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Background: Commercially available microbubbles such as Definity contain octafluoropropane encapsulated in a lipid shell. This perfluorocarbon can be compressed into liquid nanodroplets at room temperatures and activated with transthoracic diagnostic ultrasound. The aim of this study was to determine the size range and acoustic characteristics of Definity nanodroplets (DNDs) compared with Definity microbubbles (DMBs)., Methods: An in vitro flow system was used with a diagnostic ultrasound transducer (S5-1, iE33). DMBs were prepared using package insert instructions. DNDs were prepared by cooling DMBs in a -10°C to -15°C isopropyl alcohol bath before hand-pressurizing the solution. The formed DNDs were sized, diluted to 1% solutions, and infused continuously into a phosphate-buffered saline solution running within Silastic tubing. Acoustic intensity (AI) was compared with equivalent dilutions of DMBs at different mechanical indices (MIs) ranging from 0.2 to 1.4 (n = 6 comparisons at each MI) using real-time 56-Hz and triggered 2-Hz frame rates (FRs). A 3-cm-thick tissue-mimicking phantom was used to simulate transthoracic attenuation. In vivo transthoracic studies were performed in four normal pigs infused with 10% intravenous infusions of DMBs or DNDs at real-time and triggered end-systolic FRs to compare differences in myocardial and left ventricular cavity AI., Results: DNDs were smaller than DMBs and ranged in size from 50 to 1,000 nm. In vitro studies revealed that at an MI of 0.2 and an FR of 56 Hz, DMBs had high AI (37 ± 2 dB), but AI dropped to 25 ± 2 dB at an MI of 1.0 (P < .001, analysis of variance). In comparison, DNDs had virtually no AI at MIs of 0.2 to 0.6 at both triggered and 56-Hz FRs (1 ± 0 dB), but AI increased to 34 ± 2 dB at an MI of 1.4 using an FR of 56 Hz (P < .001 vs MI of 0.2). AI also persisted longer at 56 Hz with DNDs when using higher MIs. In vivo studies demonstrated higher myocardial AI for DNDs at higher MIs when using real-time FR, most likely from microvascular nanodroplet activation., Conclusion: These data indicate significant differences in acoustic responses of the commercially available DMBs when administered as an equivalent number of DNDs. The DND formulation may render them more useful for high-MI real-time imaging and other targeted transthoracic diagnostic applications., (Copyright © 2016 American Society of Echocardiography. Published by Elsevier Inc. All rights reserved.)

Contrast agents are currently being modified to combine diagnostic and therapeutic capabilities. For ultrasound (US) imaging with polymeric contrast agents, it is necessary to modify the shell to create "stealth" microbubbles but without these modifications sacrificing the agent's ability to interact with the focused US beam. We hypothesize that addition of the classic immune shielding molecule polyethylene glycol (PEG) to a polylactide (PLA) microbubble shell will affect the acoustic and physical properties of the resulting agents. In an effort to determine the best formulation to achieve a balance between stealth and acoustic activity, we compared two PEGylation techniques; addition of increasing amounts of PEG-PLA copolymer and employing incorporation of a PEG lipid (LipidPEG) into the shell. Loss of acoustic enhancement occurred in a dose-dependent manner for both types of PEGylated agents (loss of signal occurred at >5 wt% PEG-PLA and >1 wt% LipidPEG), while immune activation was also reduced in a dose-dependent manner for the PEG-PLA agents. This study shows that the balance between acoustic behavior and improved immune avoidance was scalable and successful to different degrees with both PEGylation methods, and was best achieved using for PEG-PLA at 5 wt% and for LipidPEG at 1 wt%. Studies are ongoing to evaluate the best method for the targeting and drug delivery capabilities of these agents for applications in cancer treatment. This study represents the basis for understanding the consequences of making modifications to the native polymeric shell., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This paper investigated the influence of needle gauge (19G and 27G), injection rate (0.85ml·min(-1), 3ml·min(-1)) and temperature (room temperature (RT) and body temperature (BT)) on the mean diameter, concentration, acoustic attenuation, contrast to tissue ratio (CTR) and normalised subharmonic intensity (NSI) of three ultrasound contrast agents (UCAs): Definity, SonoVue and MicroMarker (untargeted). A broadband substitution technique was used to acquire the acoustic properties over the frequency range 17-31MHz with a preclinical ultrasound scanner Vevo770 (Visualsonics, Canada). Significant differences (P<0.001-P<0.05) between typical in vitro setting (19G needle, 3ml·min(-1) at RT) and typical in vivo setting (27G needle, 0.85ml·min(-1) at BT) were found for SonoVue and MicroMarker. Moreover we found that the mean volume-based diameter and concentration of both SonoVue and Definity reduced significantly when changing from typical in vitro to in vivo experimental set-ups, while those for MicroMarker did not significantly change. From our limited measurements of Definity, we found no significant change in attenuation, CTR and NSI with needle gauge. For SonoVue, all the measured acoustic properties (attenuation, CTR and NSI) reduced significantly when changing from typical in vitro to in vivo experimental conditions, while for MicroMarker, only the NSI reduced, with attenuation and CTR increasing significantly. These differences suggest that changes in physical compression and temperature are likely to alter the shell structure of the UCAs resulting in measureable and significant changes in the physical and high frequency acoustical properties of the contrast agents under typical in vitro and preclinical in vivo experimental conditions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Microbubbles are widely used as contrast agents to improve the diagnostic capability of conventional, highly speckled, low-contrast ultrasound imaging. However, while microbubbles can be used for molecular imaging, these agents are limited to the vascular space due to their large size (> 1 μm). Smaller microbubbles are desired but their ultrasound visualization is limited due to lower echogenicity or higher resonant frequencies. Here we present nanometer scale, phase changing, blinking nanocapsules (BLInCs), which can be repeatedly optically triggered to provide transient contrast and enable background-free ultrasound imaging. In response to irradiation by near-infrared laser pulses, the BLInCs undergo cycles of rapid vaporization followed by recondensation into their native liquid state at body temperature. High frame rate ultrasound imaging measures the dynamic echogenicity changes associated with these controllable, periodic phase transitions. Using a newly developed image processing algorithm, the blinking particles are distinguished from tissue, providing a background-free image of the BLInCs while the underlying B-mode ultrasound image is used as an anatomical reference of the tissue. We demonstrate the function of BLInCs and the associated imaging technique in a tissue-mimicking phantom and in vivo for the identification of the sentinel lymph node. Our studies indicate that BLInCs may become a powerful tool to identify biological targets using a conventional ultrasound imaging system., Competing Interests: The authors declare no competing financial interests.

