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Two nonlinear interfacial elasticity models--interfacial elasticity decreasing linearly and exponentially with area fraction--are developed for the encapsulation of contrast microbubbles. The strain softening (decreasing elasticity) results from the decreasing association between the constitutive molecules of the encapsulation. The models are used to find the characteristic properties (surface tension, interfacial elasticity, interfacial viscosity and nonlinear elasticity parameters) for a commercial contrast agent. Properties are found using the ultrasound attenuation measured through a suspension of contrast agent. Dynamics of the resulting models are simulated, compared with other existing models and discussed. Imposing non-negativity on the effective surface tension (the encapsulation experiences no net compressive stress) shows "compression-only" behavior. The exponential and the quadratic (linearly varying elasticity) models result in similar behaviors. The validity of the models is investigated by comparing their predictions of the scattered nonlinear response for the contrast agent at higher excitations against experimental measurement. All models predict well the scattered fundamental response. The nonlinear strain softening included in the proposed elastic models of the encapsulation improves their ability to predict subharmonic response. They predict the threshold excitation for the initiation of subharmonic response and its subsequent saturation.

Polymer-shelled microbubbles are applied as ultrasound contrast agents. To investigate the effect of the polymer on microbubble preparation and acoustic properties, polylactides with systematic variations in molecular weight, crystallinity, and end-group hydrophobicity were used. Polymer-shelled cyclodecane filled capsules were prepared by emulsification, and the cyclodecane was removed by lyophilization to obtain hollow capsules. Complete removal of cyclodecane from the microcapsules was only achieved for short chain (about M(w) 6000) crystalline polymers. The pressure threshold for acoustic destruction of the microbubbles was found to increase with molecular weight. Noncrystalline polymers showed a higher threshold for destruction than crystalline polymers. Hydrophobically modified short chain crystalline polymers showed the steepest increase in acoustic destruction after the threshold as a function of the applied pressure, which is a favorable characteristic for ultrasound mediated drug delivery. Microcapsules made with such polymers had an inhomogeneous surface including pores through which cyclodecane was lyophilized efficiently.

Objective: The purpose of this study was to quantify perfusion in vivo using contrast-enhanced subharmonic imaging (SHI)., Methods: A modified LOGIQ 9 scanner (GE Healthcare, Milwaukee, WI) operating in gray scale SHI mode was used to measure SHI time-intensity curves in vivo. Four dogs received intravenous contrast bolus injections (dose, 0.1 mL/kg), and renal SHI was performed. After 3 contrast agent injections, a microvascular staining technique based on stable (nonradioactive) isotope-labeled microspheres (BioPhysics Assay Laboratory Inc, Worcester, MA) was used to quantify the degree of perfusion in 8 sections of each kidney. Low perfusion states were induced by ligating surgically exposed segmental renal arteries followed by contrast agent injections and microvascular staining. Digital clips were transferred to a personal computer, and SHI time-intensity curves were acquired in each section using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). Subharmonic fractional blood volumes were calculated, and the perfusion was estimated from the initial slope of the fractional blood volume uptake averaged over 3 injections. Subharmonic perfusion data were compared with the gold standard (ie, the microspheres) using linear regression analysis., Results: In vivo gray scale SHI clearly showed flow and, thus, perfusion in the kidneys with almost complete suppression of tissue signals. In total, 270 SHI time-intensity curves were acquired, which reduced to 94 perfusion estimates after averaging. Subharmonic perfusion estimates correlated significantly with microsphere results (r = 0.57; P < .0001). The best SHI perfusion estimates occurred for high perfusion states in the anterior of the kidneys (r = 0.73; P = .0001). The corresponding root mean square error was 2.4%., Conclusions: Subharmonic perfusion estimates have been obtained in vivo. The perfusion estimates were in reasonable to good agreement with a microvascular staining technique.

Zero-thickness interface models are developed to describe the encapsulation of microbubble contrast agents. Two different rheological models of the interface, Newtonian (viscous) and viscoelastic, with rheological parameters such as surface tension, surface dilatational viscosity, and surface dilatational elasticity are presented to characterize the encapsulation. The models are applied to characterize a widely used microbubble based ultrasound contrast agent. Attenuation of ultrasound passing through a solution of contrast agent is measured. The model parameters for the contrast agent are determined by matching the linearized model dynamics with measured attenuation data. The models are investigated for its ability to match with other experiments. Specifically, model predictions are compared with scattered fundamental and subharmonic responses. Experiments and model prediction results are discussed along with those obtained using an existing model [Church, J. Acoust. Soc. Am. 97, 1510 (1995) and Hoff et al., J. Acoust. Soc. Am. 107, 2272 (2000)] of contrast agents.

