Molecular and cellular mechanisms of atherogenesis and its treatment are largely being unraveled by in vitro techniques. We describe methodology to directly image macrophage cell activity in vivo in a murine model of atherosclerosis using laser scanning fluorescence microscopy (LSFM) and a macrophage-targeted, near-infrared fluorescent (NIRF) magnetofluorescent nanoparticle (MFNP). Atherosclerotic apolipoprotein E deficient (apoE -/-) mice are injected with MFNP or 0.9% saline, and wild-type mice are injected with MFNP as additional controls. After , common carotid arteries are surgically exposed and prepared for LSFM. Multichannel LSFM of MFNP-enhanced carotid atheroma ( in-plane resolution) shows a strong focal NIRF signal, with a plaque target-to-background ratio of . Minimal NIRF signal is observed in control mice. Spectrally resolved indocyanine green (ICG) fluorescence angiograms confirm the intravascular location of atheroma. On ex vivo fluorescence reflectance imaging, greater NIRF plaque signal is seen in apoE -/- MFNP mice compared to controls . The NIRF signal correlates well with immunostained macrophages, both by stained surface area and macrophage number . The validated experimental methodology thus establishes a platform for investigating macrophage activity in atherosclerosis in vivo, and has implications for the detection of clinical vulnerable plaques.