Macrophages are key cellular mediators of inflammation in atheroma and participate in all phases of atherogenesis (1, 2). The atheroma lesion is initialized by recruiting monocytes in inflamed intima. Monocytes mature into macrophages under the stimulation of overexpressed macrophage colony-stimulating factor. As cholesteryl esters gradually accumulate in cytoplasm, macrophages are converted into foam cells at the early stage of atheroma. The accumulation of foam cells leads to the formation of fatty streaks and the deposition of fibrous tissues, indicating the progression of atheroma into an intermediate stage. Fibrous caps are formed on the surface of a lipid-rich core and result in vulnerable plaque as the smooth muscle cells synthesize bulk extracellular matrix. The rupture of plaque and the calcification of vessel walls progressively occlude the lumen. All of these developmental stages are produced in apolipoprotein E–deficient (apoE) mice (3), a transgenic animal model with targeted deletion of the apoE gene. As a ligand, apoE binds to the receptors that are responsible for clearing chylomicrons and very low density lipoprotein remnants (4). A deficiency in apoE reduces diet cholesterol absorption and leads to a substantial increase in plasma cholesterol levels. ApoE mice are used in the study of spontaneous hypercholesterolemia and the subsequent development of atherosclerotic lesions (4, 5). The histopathological progression found in apoE mice is very similar to that found in humans (3). Because the arch shape and the proximity to three major arteries (the right and left common carotid arteries and the left subclavian arteries), atherosclerosis primarily occurs at the aortic arch in apoE mice (3). Dextran-coated paramagnetic nanoparticles, such as ultra-small superparamagnetic iron oxide particles (USPIO), are known to accumulate in the macrophages located in inflamed lesions and carotid artery plaques after intravenous administration (6). The active internalization mechanism may be associated with dextran receptor–mediated endocytosis (1). Intracellular dextranase cleaves the dextran coating and leaves the iron oxide to be solubilized into iron ions followed by progressive incorporation into the hemoglobin pool (6). Because of long circulation times, excellent biocompatibility, and high relaxivity, USPIO are widely used to enhance magnetic resonance imaging (MRI) contrast for imaging lesional macrophages in stroke, multiple sclerosis, atherosclerotic diseases, spinal cord injury, and brain tumors (6). Monocrystalline iron oxide nanoparticles (MION) are a special type of USPIO that contain an icosahedral core of superparamagnetic crystalline FeO (magnetite) with a diameter of ~5 nm (7). Using epichlorohydrin to cross-link the dextran coating and amine groups to functionalize the surface, MION can be converted into an aminated platform (cross-linked iron oxide (CLIO)-NH) that allows attachment of various imaging probes (8). CLIO-NH carrying fluorescent probes has demonstrated preferential uptake by lesional macrophages, concomitant with a much lower uptake by endothelial cells and smooth muscle cells (1). Cu-Labeled diethylenetriamine pentaacetic acid (DTPA)-CLIO-Vivotag 680 (VT680) is a Cu-labeled triple reporter nanoparticle (Cu-TNP) used in MRI, positron-emission tomography (PET), and optical fluorescence imaging (9). Cu-TNP consists of three probe types. A CLIO-NH forms the core of the nanoparticle, generates a T-shortening effect in MRI, and is recognizable by macrophages. Numerous complexes of Cu-DTPA are attached to the surface of CLIO-NH for PET detection. Cu is a positron-emitting radionuclide with an intermediate half-life (12.7 h) that decays by positron (β) with a branching factor of 17.4% and a maximum β energy of 0.653 MeV (10). Cu has been used as a radiotracer in PET imaging and a radiotherapy agent in cancer treatment. Five molecules of VT680, an amine-reactive N-hydroxysuccinimide (NHS) ester of a (benzyl)-indolium–derived far-red fluorescent probe that remains internalized in cells for days without interfering with cell functions, are attached to CLIO-NH for fluorescence imaging (11). Its excitation/emission peak is located at 670 ± 5 nm/688 ± 5 nm. The internalization mechanism includes diffusion into cells within minutes and covalent binding to cellular components (11). This macrophage-targeted PET/MRI/optical agent allows complementary information to be obtained by multimodal imaging (9). The PET tracer is detected at 10–10 M (10), providing a sensitivity to detection that is at least one order of magnitude higher than the 10 M in MRI (9). The spatial resolution of MRI permits mapping of the atherosclerotic vascular territories. The cell-specified optical probes explore the fate of the imaging probe at cellular levels (9, 11).