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We demonstrate a novel approach to ultrasonic detection of molecularly targeted nanoparticle contrast agent accumulation in a xenographic mouse tumor model in vivo. This is accomplished by imaging the entire mouse tumor volume at 15-minute intervals following injection of a v β 3 -targeted perfluoro-carbon core nanoparticles. Radiofrequency (RF) data were stored for off-line analysis using signal energy and a limiting form of the Renyi entropy suitable for real-time calculation, I f, ∞ . The results indicate that I f, ∞ may be used to reliably detect accumulation of targeted nanoparticles within 15 minutes of injection without the need for image subtraction, whereas signal energy images showed less sensitivity to nanoparticle accumulation. The ability to detect nanoparticle accumulation without requiring image subtraction or fixing the transducer in place are necessary prerequisites for translation of this technology to the clinic.

In several investigations of molecular imaging of angiogenic neovasculature using a targeted contrast agent, Renyi entropy (I f (r)) and a limiting form of Renyi entropy (I f, ∞ ) exhibited significantly more sensitivity to subtle changes in scattering architecture than energy-based methods. Many of these studies required the fitting of a cubic spline to backscattered waveforms prior to calculation of entropy (either I f (r) or I f, ∞ ). In this study it is shown that the robustness of I f, ∞ may be improved by using a smoothing spline. Results are presented showing the impact of different smoothing parameters. In addition, if smoothing is preceded by lowpass filtering of the waveforms, further improvements may be obtained.

This work demonstrates the utility of Shannon entropy imaging of myocardial anisotropy in mice. Conventional acoustic microscopy measurements of excised short-axis myocardial slices may be used to delineate this anisotropy ex vivo. This approach often requires processing of specular echoes, which can cause artifacts in images that obscure detailed structures near interfacial boundaries. Two different ultrasonic signal receivers were evaluated using the same raw data as input: log energy(log[E f ]) and Shannon entropy imaging( H S ), entropic imaging is least influenced by specular echoes and is able to resolve anisotropy near interfacial boundaries.

In an earlier studies we reported on the application of Renyi entropy, I f (r), r ≪ 2, for the detection of precancerous lesions, by detection subtle changes in backscattered waveforms f(t) that occured as targeted nanoparticles slowly accumulated near the lesion. In contrast, the signal energy, defined as the sum of squares of the over the same moving window, was unable to detect this change (as was conventional B-mode imaging). Although the computational effort to obtain the result precluded its clinical application with currently available equipment, the study raised the possibility of further sensitivity improvements by using values of r closer to the limiting value of 2, where I f (r) approaches infinity. The current study demonstrates that by extracting the asymptotic form of I f (r) as r → 2 there is no loss of sensitivity. However, the resulting algorithm is must faster than previous approaches and has an operation count that is suitable for implementation in a real-time imaging system.

Previously we reported on an entropy-based signal receiver and demonstrated that, in certain settings, it was more sensitive to subtle changes in scattering architecture than conventional energy-based signal characterization. In this paper we present results demonstrating further improvements in sensitivity using a signal receiver based on the Renyi-entropy.

Stem/progenitor cells participate in many pathological and regenerative processes, which can be studied in vivo with molecular imaging approaches. Labeling cells with contrast agents for non-invasive imaging and tracking typically requires long exposure times or adjunctive methods, such as electroporation or transfection agents that can compromise cell viability. We have previously utilized perfluorocarbon (PFC) nanoparticles (~200 nm) for cell tracking, but this process entailed 12 hours of incubation to achieve efficient cellular labeling. We sought to develop an approach that reduces the prolonged labeling time by enhancing PFC nanoparticle-to-cell interactions using clinical levels of ultrasound energy. Stem/progenitor (CD34+ CD133+ CD31+ ) cells were derived from human umbilical cord mononuclear cells grown in fibronectin- coated OptiCelltrade cassettes. Ultrasound (US) was applied using a medical imaging system (Acuson Sequoia) and broadband (2-3 MHz) phased array transducer, which was coupled to cells in a heated (37degC) waterbath. The transducer was advanced mechanically to expose the entire surface to calibrated levels of US energy (MI: 1.9, frequency: 2 MHz). Estimated exposure time for individual cells was ~5 min. Results: Flow cytometry revealed US application greatly improved labeling when compared with cells not exposed to US (55plusmn9% vs. 17plusmn12% respectively, p

