Monodisperse lipid-coated microbubbles are a promising avenue to unlock the full potential of ultrasound contrast agents for medical diagnosis and therapy. However, their formation by microfluidic flow-focusing is non-trivial. The lipid monolayer shell around the freshly formed bubbles is initially loosely packed, resulting in gas exchange between bubbles through Ostwald ripening, eventually leading to the formation of large, potentially thrombogenic, foam bubbles. Here, we show that by formulating a gas mixture of a low- and a high-aqueous solubility gas, a microbubble suspension can be formed that is not only monodisperse and highly stable, but it can also be synthesized without foam bubble formation at clinically relevant concentrations. The optimal gas volume fraction and resulting gas composition of the stable bubbles are modeled and were found to be in excellent agreement with the experimental data. This physics approach to an interfacial chemistry problem therefore opens a route to bedside production of stable, safe, and readily injectable monodisperse bubbles for medical applications.

Ultrasound contrast agents (UCAs) have opened up immense diagnostic possibilities by combined use of indicator dilution principles and dynamic contrast-enhanced ultrasound (DCE-US) imaging. UCAs are microbubbles encapsulated in a biocompatible shell. With a rheology comparable to that of red blood cells, UCAs provide an intravascular indicator for functional imaging of the (micro)vasculature by quantitative DCE-US. Several models of the UCA intravascular kinetics have been proposed to provide functional quantitative maps, aiding diagnosis of different pathological conditions. This article is a comprehensive review of the available methods for quantitative DCE-US imaging based on temporal, spatial and spatiotemporal analysis of the UCA kinetics. The recent introduction of novel UCAs that are targeted to specific vascular receptors has advanced DCE-US to a molecular imaging modality. In parallel, new kinetic models of increased complexity have been developed. The extraction of multiple quantitative maps, reflecting complementary variables of the underlying physiological processes, requires an integrative approach to their interpretation. A probabilistic framework based on emerging machine-learning methods represents nowadays the ultimate approach, improving the diagnostic accuracy of DCE-US imaging by optimal combination of the extracted complementary information. The current value and future perspective of all these advances are critically discussed.

Recent advances in the field of monodisperse microbubble synthesis by flow focusing allow for the production of foam-free, highly concentrated and monodisperse lipid-coated microbubble suspensions. It has been found that in vitro, such monodisperse ultrasound contrast agents (UCAs) improve the sensitivity of contrast-enhanced ultrasound imaging. Here, we present the first in vivo study in the left ventricle of rat and pig with this new monodisperse bubble agent. We systematically characterize the acoustic sensitivity and safety of the agent at an imaging frequency of 2.5 MHz as compared with three commercial polydisperse UCAs (SonoVue/Lumason, Definity/Luminity and Optison) and one research-grade polydisperse agent with the same shell composition as the monodisperse bubbles. The monodisperse microbubbles, which had a diameter of 4.2 μm, crossed the pulmonary vasculature, and their echo signal could be measured at least as long as that of the polydisperse UCAs, indicating that microfluidically formed monodisperse microbubbles are stable in vivo. Furthermore, it was found that the sensitivity of the monodisperse agent, expressed as the mean echo power per injected bubble, was at least 10 times higher than that of the polydisperse UCAs. Finally, the safety profile of the monodisperse microbubble suspension was evaluated by injecting 400 and 2000 times the imaging dose, and neither physiologic nor pathologic changes were found, which is a first indication that monodisperse lipid-coated microbubbles formed by flow focusing are safe for in vivo use. The more uniform acoustic response and corresponding increased imaging sensitivity of the monodisperse agent may boost emerging applications of microbubbles and ultrasound such as molecular imaging and therapy.

Initial reports from the 1960s describing the observations of ultrasound contrast enhancement by tiny gaseous bubbles during echocardiographic examinations prompted the development of the first ultrasound contrast agent in the 1980s. Current commercial contrast agents for echography, such as Definity, Optison, Sonazoid and SonoVue, have proven to be successful in a variety of on- and off-label clinical indications. Whereas contrast-specific technology has seen dramatic progress after the introduction of the first approved agents in the 1990s, successful clinical translation of new developments has been limited during the same period, while understanding of microbubble physical, chemical and biologic behavior has improved substantially. It is expected that for a successful development of future opportunities, such as ultrasound molecular imaging and therapeutic applications using microbubbles, new creative developments in microbubble engineering and production dedicated to further optimizing microbubble performance are required, and that they cannot rely on bubble technology developed more than 3 decades ago.

