Inflammatory responses after myocardial infarction profoundly impact tissue repair. Yet, efficient tools to serially and noninvasively assess cellular and molecular functions in postinfarct inflammation are lacking. Here we use multichannel fluorescent molecular tomography (FMT) for spatiotemporal resolution of phagocytic and proteolytic activities mediated by macrophages and neutrophils in murine infarcts. We performed FMT imaging to compare the course of efficient and impaired healing in wild-type and FXIII-/- mice, respectively. Mice subjected to coronary ligation received simultaneous injections with Prosense-680, an activatable fluorescence sensor reporting on cathepsin activity, and CLIO-VT750, a magneto-fluorescent nanoparticle for imaging of phagocyte recruitment. On FMT, Prosense-680 infarct signal was 19-fold higher than background (P<0.05). Protease activity was higher in the infarcted lateral wall than in the remote, uninjured septum on ex vivo fluorescence reflectance imaging (contrast to noise ratio 118+/-24). CLIO-VT750 FMT signal coregistered with contrast enhancement in the hypokinetic infarct on MRI. Microscopic fluorescence signal colocalized with immunoreactive staining for cathepsin, macrophages and neutrophils. Flow cytometry of digested infarcts revealed monocytes/macrophages and neutrophils as the source of the fluorescence signal. Phagocytic activity peaked on day 6, and proteolytic activity peaked on day 4 after myocardial infarction. FMT detected impaired recruitment of phagocytes and protease activity in FXIII-/- mice (P<0.05). FMT is a promising noninvasive molecular imaging approach to characterize infarct healing. Spectrally resolved imaging agents allow for simultaneous assesment of key processes of in vivo cellular functions. Specifically, we show that in vivo FMT detects impaired healing in FXIII-/- mice.