Thrombolytic agents, i.e. "clot-busters" such as tPA, have been shown to be effective therapeutics for acute ischemic stroke. However, systemic administration of such powerful agents may be complicated by increased risk of cerebral hemorrhage and expanding stroke. Targeting of clot-dissolving therapeutics has the potential to decrease the incidence of these complications while simultaneously increasing effectiveness of treatment, by concentrating the available drug at the desired site and permitting a lower overall systemic dose. We have previously demonstrated targeting of liquid perfluorocarbon nanoparticles to thrombi in vitro and in vivo, with concomitant enhancement of acoustic reflectivity from the targeted clot surfaces. In the current study, a traditional thrombolytic enzyme (streptokinase) was incorporated into fibrin-targeted nanoparticles and targeted to plasma clots in vitro to demonstrate use of the nanoparticles as both an acoustic contrast agent and an adjunctive targeted therapeutic delivery system. Human plasma clot samples targeted with streptokinase-loaded liquid perfluorocarbon nanoparticles and immersed in plasminogen/saline solution exhibited 7% loss of volume after 3 hrs exposure, as well as marked time-dependent variation in surface topography and acoustic reflectivity. Control samples exposed to either 1) targeted, streptokinase-loaded nanoparticles in the presence of saline only, or 2) targeted nanoparticles without streptokinase in the presence of plasminogen/saline solution exhibited no significant changes over time. These results suggest the potential utility of targeted nanoparticles for combined diagnosis and treatment of thrombus-initiated ischemic stroke.

The objective of this study was to assess the feasibility of image-based identification of nanoparticle targeted angiogenic neovasculature using backscattered ultrasound in several frequency ranges (7 to 15 and ~20- 35MHz). We employed a liquid-perfluorocarbon nanoparticle contrast agent to target the expression of αvβ3 in tumors implanted two different animal models. Ten K14-HPV16 transgenic mice were treated with either normal saline (n=5) or 0.3 mg/kg i.v. of αvβ3-targeted nanoparticles (n=5) and imaged dynamically for two hours using a research ultrasound imager (Vevo 660 30MHz probe) modified to store digitized RF waveforms. Data were analyzed for all mice at all times post-injection using conventional grayscale and C H (an information-theoretic quantity). These data demonstrate the ability and complementarity of information-theoretic receivers in conjunction with targeted nanoparticles to elucidate the presence of αvβ3-integrins in primordial neovasculature, particularly in acoustically unfavorable environments.

Perfluorocarbon (PFC) nanoparticles can serve as a very specific site-targeted contrast and therapeutic agent after binding to specific cellular biomarkers. Ultrasound has been used in conjunction with microbubbles to enhance the delivery of drugs and genes into the cell, mainly through the process of cavitation. We have demonstrated earlier that ultrasound at 2 MHz (1.9 MI) for 5 min can enhance the interaction of nanoparticles with targeted cells. In this study, we sought to establish that nanoparticles used in conjunction with ultrasound do not act as "cavitation" foci. Cell-culture inserts were seeded with human umbilical vein endothelial cells (HUVECs) and exposed to ultrasound in the presence of either Definity ® or PFC nanoparticles. Definity ® in conjunction with continuous wave ultrasound (2.25 MHz for 1 and 5 minutes) increased the permeability of monolayer by four to six times above the normal, decreased trans-endothelial electrical resistance (a sign of reduced membrane integrity), and decreased cell viability by ~50%. Histological evaluation demonstrated extensive disruptions of cell monolayers. Nanoparticles (both non-targeted and targeted) elicited no changes in the measured parameters under similar insonification conditions. Sonically enhanced delivery of drugs from liquid perfluorocarbon nanoparticles to targeted cells does not involve cavitation but increased lipid exchange with targeted cells.

Duchenne muscular dystrophy is a severe wasting disease, involving replacement of necrotic muscle tissue by fibrous material and fatty infiltrates. One primary animal model is the X chromosome-linked mdx strain of mice, which exhibit a clinical phenotype comparable to a mild expression of Duchenne muscular dystrophy in humans. The goals of the present work were to validate and quantify the capability of both energy and entropy metrics of radio-frequency ultrasonic backscatter to differentiate among normal, dystrophic, and steroid treated skeletal muscle in the mdx model