Objective: The purpose of this study was to evaluate the mechanical index (MI) displayed on clinical ultrasound scanners as a predictor of exposure conditions related to the destruction of sonographic microbubble contrast agents., Methods: Sonazoid (GE Healthcare, Oslo, Norway) and Optison (GE Healthcare, Princeton, NJ) microbubbles were injected into a tissue-mimicking flow phantom. Gray scale imaging was performed with 4 different scanners and 3 different transducers (3.5 MHz curved linear, 2.5 MHz convex, and 7.5 MHz linear array), and the MI displayed by the scanner was varied from 0.2 to 1.5 by changing the system output power. All other scanning parameters were kept constant. Downstream changes in echogenicity were monitored with a PowerVision 7000 scanner (Toshiba America Medical Systems, Tustin, CA) as an indirect measure of bubble destruction. Video intensity changes within the flow tube were determined as a function of MI for the different scanner/transducer combinations, and the best linear fit was determined., Results: At a displayed MI of 0.7, different scanner/transducer combinations exhibited a range in video intensity from +16% to -3% of baseline for Sonazoid and from +8% to -71% for Optison. At an MI of 0.3, reductions in video intensity of up to 32% were produced. These results indicate a wide range in bubble destruction at identical MI values. Likewise, regression analysis found no linear fits for all scanner/transducer combinations (r2 < 0.046)., Conclusions: The MI displayed on clinical ultrasound scanners does not predict the degree of microbubble destruction and should not be used by itself to define exposure conditions for destruction of microbubble contrast agents.

A new nonlinear contrast specific imaging modality, excitation-enhanced imaging (EEI) has been implemented on commercially-available scanners for real-time imaging. This novel technique employs two acoustic fields: a low-frequency, high-intensity ultrasound field (the excitation field) to actively condition contrast microbubbles, and a second lower-intensity regular imaging field applied shortly afterwards to detect enhanced contrast scattering. A Logiq 9 scanner (GE Healthcare, Milwaukee, WI) with a 3.5C curved linear array and an AN2300 digital ultrasound engine (Analogic Corporation, Peabody, MA) with a P4-2 phased array transducer (Philips Medical Systems, Bothell, WA) were modified to perform EEI on a vector-by-vector basis in fundamental and pulse inversion harmonic grayscale modes. Ultrasound contrast microbubbles within an 8 mm vessel embedded in a tissue-mimicking flow phantom (ATS Laboratories, Bridgeport, CT) were imaged in vitro. While video intensities of scattered signals from the surrounding tissue were unchanged, video intensities of echoes from contrast bubbles within the vessel were markedly enhanced. The maximum enhancement achieved was 10.4 dB in harmonic mode (mean enhancement: 6.3 dB; p = 0.0007). In conclusion, EEI may improve the sensitivity of ultrasound contrast imaging, but further work is required to assess the in vivo potential of this new technique.

Detection and measurement of blood flow in neovessels around a tumor can yield prognostic information about the tumor. Early detection and classification may help differentiate benign and malignant tumors; thus, improving patient management. This can be accomplished by injecting ultrasonic contrast agents and measuring the backscattered signals from them. Use of the subharmonic backscattered signals from these agents may be better than fundamental or second harmonic components because of the negligible subharmonics generated by the surrounding tissue. Preliminary results on the detection and measurement of subharmonic signal components up to 12 dB (at increasing pressures) from very small tubes (200 to 300 microm diameter) are reported, demonstrating the possibility and potential application of subharmonic imaging in detecting tumor angiogenesis.

A novel contrast imaging technique has been developed for use with microbubble contrast agents. It employs two acoustic fields: there is an excitation field for conditioning microbubbles and an imaging field for detecting microbubbles. The maximum increases (due to microbubble conditioning) in scattered first and second harmonic signals were 14.5 and 16 dB, respectively. This technique is unique for effectively enhancing the blood-to-tissue image contrast.

This study compared contrast-enhanced ultrasound (US) measures of tumor neovascularity with molecular markers of angiogenesis in a human melanoma xenograft model. A total of 14 mice were implanted with a human melanoma cell line (WM-9) in the thigh. After 2 to 3 weeks, a tumor, approximately 12 mm in diameter, developed. The US contrast agent Optison (Mallinckrodt, St. Louis, MO) was injected in a tail vein (dose: 0.4 to 0.6 mL/kg). Power Doppler and pulse-inversion harmonic imaging (HI) were performed with an Elegra scanner (Siemens Medical Systems, Issaquah, WA) and a 7.5 MHz linear array. Frame-rates of 30 Hz and 0.5 Hz (intermittent imaging) were used for pulse-inversion HI. After surgical removal, specimens were sectioned in the same planes as the US images. Immunohistochemical stains for endothelial cells (CD31), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and cyclooxygenase-2 (COX-2) were performed. Two observers graded the stains (for intensity and percent area), and two other observers graded the US imaging modes (for fractional tumor neovascularity) on the same scale from 0 to 3. Of the 14 mice, 4 failed for technical reasons (i.e., n = 10). Linear regressions indicated statistically significant correlations between percent area stained with COX-2 and power Doppler (r = -0.789; p < 0.01), as well as intermittent pulse-inversion HI (r = -0.795; p < 0.05). There was a trend toward significance between percent area stained with VEGF and intermittent pulse-inversion HI (r = -0.720; 0.05 < p < 0.10). No other comparisons were significant. In conclusion, contrast-enhanced US measures of tumor neovascularity in a human melanoma xenograft model appear to provide a noninvasive marker of angiogenesis corresponding to expression of COX-2. However, the sample size of this study is small and, until further studies have been conducted, these conclusions are preliminary.