08h30 Registration and poster setup + coffee 09h00 Welcome by Sabine Van Huffel 09h15 Keynote lecture 1 by by Heike Vallery Compliant Robotics for Human Augmentation: Towards Bio-Surpassing Functionality 09h45 Single slide poster presentations Part I hosted by Prof. Stefaan Vandenberghe 10h10 Keynote lecture 2 Bram Vanderborgt Actuate2Assist 10h40 Coffee break + poster session 11h10 Keynote lecture 3 by Pascal Doquet Neurostimulation: breakthrough technologies to address the patient needs 11h40 Single slide poster presentations Part II hosted by Prof. Stefaan Vandenberghe 12h05 Keynote lecture 4 by Hendrik Lambert Spinal Cord stimulation to let paralyzed people walk again: is a dream turning in reality? 12h35 Lunch + poster session + industry stands at poster booth 13h45 Parallel sessions 16u00 Poster awards and drinks 16u30 End

Monodisperse phospholipid-coated ultrasound contrast agent (UCA) microbubbles can be directly synthesized in a lab-on-a-chip flow-focusing device. However, high total lipid concentrations are required to minimize on-chip bubble coalescence. Here, we characterize the coalescence probability and the long-term size stability of microbubbles formed using DPPC and DSPC based lipid mixtures as a function of temperature. We show that the coalescence probability can be dramatically reduced by increasing the temperature during bubble formation. Moreover, it is shown that the increased coalescence stability can be explained from an exponential increase of the relative viscosity in the thin liquid film between the colliding bubbles. Furthermore, it was found that the relative viscosity of a DPPC lipid mixture is 7.6 times higher than that of a DSPC mixture and that it can be explained solely from the higher DPPC liposome concentration. Regarding long-term bubble stability, the ratio of the initial on-chip bubble size to the final stable bubble size was always found to be 2.2 for DPPC and DSPC coated bubbles with 10 mol% DPPE-PEG5000, independent of the temperature. Moreover, it was demonstrated that the microbubble suspensions formed at elevated temperatures are highly stable over a time window of 2 to 4 days when collected in a vial. All in all, this work shows that, by increasing the temperature during bubble formation from room temperature to 70 °C, the efficiency of the use of phospholipids in microbubble formation by flow-focusing can be increased by 5 times.

The acoustic response of phospholipid-coated microbubble ultrasound contrast agents (UCA) is dramatically affected by their stabilizing shell. The interfacial shell elasticity increases the resonance frequency, the shell viscosity increases damping, and the nonlinear shell viscoelasticity increases the generation of harmonic echoes that are routinely used in contrast-enhanced ultrasound imaging. To date, the surface area-dependent interfacial properties of the phospholipid coating have never been measured due to the extremely short time scales of the MHz frequencies at which the microscopic bubbles are driven. Here, we present high-precision acoustic measurements of the dilatational nonlinear viscoelastic shell properties of phospholipid-coated microbubbles. These highly accurate measurements are now accessible for the first time by tuning the surface dilatation, that is, the lipid packing density, of well-controlled monodisperse bubble suspensions through the ambient pressure. Upon compression, the shell elasticity of bubbles coated with DPPC and DPPE-PEG5000 was found to increase up to an elasticity of 0.6 N m-1 after which the monolayer collapses and the elasticity vanishes. During bubble expansion, the elasticity drops monotonically in two stages, first to an elasticity of 0.35 N m-1, and then more rapidly to zero. Integration of the elasticity vs. surface area curves showed that, indeed, a phospholipid-coated microbubble is in a tensionless state upon compression, and that it reaches the interfacial tension of the surrounding medium upon expansion. The measurements presented in this work reveal the detailed features of the nonlinear dilatational shell behavior of micron-sized lipid-coated bubbles.