Phase-shift perfluorocarbon droplets are designed to convert from the liquid to the gas state by the external application of acoustic or optical energy. Although droplet vaporization has been investigated extensively at ultrasonic frequencies between 1 and 10 MHz, few studies have characterized performance at the higher frequencies commonly used in small animal imaging. In this study, we use standard B-mode imaging sequences on a pre-clinical ultrasound platform to both image and activate sub-micron decafluorobutane droplet populations in vitro and in vivo at center frequencies in the range of 20-40 MHz. Results show that droplets remain stable against vaporization at low imaging pressures but are vaporized at peak negative pressures near 3.5 MPa at the three frequencies tested. This study also found that a small number of size outliers present in the distribution can greatly influence droplet performance. Removal of these outliers results in a more accurate assessment of the vaporization threshold and produces free-flowing microbubbles upon vaporization in the mouse kidney., (Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Angioplasty and stenting of a stenosed artery enable acute restoration of blood flow. However, restenosis or a lack of re-endothelization can subsequently occur depending on the stent type. Cavitation-mediated drug delivery is a potential therapy for these conditions, but requires that particular types of cavitation be induced by ultrasound insonation. Because of the heterogeneity of tissue and stochastic nature of cavitation, feedback mechanisms are needed to determine whether the sustained bubble activity is induced. The objective of this study was to determine the feasibility of passive cavitation imaging through a metal stent in a flow phantom and an animal model. In this study, an endovascular stent was deployed in a flow phantom and in porcine femoral arteries. Fluorophore-labeled echogenic liposomes, a theragnostic ultrasound contrast agent, were injected proximal to the stent. Cavitation images were obtained by passively recording and beamforming the acoustic emissions from echogenic liposomes insonified with a low-frequency (500 kHz) transducer. In vitro experiments revealed that the signal-to-noise ratio for detecting stable cavitation activity through the stent was greater than 8 dB. The stent did not significantly reduce the signal-to-noise ratio. Trans-stent cavitation activity was also detected in vivo via passive cavitation imaging when echogenic liposomes were insonified by the 500-kHz transducer. When stable cavitation was detected, delivery of the fluorophore into the arterial wall was observed. Increased echogenicity within the stent was also observed when echogenic liposomes were administered. Thus, both B-mode ultrasound imaging and cavitation imaging are feasible in the presence of an endovascular stent in vivo. Demonstration of this capability supports future studies to monitor restenosis with contrast-enhanced ultrasound and pursue image-guided ultrasound-mediated drug delivery to inhibit restenosis., (Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Characterizing the non-linear response of microbubble contrast agents is important for their efficacious use in imaging and therapy. In this article, we report that the subharmonic and ultraharmonic response of lipid-shelled microbubble contrast agents exhibits a strong temporal dependence. We characterized non-linear emissions from Targestar-p microbubbles (Targeson Inc., San Diego, CA, USA) periodically for 60 min, at 10 MHz excitation frequency. The results revealed a considerable increase in the subharmonic and ultraharmonic response (nearly 12-15 and 5-8 dB) after 5-10 min of agent preparation. However, the fundamental and the harmonic response remained almost unchanged in this period. During the next 50 min, the subharmonic, fundamental, ultraharmonic, and harmonic responses decreased steadily by 2-5 dB. The temporal changes in the non-linear behavior of the agent appeared to be primarily mediated by gas-exchange through the microbubble shell; temperature and prior acoustic excitation based mechanisms were ruled out. Further, there was no measurable change in the agent size distribution by static diffusion. We envisage that these findings will help obtain reproducible measurements from agent characterization, non-linear imaging, and fluid-pressure sensing. These findings also suggest the possibility for improving non-linear imaging by careful design of ultrasound contrast agents., (Published by Elsevier Inc.)