Lipidomics (the system-level analysis of lipids and their interacting moieties) is an emerging field in biomedical research that seeks to identify lipids as molecular targets for diagnosis and therapy. However, the delivery of lipids to pathological cells depends on the biochemical characteristics of the lipid including the overall charge, size of the polar head groups and length of fatty -acid side chains (Struck and Pagano, 1980). We have reported previously that focused ultrasound energy produced by clinical phase array imagers (2 MHz, 1.9 MI) can augment lipid delivery to cancer cells in vitro with the use of perfluorocarbon nanoparticles as non-cavitating carriers, which comprise a perfluorocarbon core and an outer lipid surfactant monolayer (Crowder et al, 2005). Accordingly, we now seek to establish the role of non-cavitational ultrasound in facilitating the delivery of lipids with a surrogate bulky polar head group. PFOB nanoparticles were synthesized by incorporating a bulky (1.4 nm) polar head group nanogold-DPPE (Nanoprobes, NY) at 0.172 mol% in the lipid monolayer. C-32 melanoma cells were grown on cell culture inserts and incubated with nanogold particles for 30 min followed by insonication with continuous wave ultrasound (2.25 MHz, 5 min, 0.6 MPa) and further incubation for 1 hour at 37 degC. The cells were then prefixed with glutaraldehyde, silver enhanced with HQ silver Enhancement Kit (Nanoprobes, NY) and fixed with sodium thiosulphate. After postfixation with osmium tetroxide the cells were treated with uranyl acetate, embedded in epoxy resin, thin-sectioned and stained with lead citrate. Sections 60 nm thick were examined with a Hitachi H-600 electron microscope. C-32 cells not exposed to ultrasound showed silver-enhanced gold particles partitioned only in the outer bilayer of the cell membrane and no evidence of intracellular nanogold. However, the cells exposed to ultrasound showed numerous nanogold-DPPE components inside the cell polarized to one end of intracellular vesicles. These observations are consistent with a mechanism that suggests ultrasound is capable of stimulating lipid internalization, sorting and intracellular trafficking. We surmise that non-cavitational ultrasound may facilitate the lipid uptake even under conditions not conducive to lipid transport by increasing the lipid diffusion coefficients. Therefore, adjunctive ultrasound could be used to increase the range of lipids delivered from perfluorocarbon nanoparticles to targeted cells, which might otherwise remain confined to the outer bilayer leaflet due to biophysical constraints

Angiogenesis has been postulated as an important marker for the early detection of cancer. The proteins associated with new vessels are sub-resolution for ultrasonic imaging, necessitating the use of contrast agents. In this work we use a liquid, perfluorocarbon nanoparticle previously shown to enhance specific targets in in vitro and in situ settings. Previous studies focused on the use of conventional signal analysis techniques including signal amplitude, signal energy, and spectral analysis. To explore the possibility of further increasing contrast between targeted bio-markers and untargeted tissue, we applied concepts from measure-theoretic (e.g., information theory, thermodynamics) and topological dynamics. Specifically, Shannon entropy, H/sub S/, its continuous limit, H/sub C/, and three quantities obtained using analogies with thermodynamics: C/sub v/, E/sub th/, which have been described elsewhere. We describe the outcome of employing these types of dynamical quantities to ultrasonic data acquired in vivo using New Zealand white rabbits implanted with VX2-tumors and then exposed over the course of two hours to /spl alpha//sub v//spl beta//sub 3/ integrin-targeted liquid perfluorocarbon nanoparticles.

Liquid perfluorocarbon nanoparticle contrast agents can be used to target specific tissue types through incorporation of appropriate ligands into the nanoparticles' outer lipid monolayer. In this study we sought to characterize the specificity of targeting of perfluorooctyl bromide (PFOB) nanoparticles to tissue factor, a transmembrane glycoprotein expressed by smooth muscle cells as part of inflammatory response after vessel injury (e.g., angioplasty). Porcine smooth muscle cells constitutively expressing tissue factor on the cell surface were cultured on microporous membranes and targeted with PFOB nanoparticles by avidin-biotin binding techniques. Treatment groups included: 1) "targeted" cell samples exposed sequentially to biotinylated tissue factor antibody/avidin/biotinlyated nanoparticles, 2) "non-targeted" smooth muscle cells exposed to avidin/biotinlyated nanoparticles; 3) "control" smooth muscle cells left untreated. Ultrasonic imaging and quantification of surface reflectivity for each cell group was accomplished by scanning a 25MHz transducer in a grid over the membrane inserts and analyzing the reflected RF signal from the cell-covered surfaces. Total PFOB content for each sample (directly related to nanoparticle concentration) was determined independently by gas chromatography. Targeted cells exhibited significantly enhanced surface reflectivity (-22.6/spl plusmn/2.0 dB referenced to steel reflector, p