Objective: To determine whether a contrast agent enhances sonographic detection of bleeding sites in the abdomen and whether contrast-enhanced three-dimensional sonography provides additional information compared with contrast-enhanced two-dimensional sonography., Methods: Bleeding sites were created within the livers (n = 3), spleens (n = 5), and kidneys (n = 3) of 3 dogs. A sonographic contrast agent with vascular and parenchymal enhancement capabilities was administered intravenously at a dose of 0.02 mL/kg. Before and after each contrast agent injection, the bleeding sites were imaged with two- and three-dimensional sonography in gray scale harmonic imaging and color flow modes. Sonographic findings were compared with gross pathologic findings., Results: Noncontrast-enhanced sonography was not able to show the specific location of the active bleeding in any of the organs evaluated. The contrast agent enhanced the sonographic detection of blood flow in normal vessels and extravasated blood from damaged vessels or organs in all cases. Intrasplenic and intrahepatic hematomas were better identified on delayed imaging sequences because there was marked enhancement of the normal parenchyma, whereas the hematomas remained unenhanced. Reconstructed three-dimensional sonography showed spatial relationships of the bleeding sites and surrounding structures. Gross pathologic findings were consistent with the contrast-enhanced sonographic results., Conclusions: Contrast-enhanced sonography improves the detection and evaluation of abdominal bleeding sites. Contrast-enhanced three-dimensional sonography appears to provide additional information when compared with two-dimensional sonography.

The destruction of insonified Sonazoid microbubbles and its association with inertial cavitation in vitro utilizing an active acoustic detector was investigated. The experimental observation indicated that contrast microbubbles could be damaged at moderate acoustic pressures of 0.6-1.6 MPa (0.4-1.0 in mechanical index, MI). A damaged bubble could be dissolved into the medium on the order of 1 ms, implying that the destruction at moderate pressures is a relatively slow (relative to inertial bubble collapse), nonviolent dissolution process following the disruption of encapsulating surface materials. Inertial cavitation events in the presence of contrast microbubbles were observed using multiple highly intense ultrasound (US) pulses (>1.6 MPa). This observation suggested that intense US might disintegrate contrast microbubbles, and fragments of disintegrated microbubbles could be activated by an upcoming highly intense imaging pulse. The above results imply that inertial cavitation is unlikely to take place in the presence of Sonazoid contrast microbubbles when exposed to diagnostic US with an MI <1.

Ultrasound contrast agents promise to improve the sensitivity and specificity of diagnostic ultrasound imaging. It is of great importance to adapt ultrasound equipment for optimal use with contrast agents e.g., by exploiting the nonlinear properties of the contrast microbubbles. Harmonic imaging is one technique that has been extensively studied and is commercially available. However, harmonic imaging is associated with problems, due to second harmonic generation and accumulation within the tissue itself. Given the lack of subharmonic generation in tissue, one alternative is the creation of subharmonic images by transmitting at the fundamental frequency (fo) and receiving at the subharmonic (fo/2). Subharmonic imaging should have a much better lateral resolution and may be suitable for scanning deep-lying structures owing to the higher transmit frequency and the much smaller attenuation of scattered subharmonic signals. In this paper, we will review different aspects of subharmonic imaging including implementation, in-vitro gray-scale imaging and subharmonic aided pressure estimation.

The nonlinear properties of microbubble contrast agents have been used to create contrast-specific imaging modalities such as harmonic imaging and subharmonic imaging. Thus, a better understanding of the nonlinear performance of contrast microbubbles may enhance the diagnostic capabilities of medical ultrasound (US) imaging. The first and second harmonic, the 1/2 order subharmonic and the 3/2 order ultraharmonic components in spectra of scattered signals from Optison microbubbles insonified at 2 and 4 MHz have been investigated using an in vitro laboratory pulse-echo system. The development of these signal components over time is quite different for 2-MHz insonification compared to 4-MHz insonification. Scattered subharmonic and ultraharmonic signals are much more time-dependent than first and second harmonic echoes. The dependence of the first and second harmonic, subharmonic and ultraharmonic components on acoustic pressure for 2-MHz insonification is similar to that for 4-MHz insonification. The first and second harmonic components increase linearly with acoustic pressure (in double logarithmic scales) and the subharmonic and ultraharmonic amplitudes undergo rapid growths in the intermediate acoustic pressure range and much slower increases at both lower and higher acoustic pressures.