The acoustic properties of two clinical (Definity, Lantheus Medical Imaging, North Billerica, MA, USA; SonoVue, Bracco S.P.A., Milan, Italy) and one pre-clinical (MicroMarker, untargeted, Bracco, Geneva, Switzerland; VisualSonics, Toronto, ON, Canada) ultrasound contrast agent were characterized using a broadband substitution technique over the ultrasound frequency range 12-43 MHz at 20 ± 1°C. At the same number concentration, the acoustic attenuation and contrast-to-tissue ratio of the three native ultrasound contrast agents are comparable at frequencies below 30 MHz, though their size distributions and encapsulated gases and shells differ. At frequencies above 30 MHz, native MicroMarker has higher attenuation values and contrast-to-tissue ratios than native Definity and SonoVue. Decantation was found to be an effective method to alter the size distribution and concentration of native clinical microbubble populations, enabling further contrast enhancement for specific pre-clinical applications., (Copyright © 2014 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

We report the first direct observation for a single stable sonoluminescing bubble of a shape instability. Furthermore we show that stable saturation of the shape distortion caused by the instability for a certain range of parameters is experimentally possible and furthermore is directly linked to the curious phenomenon of flash by flash period doubling of the sonoluminescent emission as the afterbounce instability causing the shape distortion is always period doubled whenever the emission is & vice versa., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Liquid-core nanoparticles are promising candidates for targeted ultrasound-controlled therapy, but their acoustic detection remains challenging. High-frequency (20 to 40 MHz) tone burst sequences were implemented with a programmable ultrasound biomicroscope to characterize acoustic response from perfluorooctyl bromide-core nanoparticles with thick poly(lactide-coglycolide) (PLGA) shells. Radio-frequency signals were acquired from flowing solutions of nanoparticles with two different shell-thickness-to-particle-radius ratios, solid PLGA nanoparticles, and latex nanobeads (linear controls). Normalized fundamental (20 MHz) and second-harmonic power spectral density (PSD) increased with particle concentration and was highest for the thinnest shelled particles. The second- harmonic PSD was detectable from the nanoparticles for peak rarefactional pressures (PRP) from 0.97 to 2.01 MPa at 23 cycles and for tone bursts from 11 to 23 cycles at 2.01 MPa. Their second-harmonic¿to¿fundamental ratio increased as a function of PRP and number of cycles. Within the same PRP and cycle ranges, the second-harmonic¿to¿fundamental ratios from matched concentration solutions of latex nanobeads and solid PLGA nanoparticles was more weakly detectable but also increased with PRP and number of cycles. Nanoparticles were detectable under flow conditions in vitro using the contrast agent mode of a high-frequency commercial scanner. These results characterize linear acoustic response from the nanoparticles (20 to 40 MHz) and demonstrate potential for their highfrequency detection.

Ultrasound (US) scanners typically apply lossy, non-linear modifications to the US data for visualization purposes. The resulting images are then stored as compressed video data. Some system manufacturers provide dedicated software for quantification purposes to eliminate such processing distortions, at least partially. This is currently the recommended approach for quantitatively assessing changes in contrast-agent concentration from clinical data. However, the machine-specific access to US data and the limited set of analysis functionalities offered by each dedicated-software package make it difficult to perform comparable analyses with different US systems. The objective of this work was to establish if linearization of compressed video images obtained with an arbitrary US system can provide an alternative to dedicated-software analysis of machine-specific files for the estimation of echo-power. For this purpose, an Aplio 50 system (Toshiba Medical Systems, Tochigi, Japan), coupled with dedicated CHI-Q (Contrast Harmonic Imaging Quantification) software by Toshiba Medical Systems, was used. Results were compared with two approaches that apply algorithms to estimate relative echo-power from compressed video images: commercially available VueBox software by Bracco Suisse SA (Geneva, Switzerland) and in-laboratory software called PixPower. The echo-power estimated by CHI-Q analysis indicated a strong linear relationship versus agent concentration in vitro (R(2) ≥ 0.9996) for dynamic range (DR) settings of DR60 and DR80, with slopes between 9.22 and 9.57 dB/decade (p = 0.05). These values approach the theoretically predicted dependence of 10.0 dB/decade (equivalent to 3 dB for each concentration doubling). Echo-power estimations obtained from compressed video images with VueBox and PixPower also exhibited strong linear proportionality with concentration (R(2) ≥ 0.9996), with slopes between 9.30 and 9.68 dB/decade (p = 0.05). On an independent in vivo data set (N = 24), the difference in echo-power estimation between CHI-Q and each of the other two approaches was calculated after excluding regions that contain pixels affected by saturated or thresholded pixel values. The mean difference in estimates (expressed in decibels) was -0.25 dB between VueBox and CHI-Q (95% confidence interval: -0.75 to 0.26 dB) and -0.17 dB between PixPower and CHI-Q (95% confidence interval: -0.67 to 0.13 dB). To achieve linearization of data, one of the approaches (VueBox) requires calibration files provided by the software manufacturer for each machine type and setting. The other (PixPower) requires empirical correction of the imaging dynamic range based on ground truth data. These requirements could potentially be removed if US system manufacturers were willing to make relevant information on the applied processing publically available. Reliable echo-power estimation from linearized data would facilitate inclusion of different US systems in multicentric studies and more widespread implementation of emerging techniques for quantitative analysis of contrast ultrasound., (2013 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved)