Advances in molecular biology and cellular biochemistry are providing new opportunities for diagnostic medical imaging. Molecular imaging provides an opportunity to "see" the earliest biochemical signatures of disease in vivo analogous to microscopic detection of epitopes with immunohistochemistry techniques. Previously the province of nuclear medicine, today numerous, highly active research programs can be found for all clinically relevant imaging modalities. In many instances, these emerging site-directed imaging agents also incorporate therapeutic agents for drug delivery, allowing noninvasive confirmation and quantification of the targeted therapeutic dose. We have developed a novel multi-modal site-targeted contrast agent for sensitive and specific imaging of molecular epitopes and local therapy, which illustrates the key features of these emerging platform technologies. Targeted nanoparticles are applicable to ultrasound, nuclear, CT, and magnetic resonance imaging and have been used to detect a variety of epitopes expressed on angiogenic vessels, thrombus, and within vascular walls. Moreover, this particular targeted agent can deposit a therapeutic payload through a unique mechanism termed "contact-facilitated delivery". The combined benefits of molecular imaging and therapeutic systems are merging and will potentially alter many current clinical paradigms in the next decade.

Our laboratory previously has demonstrated the ability of liquid perfluoro-carbon (PFC) nanoparticles to serve as site-targeted contrast and therapeutic agents by specifically binding molecular markers. In this study, we sought to enhance the delivery of targeted PFC nanoparticles to cells expressing the integrin /spl alpha//sub v//spl beta//sub 3/ using clinical levels of ultrasound energy. Using an Acuson Sequoia imager, we demonstrate that ultrasound applied at 2MHz and 1.9 mechanical index (MI) for a five minute duration enhances the cellular interaction of targeted perfluorocarbon nanoparticles by melanoma cancer cells in vitro without any untoward effect from the ultrasound energy itself upon either the cells or the perfluorocarbon nanoparticles. Employing a custom designed specimen chamber, we have visualized a fundamental physical mechanism for this effect, that of acoustic radiation force (primary and secondary) on the particles, that may contribute to enhanced delivery. Accordingly, our study shows that enhancement of cellular interaction with targeted nanoparticles is feasible by noncavitational mechanisms. Ultrasonically-enhanced delivery of tracers or drugs to a wide variety of pathologic tissues may be useful for augmenting drug delivery after targeting, while limiting untoward effects on other tissues.

The ability to enhance specifically molecular markers of pathology with ultrasound has been previously demonstrated by our group employing a nanoparticle contrast agent. One of the advantages of this agent is it's relative non-echogenicity in the blood pool that allows increased contrast-to-noise between the blood pool and the bound, site-targeted agent. We sought to define the contrast agent concentration and acoustic parameters necessary to detect contrast enhancement in the blood so that molecular contrast enhancement could be more precisely defined. This study addresses two potential mechanisms that have been proposed for backscatter from the nanoparticle contrast agent in the blood pool - concentration-related scattering and phase conversion from liquid to gaseous perfluorocarbon. The nanoparticles were produced by methods currently standard in our laboratory using perfluorooctyl bromide (PFOB: b.p. 142/spl deg/C) as the major component. Particle size was measured at 200/spl plusmn/30nm. Attenuation coefficient and backscatter of the agent were measured in whole porcine blood (hct 40%) and porcine plasma maintained at 37/spl deg/C. Specimens were insonified using a broadband, single element transducer (5 MHz, 2.54 cm diameter, 5.08 cm focal length). Acoustic pulses with usable bandwidth of 1.5 to 10 MHz, a repetition rate of 1kHz, and peak negative pressure of 3.9, 2.7, 1.5, and 0.8MPa (equivalent to M.I. of: 1.7, 1.2, 0.67, 0.36) were used to measure of attenuation coefficient and backscatter of nanoparticles at concentrations of 0.26, 0.51, 1.02, 2.04, 4.08/spl times/10/sup 14/ particles/mL while suspended in either whole porcine blood or porcine plasma. The attenuation coefficient was linear at all concentrations and power levels and shows no evidence of a resonant peak characteristic of liquid-to-gas phase conversion. The back-scatter coefficient in plasma increased with concentration. However, in blood, backscatter was only significantly different from baseline at 2.04/spl times/l0/sup 14/ particles/mL and above (8x the maximum anticipated dose). These data indicate that phase conversion of PFOB to gas is not the source of the contrast in molecular imaging with site targeted nanoparticles.