Two surfactant-based contrast agents, ST44 and ST68, were produced according to US Patent # 5,352,436 and filled with either air, C4F10 (perfluorobutane) or SF6 (sulfur hexaflouride). Ten rabbits received i.v. injections of each agent/gas combination with 5 repetitions of each dose (range: 0.005-0.13 mL/kg). A custom-made 10-MHz cuff transducer was placed around the surgically exposed distal aorta and audio Doppler signals were acquired in vivo. Quantitative in vivo dose responses were calculated off-line using spectral power analysis and compared to a theoretical model of microbubble dissolution and enhancement. For qualitative comparisons, 10 rabbits were imaged pre- and postcontrast administration (dose: 0.1 mL/kg) in gray-scale and colour. All agent/gas combinations produced marked Doppler enhancement with air bubbles enhancing least of all (p < 0.0001) and ST68-SF6 best of all (maximum: 27.6 +/- 2.04 dB; p < 0.012). There were no significant differences between other agent/gas combinations (0.30 < p < 0.70). Theoretical enhancement was within 1 order of magnitude of the experimental observations (i.e., deviations of up to 10 dB). The duration of contrast enhancement was 1-2 min for air-filled bubbles, 3-5 min for SF6-filled bubbles and more than 7 min for C4F10-filled bubbles. In conclusion, ST68-SF6 microbubbles produced most in vivo enhancement of the agent/gas combinations studied. Theory matched the measurements within an order of magnitude.

The subharmonic emission from insonified contrast microbubbles was used to create a new imaging modality called Subharmonic Imaging. The subharmonic response of contrast microbubbles to ultrasound pulses was first investigated for determining adequate acoustic transmit parameters. Subharmonic A-lines and gray scale images were then obtained using a laboratory pulse-echo system in vitro and a modified ultrasound scanner in vivo. Excellent suppression of all backscattered signals other than from contrast microbubbles was achieved for subharmonic A-lines in vitro while further optimization is required for in vivo gray scale subharmonic images.

Noninvasive pressure estimation in heart cavities and in major vessels would provide clinicians with a valuable tool for assessing patients with heart and vascular diseases. Some microbubble-based ultrasound contrast agents are particularly well suited for pressure measurements because their substantial compressibility enables microbubbles to vary significantly in size in response to changes in pressure. Pressure changes should then affect reflectivity of microbubbles after intravenous injection of a contrast agent. This has been demonstrated with a galactose-based contrast agent using 2.0-MHz ultrasound tone bursts. Preliminary results indicate that, over the pressure range of 0-186 mmHg, the subharmonic amplitude of scattered signals decreases by as much as 10 dB under optimal acoustic settings and the first and second harmonic amplitudes decrease by less than 3 dB. An excellent correlation between the subharmonic amplitude and the hydrostatic pressure suggests that the subharmonic signal may be utilized for noninvasive detection of pressure changes.

Purpose: To evaluate the recently developed ultrasonographic (US) contrast agent SHU 563A, which is specifically taken up by the reticuloendothelial system (RES)., Materials and Methods: Color Doppler imaging (CDI) was performed in a gel phantom, with SHU 563A microbubbles in stationary suspension. CDI was performed in vivo in five woodchucks with natural hepatomas and in 12 rabbits before and after intravenous bolus injections of SHU 563A (0.16-0.48 mL/kg). After a 15-135-minute delay, the liver and spleen were scanned again, and the image findings were compared with pathologic analysis results., Results: Phantom CDI demonstrated a random mosaic color pattern in spite of the lack of flow. This phenomenon, which is associated with bubble rupture, is termed induced acoustic emission. In vivo, delayed imaging demonstrated acoustic emission signals in normal parenchyma, whereas no mosaic color was seen in regions lacking reticuloendothelial cells (e.g., tumors). Four of 12 VX-2 tumors detected with pathologic analysis were detected with US alone; the remaining eight tumors were detected by using US with contrast agent (100%, P = .0078). Nine of 20 hepatomas were detected at baseline US, whereas 17 were detected after administration of SHU 563A (P = .0215). Acoustic emission enabled detection of hepatic tumors as small as 3 mm in diameter., Conclusion: CDI with SHU 563A demonstrates a random mosaic color pattern, even without flow. The characteristic appearance of acoustic emission signals provides a distinctive method of visualizing normal hepatic tissues and substantially improves the detectability of hepatic tumors.