CT data. The incorporation of SPECT data as a priori information in the reconstruction of FMT data integrated both optical and nuclear contrasts.RESULTSAccurate depth localization of phantoms with different thicknesses was accomplished with an average center-of-mass error of 4.1 ± 2.1 mm between FMT and SPECT measurements. During in vivo tests, fluorescence and radioactivity from the MOMIA were colocalized in spatially coincident regions with an average center-of-mass error of 2.68 ± 1.0 mm between FMT and SPECT for axillary lymph node localization. Intravital imaging with surgical exposure of the lymph node validated the localization of the optical contrast.CONCLUSIONThe feasibility of integrating a fiber-based, video-rate FMT system with a commercial preclinical SPECT/CT platform was established. These coregistered FMT and SPECT/CT results with MOMIAs may facilitate the development of the next generation of preclinical and clinical multimodal optical-nuclear platforms for a broad array of imaging applications and help elucidate the underlying biologic processes relevant to cancer diagnosis and therapy monitoring.