Previously we reported that useful ultrasound contrast enhancement could be obtained using a liquid emulsion contrast agent developed in our laboratory. Although the physical basis for this enhancement was not completely characterized, the effect was reproducible in vivo and in vitro. We now hypothesize that microbubble formation does not play a significant role in the acoustic behavior of these contrast agents. Otherwise, attenuation and backscatter would be significantly affected by changes in hydrostatic pressure and acoustic pressure, and would show evidence of scattering agent destruction as the duration of ultrasound exposure increased. Both low power (0.65 MPa) unipolar pulses and high-power (3 MPa) unipolar pulses were used to measure the attenuation coefficient of Optison and that of the emulsion. The coefficient was also measured using a high-power unipolar pulse preceded by 5 cycles of a 1 MHz sine-wave of the same amplitude, emitted at a high repetition frequency (5 kHz). Both specimens were held in a pressurized chamber in a water bath maintained at temperatures ranging from 37/spl deg/ to 50/spl deg/C. The emulsion nanoparticles were produced by methods standard in our laboratory using perfluorooctyl bromide (PFOB: boiling point 142/spl deg/C) as the major component. Particle size was measured at 276 nm. We varied ambient hydrostatic pressure (-50 to 200 mm Hg in 50 mm steps), duration of exposure to the acoustic field (2, 20, 40, 80 s), and peak positive and negative acoustic pressure (0.65 to 3.0 MPa).

The authors have developed a nongaseous, ligand-targeted perfluorocarbon nanoparticle emulsion which can acoustically enhance the presence of molecular epitopes on tissue surfaces. In a two-part study, they first demonstrate how perfluorocarbons with varying phase velocities impact the acoustic reflectivity of plasma clots targeted with an anti-fibrin perfluorocarbon nanoparticle in vitro. The targeted perfluorocarbon contrast agents increased backscattered power from 18.2 to 23.4 dB with the greatest enhancement noted for formulations with low acoustic impedance. Porcine carotid arteries were injured by percutaneous balloon angioplasty and treated in situ with a tissue factor-targeted or control nanoparticulate immunoemulsion. Intravascular ultrasound images (30 MHz) of the arteries before and after application of contrast agent demonstrated the acoustic localization and enhancement of stretch-induced tissue factor expression within the carotid media, which was not apparent in the control vessels.

The goal of myocardial ultrasonic tissue characterization is to complement two-dimensional and Doppler echocardiography by providing information (such as assessment of regional viability based on localized values of backscatter) beyond that derived from an assessment of myocardial dimensions and motion. Quantitative backscatter imaging can be subdivided into three broad areas: (1) direct applications, in which specific pathologies are identified and monitored, (2) indirect applications, in which quantitative techniques designed for use in tissue characterization serve to expand the role of echocardiography, and (3) contributions to the understanding of cardiac structure and function.

Progressive collagen cross-linking is a feature of many cardiovascular conditions (e.g., aging, diabetes, remodeling) and may contribute to impaired ventricular relaxation. However, it is not known whether cross-linking affects anisotropic tissue material properties more in one direction than another. We hypothesized that biological tissue comprising a highly ordered collagen matrix (porcine tendon) would manifest a directional dependence of the physical effects of cross-links that could be quantitatively delineated with ultrasound. Fresh porcine flexor tendons were sliced in 2-mm planes parallel (n=7) and perpendicular (n=7) to the tissue fiber axis for attenuation measurements (1-7 MHz). Tendons subjected to cross-linking treatment were immersed in 10% formalin, and control tendons in normal saline solution. Attenuation measurements were made immediately after immersion for both tendon groups at numerous intervals for 1 hr. The attenuation coefficient of the samples insonified perpendicular to the collagen fibers increased after formalin fixation over 1-hr period, while attenuation for parallel measurements decreased at selected frequencies during the first 20 min and subsequently increased modestly by 1 hr. Although the molecular mechanism responsible for the unexpected decrease in attenuation in the parallel direction as compared with the increase in the perpendicular direction is not clear, it may reflect rapid alterations in directionally dependent viscous and/or elastic components that contribute to material properties of well-ordered biological tissues.