Experiments and theory were undertaken on the destruction of ultrasound contrast agent microbubbles on needle injection, with the aim of predicting agent loss during in vivo studies. Agents were expelled through a variety of syringe and needle combinations, subjecting the microbubbles to a range of pressure drops. Imaging of the bubbles identified cases where bubbles were destroyed and the extent of destruction. Fluid-dynamic calculations determined the pressure drop for each syringe and needle combination. It was found that agent destruction occurred at a critical pressure drop that depended only on the type of microbubble. Protein-shelled microbubbles (sonicated bovine serum albumin) were virtually all destroyed above their critical pressure drop of 109 ± 7 kPa Two types of lipid-shelled microbubbles were found to have a pressure drop threshold above which more than 50% of the microbubbles were destroyed. The commercial lipid-shelled agent Definity was found to have a critical pressure drop for destruction of 230 ± 10 kPa; for a previously published lipid-shelled agent, this value was 150 ± 40 kPa. It is recommended that attention to the predictions of a simple formula could preclude unnecessary destruction of microbubble contrast agent during in vivo injections. This approach may also preclude undesirable release of drug or gene payloads in targeted microbubble therapies. Example values of appropriate injection rates for various agents and conditions are given., (2013 World Federation for Ultrasound in Medicine & Biology. All rights reserved)

In designing targeted contrast agent materials for imaging, the need to present a targeting ligand for recognition and binding by the target is counterbalanced by the need to minimize interactions with plasma components and to avoid recognition by the immune system. We have previously reported on a microbubble imaging probe for ultrasound molecular imaging that uses a buried-ligand surface architecture to minimize unwanted interactions and immunogenicity. Here we examine for the first time the utility of this approach for in vivo molecular imaging. In accordance with previous results, we showed a threefold increase in circulation persistence through the tumor of a fibrosarcoma model in comparison with controls. The buried-ligand microbubbles were then activated for targeted adhesion through the application of noninvasive ultrasound radiation forces applied specifically to the tumor region. Using a clinical ultrasound scanner, microbubbles were activated, imaged, and silenced. The results showed visually conspicuous images of tumor neovasculature and a twofold increase in ultrasound radiation force enhancement of acoustic contrast intensity for buried-ligand microbubbles, whereas no such increase was found for exposed-ligand microbubbles. We therefore conclude that the use of acoustically active buried-ligand microbubbles for ultrasound molecular imaging bridges the demand for low immunogenicity with the necessity of maintaining targeting efficacy and imaging conspicuity in vivo.

Background and Objective: Acoustic scattering properties of ultrasound contrast agents are useful in extending existing or developing new techniques for biomedical imaging applications. A useful first step in this direction is to investigate the acoustic scattering of a new class of acoustic beams, known as helicoidal high-order Bessel beams, to improve the understanding of their scattering characteristics by an ultrasound contrast agent, which at present is very limited., Method: The transverse acoustic scattering of a commercially available albuminoidal ultrasound contrast agent shell filled with air or a denser gas such as perfluoropropane and placed in a helicoidal Bessel beam of any order is examined numerically. The shell is assumed to possess an outer radius a=3.5 microns and a thickness of approximately 105 nm. Moduli of the total and resonance transverse acoustic scattering form functions are numerically evaluated in the bandwidth 0

Microbubbles and cavitation are playing an increasingly significant role in both diagnostic and therapeutic applications of ultrasound. Microbubble ultrasound contrast agents have been in clinical use now for more than two decades, stimulating the development of a range of new contrast-specific imaging techniques which offer substantial benefits in echocardiography, microcirculatory imaging, and more recently, quantitative and molecular imaging. In drug delivery and gene therapy, microbubbles are being investigated/developed as vehicles which can be loaded with the required therapeutic agent, traced to the target site using diagnostic ultrasound, and then destroyed with ultrasound of higher intensity energy burst to release the material locally, thus avoiding side effects associated with systemic administration, e.g. of toxic chemotherapy. It has moreover been shown that the motion of the microbubbles increases the permeability of both individual cell membranes and the endothelium, thus enhancing therapeutic uptake, and can locally increase the activity of drugs by enhancing their transport across biologically inaccessible interfaces such as blood clots or solid tumours. In high-intensity focused ultrasound (HIFU) surgery and lithotripsy, controlled cavitation is being investigated as a means of increasing the speed and efficacy of the treatment. The aim of this paper is both to describe the key features of the physical behaviour of acoustically driven bubbles which underlie their effectiveness in biomedical applications and to review the current state of the art.

