Positive Contrast Magnetic Resonance Imaging of Cells Labeled with Magnetic Nanoparticles Charles H. Cunningham, 1 * Takayasu Arai, 2 Phillip C. Yang, 2 Michael V. McConnell, 2 John M. Pauly, 1 and Steven M. Conolly 1 Contrast agents incorporating superparamagnetic iron-oxide nanoparticles have shown promise as a means to visualize labeled cells using MRI. Labeled cells cause significant signal dephasing due to the magnetic field inhomogeneity induced in water molecules near the cell. With the resulting signal void as the means for detection, the particles behave as a negative contrast agent, which can suffer from partial-volume effects. In this paper, a new method is described for imaging labeled cells with positive contrast. Spectrally selective RF pulses are used to excite and refocus the off-resonance water surrounding the labeled cells so that only the fluid and tissue immediately ad- jacent to the labeled cells are visible in the image. Phantom, in vitro, and in vivo experiments show the feasibility of the new method. A significant linear correlation (r  0.87, P < 0.005) between the estimated number of cells and the signal was observed. Magn Reson Med 53:999 –1005, 2005. © 2005 Wiley- Liss, Inc. Key words: paramagnetic contrast agent; SPIO; nanoparticle; selective excitation; ferumoxides Contrast agents incorporating superparamagnetic iron-ox- ide (SPIO) nanoparticles (1,2) have shown much promise as a means to visualize labeled cells using MRI (3–5). The small size of the particles (100 nm) facilitates transport across cell membranes, and the low toxicity allows for large iron loads without significant cell death [e.g., 25 pg/cell (4)]. Labeling can be performed by incubating cells of interest (e.g., embryonic stem cells) with the con- trast agent in vitro, so that they can be monitored in vivo using MRI (6). Cells such as macrophages can be labeled in vivo by introducing the contrast agent into the blood- stream, with the uptake of the agent occurring by phago- cytosis, which has been used to image atherosclerosis and other inflammatory processes (7–9). In more advanced schemes, SPIO–protein complexes that bind to the recep- tors on specific cells have been used (10,11). Cells loaded with SPIO cause significant signal dephas- ing due to the magnetic field inhomogeneity induced in water molecules near the cell. These manifest as signal voids in the image. With the signal void as the means for detection, the particles behave as a negative contrast agent, as opposed to positive contrast agents such as gadolinium chelates that brighten the local signal intensity by short- ening T 1 . A fundamental drawback of negative contrast agents is that the agent cannot be distinguished from a void in the image. Moreover, negative contrast agents suf- fer from partial-volume effects, where the ability to detect a void depends critically on the resolution of the image; voxel size must be smaller than the void volume for reli- able visualization. While it is possible to achieve positive contrast with SPIOs by employing T 1 weighting (12), this is only possible with the smaller size particles (10 –50 nm) and can be inefficient because of competing T 1 and T 2 * effects. In this paper, we describe a new method for imaging cells labeled with SPIO agents with positive contrast. In the new method, spectrally selective RF pulses are used to excite and refocus the off-resonance water surrounding the labeled cells, while suppressing on-resonance signal, so that only the fluid and tissue immediately adjacent to the labeled cells are visible in the image. The spins contribut- ing signal are similar to those imaged with an alternative positive-contrast method (13–15) but the mechanism is different. The pulse sequence design and tradeoffs are described. We also include the results from preliminary phantom, in vitro, and in vivo experiments. THEORY A collection of labeled cells will cast a field pattern in the water molecules immediately surrounding the cells. The field pattern can be approximated by a dipole field from a magnetized sphere (16,17). The dipole pattern demon- strates a classic field cross pattern, in which the local B z field is enhanced in the north and south poles and sup- pressed along the equator (18). The polarity of the field perturbation would be reversed for a diamagnetic agent. The dipole field pattern intensity falls off quickly. The field perturbation varies as B z  r ,  = B 0 3  a r  3  3cos 2  - 1, [1] where  is the difference in bulk magnetic susceptibility between the sphere and surroundings, a is the radius of the sphere, r is the distance from the sphere center, and  is the angle relative to the main field, B 0 [18]. Hence, the field pattern from a smaller collection of cells will fall off more steeply than that from a larger collection. In practice, ag- glomerations of labeled cells may not be spherical, but this theory can be applied to the general case by summing the patterns from a group of spheres. 1 Department of Electrical Engineering, Stanford University, Stanford CA, Cal- ifornia, USA 2 Department of Medicine, Stanford University, Stanford, California, USA Grant sponsor: NIH; Grant numbers: R01EB002992, R21EB002969, R01HL067161, R33EB00777, R01EB00346, R24CA092862-03, R01HL61864; Grant sponsor: Donald W. Reynolds Cardiovascular Clinical Research Center, Stanford University. *Correspondence to: Charles H. Cunningham, 212 Packard EE, 650 Serra Mall, Stanford, CA 94305, USA. E-mail: chuck.cunningham@stanford.edu Received 18 October 2004; accepted 22 December 2004 DOI 10.1002/mrm.20477 Published online in Wiley InterScience (www.interscience.wiley.com). Magnetic Resonance in Medicine 53:999 –1005 (2005) © 2005 Wiley-Liss, Inc. 999