In vivo Time-Resolved Wide-Field Molecular Optical Tomography (Parent R01) ABSTRACT There is still great need in better characterizing new targeted therapies in vivo, especially prior to clinical translation. In this regard, preclinical molecular imaging is a central tool in the targeted drug development pipeline. However, there is still a lack of integrated imaging platforms that can enable longitudinal (multiple time points) and spatially-resolved monitoring of complex fingerprints including molecular, metabolic and functional signatures in the same tumor/subject. This new integrated multiplexing imaging platform will play a crucial role in the development of the next generation of targeted drugs and elucidating (multi-) drug resistance mechanisms. Recently, we have demonstrated the unique capabilities of optical imaging in quantifying receptor-target engagement in live subjects by leveraging fluorescence lifetime. This outstanding achievement was realized thanks to the combination of instrumental, algorithmic and biochemical innovations to enable, for the first time, whole-body time-resolved optical imaging based on structured light for 2D or 3D Förster resonance energy transfer (FRET) imaging in live subjects. Herein, using the RFA-CA-20-021, Revision Applications for Incorporation of NCI-Supported Technology to Accelerate Cancer Research, we propose to expand on the parent R01 and employ the IMAT-funded meditope antibody technology to quantitatively monitor in vivo and non- invasively the efficacy of HER2 receptor-targeted drug delivery systems using macroscopy fluorescence lifetime imaging (MFLI)-FRET imaging. We will establish and optimize an antibody-to-target imaging pipeline to quantitatively monitor the binding of near-infrared labeled anti-HER2 meditope antibodies to their respective targets (target engagement) in heterogeneous breast tumor microenvironments.