Ultrasound contrast agents (UCA) populations are typically polydisperse and contain microbubbles with radii over a given range. Although the behavior of microbubbles of certain sizes might be masked by the behavior of others, the acoustic characterization of UCA is typically made on full populations. In this paper, we have combined acoustic and optical methods to investigate the response of isolated lipid-shelled microbubbles to low-pressure (49 and 62 kPa peak negative pressure) ultrasound tone bursts. These bursts induced slow deflation of the microbubbles. The experimental setup included a microscope connected to a fast camera acquiring one frame per pulse transmitted by a single-element transducer. The behavior of each bubble was measured at multiple frequencies, by cyclically changing the transmission frequency over the range of 2 to 4 MHz during subsequent pulse repetition intervals. The bubble echoes were captured by a second transducer and coherently recorded. More than 50 individual microbubbles were observed. Microbubbles with radii larger than 3 mum did not experience any size reduction. Smaller bubbles slowly deflated, generally until the radius reached a value around 1.4 microm, independent of the initial microbubble size. The detected pressure amplitude backscattered at 2.5 cm distance was very low, decreasing from about 5 Pa down to 1 Pa at 2 MHz as the bubbles deflated. The resonant radius was evaluated from the echo amplitude normalized with respect to the geometrical cross section. At 2-MHz excitation, deflating microbubbles showed highest normalized echo when the radius was 2.2 microm while at higher excitation frequencies, the resonant radius was lower. The relative phase shift of the echo during the deflation process was also measured. It was found to exceed pi/2 in all cases. A heuristic procedure based on the analysis of multiple bubbles of a same population was used to estimate the undamped natural frequency. It was found that a microbubble of 1.7 microm has an undamped natural frequency of 2 MHz. The difference between this size and the resonant radius is discussed as indicative of significant damping.

We focus on a single cavitation bubble driven by ultrasound, a system which is a specimen of forced nonlinear oscillators and is characterized by its extreme sensitivity to the initial conditions. The driven radial oscillations of the bubble are considered to be implicated by the principles of chaos physics and owing to specific ranges of control parameters, can be periodic or chaotic. Despite the growing number of investigations on its dynamics, there is not yet an inclusive yardstick to sort the dynamical behavior of the bubble into classes; also, the response oscillations are so complex that long term prediction on the behavior becomes difficult to accomplish. In this study, the nonlinear dynamics of a bubble oscillator was treated numerically and the simulations were proceeded with bifurcation diagrams. The calculated bifurcation diagrams were compared in an attempt to classify the bubble dynamic characteristics when varying the control parameters. The comparison reveals distinctive bifurcation patterns as a consequence of driving the systems with unequal ratios of R(0)lambda (where R(0) is the bubble initial radius and lambda is the wavelength of the driving ultrasonic wave). Results indicated that systems having the equal ratio of R(0)lambda, share remarkable similarities in their bifurcating behavior and can be classified under a unit category.

Radial modulation imaging is a new promising technique to improve contrast-enhanced ultrasound images. The method is based on dual-frequency insonation of contrast agent microbubbles. A low-frequency (LF) pulse is used to modulate the responses of the microbubbles to a high-frequency (HF) imaging pulse. Inverting the LF pulse induces amplitude and phase differences in the HF response of contrast agent microbubbles, which can be detected using Doppler techniques. Although the technique has been successfully implemented, no consensus persists on parameter choice and resulting effects. In a separate study, "compression-only" behavior of coated microbubbles was observed. Compression-only behavior could be beneficial for radial modulation imaging. This was investigated using high-speed camera recordings and simulations. We recorded the vibrations of 78 single microbubbles in a dual-frequency ultrasound field. The results showed that the LF pulse induced significant compression-only behavior, which for microbubble sizes below and at HF resonance resulted in high radial amplitude modulation. It, however, also appeared that, for radial modulation imaging, microbubble size is more important than resonance and compression-only effects.

Sub-harmonic imaging using encapsulated microbubbles (EMs) improves the contrast of ultrasound imaging by taking advantage of increased contrast to the tissue signal. A dual-frequency excitation technique (DFET) is proposed for enhancing the sub-harmonic emission from EMs as compared with the conventional single frequency sinusoidal excitation technique (SFSET). This study includes theoretical simulation and in vitro experimental verification. A dual-frequency signal (2 and 4 MHz) is used to insonate EMs developed in our laboratory. Both theoretical and experimental studies indicate that the DFET may be able to improve the amplitude of the sub-harmonic component up to 13 dB over the SFSET. Increasing the value of the pulse repetition frequency or the number of cycles of ultrasound tone burst in the application of the DFET may increase the sub-harmonic emission. Furthermore, it is confirmed that the amplitude ratio of the second frequency (4 MHz) to the first frequency (2 MHz) and phase shift of the second frequency with respect to the first frequency also play an important role in sub-harmonic emission. A ratio of 0.5 and a phase shift around 180 degrees are found to be the optimum values.

