Proteomic and genomic approaches provide increasingly comprehensive snapshots of biological networks. Often however, the molecular players in these networks are temporally and spatially very dynamic, and their precise mapping along the time-space continuum can become cost prohibitive or be sample limited. Noninvasive molecular imaging has the potential to longitudinally interrogate specific cellular and molecular hubs in vivo. Until recently, most macroscopic imaging techniques have been limited to display only one molecular player at any given time. We have developed quantitative 4-channel FMT-CT (Q4-FMT) to simultaneously map small clusters of biological targets in vivo. The technology is based on reconstruction of optical photons in transillumination geometry and fusion of 3D fluorescence data-sets with anatomic CT. We have recently adapted reconstruction algorithms to different wavelengths, effectively yielding 4 separate read-out channels. Here we propose to biologically validate and apply Q4-FMT for non-invasive interrogation of a biomarker network in wound healing models following myocardial infarction. We will use a set of imaging agents that interrogate key wound healing biomarkers: a) fluorescent nanoparticles to probe phagocytic activity, b) activatable optical agents to measure protease activity, c) tagged transglutaminase peptide substrates to report on extracellular matrix crosslinking, and d) integrin-targeted nanoparticles reporting on angiogenesis. First, systematic phantom experiments will address system performance in all channels, channel crosstalk, sensitivity, and precision of fluorochrome quantitation and image fusion. Second, in vivo imaging results will be quantitated and justaxposed to results from traditional, accepted gold standards such as flow cytometric analysis of cell suspensions from infarcts, genomic and proteomic data, and to immunoreactive histology. Finally, we will use Q4- FMT to assess therapeutic implications of monocyte subset recruitment and function on the above biomarkers during myocardial infarction in a mouse model of coronary ligation. This is important because timely detection of impaired healing would allow to intervene therapeutically to prevent heart failure. Using lipidoid-delivered siRNA we will silence CCR2. This experimental therapy targets the chemokine receptor that governs recruitment of inflammatory Ly-6Chi but not that of reparative Ly6Clo monocytes, while we will image infarct healing serially with Q4-FMT. We hypothesize that effects of modulating inflammatory monocyte recruitment can be monitored by Q4-FMT in realtime, enhances myocardial repair, and provides novel therapeutic strategies for infarct patients. PUBLIC HEALTH RELEVANCE: Our goal is to establish and validate Q4-FMT, which combines simultaneous 4-channel fluorescence molecular tomography with anatomical CT imaging, allowing to image a cluster of biomarkers simultaneously. We propose to validate Q4-FMT against established ex vivo gold standards and to apply it to study tissue repair after myocardial infarction to identify novel therapeutic targets for enhancement of myocardial repair and prevention of heart failure.