Preclinical optical imaging systems enable investigation of biomedical concerns such as cancer cardiovascular disease and neurology, non-invasively in animal models. Research findings from basic biological research conducted in test tubes and under the microscope can be expanded into living tissue. Preclinical optical imaging can also be used to investigate the effects of new drugs and other interventions which can provide direct benefit to human medicine. Washington University in St. Louis is actively engaged in biological imaging at different levels, ranging from cellular and small animals to humans. Diffuse optical imaging enables greater depth sensitivity for fluorescence detection than conventional planar imaging systems, but until recently have been too slow and complicated for general use. We propose to acquire upgrade the current time-domain diffuse optical imaging system. The current system has provided groundbreaking optical imaging findings, but is outdated by slow acquisition speed, limited excitation and detection capabilities and a complex user interface. These attributes limit the user base and thus potential research progress is lost. The requested system includes a tunable laser for selection of excitation wavelengths from 480-780 nm providing the capability to detect all commonly used fluorophores. Unlike other optical imaging techniques, the time domain technology used in the requested system provides more accurate recovery of depth and relative fluorophore concentration and enables true 3D representations of fluorophore distribution. The 3D capabilities are extended by enhanced software for reconstruction and fusion with other 3D modalities including CT, MRI, SPECT, PET and PAT. This new system has been improved significantly in key aspects that broaden its field of applications, enhancing current research and attracting users new to optical imaging. Relevance: Upgrade to the new time-domain diffuse optical imaging system will broaden the current applications and user base as well as enhance the funded research of the current users. The enhancements will result in more efficient research progress in molecular-targeted diagnostic agent development, optical imaging system validation and basic biomedical sciences.