Purpose: To investigate the behavior of perflubutane microbubbles outside and inside Kupffer cells during diagnostic ultrasound (US) examination, and to determine the thresholds of the acoustic pressure of different kinds of behavior., Methods: Acoustic behavior of perflubutane microbubbles inside and outside Kupffer cells in an acoustic field induced by a clinical US transducer and equipment was optically observed in vitro. The acoustic pressure was measured simultaneously by a calibrated hydrophone and an oscilloscope., Results: The acoustic behavior of microbubbles was optically categorized as stabilization, oscillation, transposition, shrinkage, and destruction. The mechanical index (MI) displayed on the US equipment correlated well with the acoustic pressure at the level of microbubbles measured hydrophonically. At a frame rate of 15 Hz with a frequency of 3.5 MHz and pulse repetition frequency of 3 KHz, the thresholds in term of MI for free microbubbles to begin oscillation, reach best oscillation, transposition, shrinkage, and destruction were 0.21, 0.44, 0.53, 0.75, and 1.03, respectively. Although adherent and phagocytosed microbubbles showed more stability enduring insonation compared with free microbubbles, the thresholds of shrinkage and destruction were MI 1.03 and 1.18 for adherent microbubbles, and 1.18 and 1.37 for phagocytosed microbubbles, respectively. Neither oscillation nor transposition of microbubbles inside Kupffer cells was observed microscopically. No cell damage because of microbubbles destruction was found in the present study., Conclusion: Perflubutane microbubbles outside and inside Kupffer cells respond to external US insonation with same parameters of a clinical contrast-enhanced US study according to the acoustic pressure. Free microbubbles behave as stabilization, oscillation, transposition, shrinkage, and destruction under insonation. The adherent and phagocytosed microbubbles are more stable under insonation than free microbubbles, but still respond showing shrinkage and destruction when MI is over 1.03.

Stimulating high-frequency nonlinear oscillations of ultrasound contrast agents is helpful to distinguish microbubbles from background tissues. Nevertheless, inefficiency of such oscillations from most commercially available contrast agents and intense attenuation of the resultant high-frequency harmonics limit microbubble detection with high-frequency ultrasound. To avoid this high-frequency nature, we devised and explored a dual-frequency difference excitation technique to induce efficiently low-frequency, rather than high-frequency, nonlinear scattering from microbubbles by using high-frequency ultrasound. The proposed excitation pulse is comprised of 2 high-frequency sinusoids with frequency difference subject to the microbubble resonance frequency. Its envelope, with frequency being the difference between the 2 frequencies, is used to stimulate nonlinear oscillation of microbubbles for the consonant low-frequency harmonic generation, whereas high-imaging resolution is retained because of narrow high-frequency transmit beams. Hydrophone measurements and phantom experiments of speckle-generating flow phantoms were performed to demonstrate the efficacy of the proposed technique. The results show that, especially when the envelope frequency is near the microbubbleiquests resonance frequency, the envelope of the proposed excitation pulse can induce significant nonlinear scattering from microbubbles, the induced nonlinear responses tend to increase with the pulse pressures, and up to 26 dB and 36 dB contrast-to-tissue ratios with second- and fourth-order nonlinear responses, respectively, can be obtained. Potential applications of this method include microbubble fragmentation and cavitation with high-frequency ultrasound.

Abstract-Modified Rayleigh-Plesset models are commonly used to characterize the acoustic response of microbubbles under ultrasound exposure. In most instances these models have been parameterized through acoustic measurements taken from bulk suspensions of microbubbles. The aim of this study was to parameterize the Hoff model for the commercial contrast agent SonoVue using optically observed oscillations from individual microbubbles recorded with a high-speed camera. The shell elasticity model term was tuned to fit simulation data to the measured oscillations while the shell viscosity parameter was held constant at 1 Pa??s. The results demonstrate a limited ability of the model to predict the microbubble behavior. The shell elasticity parameter was found to vary proportionally between 10 and 80 MPa with the initial microbubble diameter, implying the viscoelastic shell terms are not a constant property of the shell material. Further analysis using a moving window optimization to probe the microbubble responses suggests that the elasticity of the shell can increase by up to 50% over the course of insonation, particularly for microbubbles oscillating nearer to their resonant frequency. Microbubble oscillations were modeled more successfully by incorporating a varying elasticity term into the model.