Previously we reported that useful ultrasound contrast enhancement could be obtained using a liquid emulsion contrast agent developed in our laboratory. The physical basis for this enhancement was unknown at the time of our report, although the effect was reproducible, in vivo and in vitro. We hypothesize that microbubble formation does not play a significant role in the acoustic behavior of these contrast agents, otherwise attenuation and backscatter would be. significantly affected by changes in hydrostatic pressure, acoustic pressure, and show evidence of scattering agent destruction as the duration of ultrasound exposure increased. Measurements of attenuation coefficient and backscatter of Optison and the emulsion were made in a water bath maintained at temperatures ranging from 37/spl deg/ to 50/spl deg/C. The emulsion nanoparticles were produced by methods standard in our laboratory using perfluorooctyl bromide (PFOB: boiling point 142/spl deg/C) or perfluorooctane (PFO: boiling point 99/spl deg/C) as the major component. Particle size was measured at 435 and 375 /spl plusmn/ 30 nm respectively. We varied ambient hydrostatic pressure (-50 to 200 mm Hg in 50 mm steps), duration of exposure to insonifying acoustic field (2, 20, 40, 80 s), peak positive and negative acoustic pressure (0.038 to 3.0 MPa).

The emerging field of "molecular imaging" refers to in vivo diagnosis of complex pathological processes by specific detection of unique molecular signatures of disease states either at the cellular, subcellular, or gene level. Identification of specific molecular epitopes causally associated with pathologies can be accomplished with the use of various novel targeted contrast agents, which permit conjunctive delivery and local deposition of therapeutic agents directly at the site of disease. Combining imaging with drug delivery provides verification of drug delivery and quantification of treatment, serving in effect as a new paradigm for rational drug dosing. Important potential clinical applications for targeted contrast agents include: (1) characterization of vulnerable plaques by detection of microscopic deposits of fibrin in plaque fissures prior to rupture. (2) early detection and treatment of solid tumors and metastases (colon, breast, prostate, etc.) by localization of angiogenesis. (3) detection of early atherosclerosis by assessment of epitopes related to macrophage trafficking (e.g., VCAM), and (4) diagnosis and treatment of inflammatory pathologies (infection, arthritis, myocarditis, transplant rejection, etc.) by delineation of epitopes that are markers for inflammatory cell recruitment (adhesion molecules, selectins, etc.) We provide a brief review of the field and discuss specific applications.

Molecular imaging with targeted contrast agents is emerging as an important technological advance for diagnostic ultrasound. In vivo, these agents must bind to and acoustically enhance their targets before the agents are cleared or destroyed. In this study, we present results using a system that was designed and constructed for visualization of the evolution of acoustic contrast enhancement. To evaluate the system, two targets were examined: avidin-complexed nitrocellulose membrane and human plasma clots. The time evolution of enhancement was visualized in 10-minute increments for one hour. A monotonic increase was observed in ultrasonic reflection enhancement from specially treated nitrocellulose membranes for targeted emulsions containing perfluorooctylbromide (1.3/spl plusmn/0.3 dB) and perfluorooctane (2.6/spl plusmn/0.5 dB) within the first 60 minutes of imaging. In comparison, the inherently non-echogenic plasma clots showed a substantial increase of 12.0/spl plusmn/0.9 dB when targeted with a perfluorooctane emulsion. This study demonstrates the concept of molecular imaging and provides the first quantifiable time evolution report of the binding of a site-targeted ultrasonic contrast agent.

Improved echocardiographic endocardial border delineation aids in the assessment of ventricular function and wall motion abnormalities. Microbubble-based contrast agents provide excellent echogenicity but a short-lived effect. In contrast, liquid perfluorocarbon (PFC) nanoparticles exhibit long circulatory times and are stable to insonification but have poor echogenicity. Ideally, a contrast agent should demonstrate long circulatory persistence, resistance to ultrasonic destruction, while displaying significant echogenicity. This study demonstrates the effectiveness of biocompatible, liquid PFC nanoparticles as a blood pool contrast agent. When imaged with power Doppler harmonic imaging (PDHI), this agent exhibited marked blood pool enhancement lasting over 1 hour. Furthermore, the contrast enhancement was achieved at doses of 0.7% blood volume. In order to investigate the influence of particle size on blood pool contrast effect, liquid PFC nanoparticles were formulated with diameters of 232 and 465 nm. The nanoparticles were then administered to canines (N=10), with 7 canines receiving the 232 nm nanoparticles and the remainder receiving the 465 nm nanoparticles at a dose of 0.5 ml/kg. PDHI revealed marked blood pool enhancement in all three canines receiving the 465 nm nanoparticles but minimal contrast effect in the canines receiving the 232 nm nanoparticles. This data suggests that PFC nanoparticles in conjunction with PDHI may serve as an important adjunct for the assessment of ventricular function and wall motion abnormalities.