Objectives: We sought to characterize the acoustical behavior of the experimental ultrasound contrast agent BR14 by determining the acoustic pressure threshold above which nonlinear oscillation becomes significant and investigating microbubble destruction mechanisms., Materials and Methods: We used a custom-designed in vitro setup to conduct broadband attenuation measurements at 3.5 MHz varying acoustic pressure (range, 50-190 kPa). We also performed granulometric analyses on contrast agent solutions to accurately measure microbubble size distribution and to evaluate insonification effects., Results: Attenuation did not depend on acoustic pressure less than 100 kPa, indicating this pressure as the threshold for the appearance of microbubble nonlinear behavior. At the lowest excitation amplitude, attenuation increased during insonification, while, at higher excitation levels, the attenuation decreased over time, indicating microbubble destruction. The destruction rate changed with pressure amplitude suggesting different destruction mechanisms, as it was confirmed by granulometric analysis., Conclusions: Microbubbles showed a linear behavior until 100 kPa, whereas beyond this value significant nonlinearities occurred. Observed destruction phenomena seem to be mainly due to gas diffusion and bubble fragmentation mechanisms.

Hyperthermia (HT) is a therapeutic strategy based on the selective damaging of tumoral cells when heated at temperatures in the range 41-45 degrees C. We are currently investigating the feasibility of Ultrasound (US) imaging to perform a non-invasive, efficient and cost effective temperature monitoring of heated tissues. Commercial US contrast agents (Sonovue, Bracco), consisting in microbubbles of SF(6) coated with a phospholipidic shell, greatly improve the US echo signal from tissues. Further investigations have been performed, consisting in physico-chemical and US-imaging characterization. In conclusion, we demonstrate that Sonovue microbubbles reach their maximal diameter at 40 degrees C, and then a sharp decrease is observed, possible due to the occurrence of gel-sol transition of the phospholipidic shell. At the same temperature the maximal backscattering intensity is predicted and actually experimentally observed. Sonovue, as well as other contrast agents based only on phospholipids, are, therefore, not suitable for use as non-invasive temperature monitoring medium since it is sensitive to temperatures below the hyperthermic range. Although microbubbles are in principle thermally effective, other coating materials should be investigated in order to increase their operative thermal range.

In this paper, the multiple scattering of interacting encapsulated microbubbles in suspensions is calculated using two novel approaches--Kargl's effective medium approach and Ye and Ding's approach of 2nd-order correction. Two types of contrast agents with bubbles of different sizes and concentrations are chosen for our numerical simulations. One is Albunex, which is depicted by Church and has a size range of several microns, and the other is sodium laureate solution (before fractionation) described by Soetanto and Chan and has an average size of 35.5 microm. The numerical results from these two approaches are compared with that from the linear approximation. It is found that the multiple scattering effects on attenuation and dispersion of sound in suspensions are evident in the cases of high bubble volume fractions, basically greater than the order of 1 x 10(-4), and much more obvious for larger bubbles with average size of tens of microns.

Microbubble contrast agents produce nonlinear echoes under ultrasound insonation, and current imaging techniques detect these nonlinear echoes to generate contrast agent images accordingly. For these techniques, there is a potential problem in that bubbles along the ultrasound transmission path between transducer and target can alter the ultrasound transmission nonlinearly and contribute to the nonlinear echoes. This can lead to imaging artefacts, especially in regions at depth. In this paper we provide insight, through both simulation and experimental measurement, into the nonlinear propagation caused by microbubbles and the implications for current imaging techniques. A series of investigations at frequencies below, at, and above the resonance frequency of microbubbles were performed. Three specific effects on the pulse propagation (i.e., amplitude attenuation, phase changes, and harmonic generation) were studied. It was found that all these effects are dependent on the initial pulse amplitude, and their dependence on the initial phase of the pulse is shown to be insignificant. Two types of imaging errors are shown to result from this nonlinear propagation: first, that tissue can be misclassified as microbubbles; second, the concentration of microbubbles in the image can be misrepresented. It is found that these imaging errors are significant for all three pulse frequencies when the pulses transmit through a microbubble suspension of 6 cm in path length. It also is found that the first type of error is larger at the bubble resonance frequency.

In this study, the destruction of the contrast agent Sonazoid (GE Healthcare, Oslo, Norway) was measured in vitro as a function of centre frequency (2-3 MHz), acoustic amplitude (0.66-1.6 MPa), pulse length (2-16 cycles) and PRF (0.5-8.0 kHz). Up to 82% of microbubbles were destroyed after exposure to a single 1.6 MPa acoustic pulse (16 cycles, 2.5 MHz and PRF of 1.0 kHz), while at a low amplitude of 0.66 MPa, fractional destruction increased gradually from 0 to 40% after exposure to 9 (identical) pulses. Fractional destruction increased from approximately 8 to 66% as pulse length was changed from 2 to 16 cycles following exposure to a single 2.5 MHz, 1.3 MPa pulse. As the PRF was increased from 0.5 to 8.0 kHz, shorter exposure time intervals (from 4.8 to 1.2 ms) were needed to achieve the same fractional destruction of 80%. Conversely, as the transmit frequency was increased from 2 to 3 MHz the fractional destruction decreased (by more than half within the first 3 pulses). The influence of changes in acoustic pressure and duty cycle on the destruction of Sonazoid microbubbles was highly statistically significant (p < or = 0.01) with a threshold around 0.67 MPa for a duty cycle of 0.0064. In conclusion, the fractional destruction increases with the duty cycle and the acoustic pressure amplitude and decreases with ultrasonic transmit frequency. Better understanding of the influence of the ultrasound transmit parameters on the destruction of contrast microbubbles should help improve existing contrast-assisted imaging modalities and may help develop new techniques for better use of contrast agents.

Ultrasound contrast agents (UCA), created originally for visualization and diagnostic purposes, recently have been suggested as efficient enhancers of ultrasonic power deposition in tissue. The ultrasonic energy absorption by the contrast agents, considered as problematic in diagnostic imaging, might have beneficial impact in therapeutic applications such as targeted hyperthermia-based or ablation treatments. Introduction of gas microbubbles into the tissue to be treated can improve the effectiveness of current treatments by limiting the temperature rise to the treated site and minimizing the damage to the surrounding healthy tissues. To this end, proper assessment of the governing parameters of energy absorption by ultrasonically induced stabilized bubbles is important for both diagnostic and therapeutic ultrasound applications. The current study was designed to predict theoretically and measure experimentally the dissipation and heating effects of encapsulated UCA in a well-controlled and calibrated environment. The ultrasonic effects of the microbubble concentration, transmitted intensity, and frequency on power dissipation and stability of the UCA have been studied. The maximal temperature elevation obtained during 300 s experiments was 21 degrees C, in a 10 ml volume target containing UCA, insonifled by unfocused 3.2 MHz continuous wave (CW) at spatial average intensity of 1.1 W/cm2 (182 kPa). The results also suggest that higher frequencies are more efficiently absorbed by commonly used UCA. In particular, for spatial average intensity of 1.1 W/cm2 and concentration of 5 x 10(6) microspheres/cm3, no significant reduction of UCA absorption was noticed during the first 150 s for insonation at 3.2 MHz and the first 100 s for insonation at 1 MHz. In addition, when lower average intensity of 0.5 W/cm2 (160 kPa) at 3.2 MHz was used, the UCA absorptivity sustained for almost 200 s. Thus, when properly activated, UCA may be suitable for localized hyperthermic therapies.

Ultrasound contrast agents enhance echoes from the microvasculature and enable the visualization of flow in smaller vessels. Here, we optically and acoustically investigate microbubble oscillation and echoes following insonation with a 10 MHz center frequency pulse. A high-speed camera system with a temporal resolution of 10 ns, which provides two-dimensional (2-D) frame images and streak images, is used in optical experiments. Two confocally aligned transducers, transmitting at 10 MHz and receiving at 5 MHz, are used in acoustical experiments in order to detect subharmonic components. Results of a numerical evaluation of the modified Rayleigh-Plesset equation are used to predict the dynamics of a microbubble and are compared to results of in vitro experiments. From the optical observations of a single microbubble, nonlinear oscillation, destruction, and radiation force are observed. The maximum bubble expansion, resulting from insonation with a 20-cycle, 10-MHz linear chirp with a peak negative pressure of 3.5 MPa, has been evaluated. For an initial diameter ranging from 1.5 to 5 microm, a maximum diameter less than 8 microm is produced during insonation. Optical and acoustical experiments provide insight into the mechanisms of destruction, including fragmentation and active diffusion. High-frequency pulse transmission may provide the opportunity to detect contrast echoes resulting from a single pulse, may be robust in the presence of tissue motion, and may provide the opportunity to incorporate high-frequency ultrasound into destruction-replenishment techniques.

Previous studies have revealed that hard-shelled submicron contrast agents exhibit large relative expansions and strong acoustical echoes that can be observed experimentally, and predicted by theoretical simulations. In this paper, we study harmonic imaging and pulse-pair imaging techniques designed to assist in the differentiation of these contrast agents from tissue. For harmonic imaging, we apply a high-sensitivity, narrowband strategy that differentiates the microbubble from tissue based on the generation of strong harmonic echoes. For pulse-pair imaging, we apply high spatial resolution, wideband strategies using phase inversion, which relies on the frequency differences observed in response to phase-inverted pulses, and signal subtraction, which takes advantage of the amplitude differences in response to identical pulses. The bubble-to-phantom signal amplitude ratio in the absence of motion approaches 20 dB using phase inversion and 30 dB using signal subtraction; both techniques are robust for up to 50 microm of simulated motion. With the experience gained in these studies, we hope to advance the development of multi-pulse or shaped-pulse techniques that are optimized for specific clinical applications.

In a previous study, quantitative measurement of nonlinear scattering revealed some quantitative discrepancies with a model for ultrasound scattering by a population ensemble of microbubbles. This study is designed to investigate the effect of the shell on single-pulse scattering by a population of contrast microbubbles. Nonlinear scattering was measured shortly (15 micros) after exposure to a previous ultrasound pulse with variable intensity. The short time delay eliminated the influence of gas diffusion and focused the study on the consequence of ultrasound exposure. The results suggest that single-pulse scattering is dependent on the disruption properties of the bubble shell and confirm the significance of the shell properties.