Cancer growth requires angiogenesis; imaging of angiogenesis may thus improve cancer diagnostics and therapy monitoring. Dynamic contrast enhanced ultrasound (DCE-US) permits imaging angiogenesis at the molecular level by using novel targeted ultrasound contrast agents (tUCA). These agents consist of functionalized microbubbles obtained by engineering their shell with targeting ligands able to bind specific biomarkers, over-expressed in tumor angiogenic vasculature. Quantification of binding may thus provide an indirect way of quantifying angiogenesis. Recently, we proposed the first-pass binding (FPB) model to describe the binding kinetics of tUCA. Fitting DCE-US time-intensity curves (TICs) by the FPB model enables quantification of binding by the estimation of the binding rate Kb. After showing the feasibility of the method for angiogenesis imaging in prostate-tumor bearing rats, and performing a preliminary validation for anti-angiogenesis therapy monitoring in colon cancer-bearing mice, in this work we investigated the validity of the proposed model by comparing Kb estimates in rats injected with non-targeted UCAs (Sonovue) and tUCAs (BR55). Significantly lower values of Kb were found for Sonovue compared to BR55, with no significant difference between cancer and healthy prostate for Sonovue.

Ultrasound molecular imaging (USMI) is an emerging technique to monitor diseases at the molecular level by the use of novel targeted ultrasound contrast agents (tUCA). These consist of microbubbles functionalized with targeting ligands with high-affinity for molecular markers of specific disease processes, such as cancer-related angiogenesis. Among the molecular markers of angiogenesis, the vascular endothelial growth factor receptor 2 (VEGFR2) is recognized to play a major role. In response, the clinical-grade tUCA BR55 was recently developed, consisting of VEGFR2-targeting microbubbles which can flow through the entire circulation and accumulate where VEGFR2 is over-expressed, thus causing selective enhancement in areas of active angiogenesis. Discrimination between bound and free microbubbles is crucial to assess cancer angiogenesis. Currently, this is done non-quantitatively by looking at the late enhancement, about 10 min after injection, or by calculation of the differential targeted enhancement, requiring the application of a high-pressure ultrasound (US) burst to destroy all the microbubbles in the acoustic field and isolate the signal coming only from bound microbubbles. In this work, we propose a novel method based on mathematical modeling of the binding kinetics during the tUCA first pass, thus reducing the acquisition time and with no need for a destructive US burst. Fitting time-intensity curves measured with USMI by the proposed model enables the assessment of cancer angiogenesis at both the vascular and molecular levels. This is achieved by estimation of quantitative parameters related to the microvascular architecture and microbubble binding. The proposed method was tested in 11 prostate-tumor bearing rats by performing USMI after injection of BR55, and showed good agreement with current USMI methods. The novel information provided by the proposed method, possibly combined with the current non-quantitative methods, may bring deeper insight into cancer angiogenesis, and thus potentially improve cancer diagnosis and management.

Introduction The recognition of the key role of angiogenesis for cancer growth has opened new frontiers in oncology1. In cancer therapy, the development of novel drugs aimed at blocking angiogenic processes has spurred the need for imaging biomarkers able to assess and predict the response to therapy. In response, targeted contrast agents have been developed providing selective enhancement in areas of active angiogenesis by binding to specific molecules over-expressed in angiogenic tumor vasculature. In the context of ultrasound molecular imaging (USMI), the first and currently only clinical-translatable targeted ultrasound contrast agent (tUCA) is obtained from conventional UCA by decoration of the microbubble shell with ligands targeting the vascular endothelial growth receptor factor 2 (VEGFR2), over-expressed in several solid tumors1. Assessment of the level of binding has been adopted as an indirect way of quantifying angiogenesis. Semi-quantitatively, this is done by looking at the late-enhancement (LE) several minutes after injection, often in conjunction with the application of a high-pressure US burst to derive the differential targeted enhancement (dTE), i.e., the difference in the acoustic signal before and after the application of the high-pressure burst1,2. For quantitative assessment, we recently proposed pharmacokinetic modeling of the binding kinetics of tUCA by the first-pass binding model (FPB), enabling quantification of angiogenesis by estimation of the microbubble binding rate (Kb)3. Here, we show the feasibility of the method for angiogenesis imaging in 11 rat models of prostate cancer, and for therapy monitoring of colorectal cancer in 17 mice undergoing anti-angiogenic treatment. Methods Time-intensity curves extracted at each pixel/voxel of USMI loops were fitted by the FPB model for estimation of the microbubble binding rate Kb. This was compared to semi-quantitative assessment by LE and dTE for: (i) angiogenesis imaging of prostate cancer by comparing parameter values in cancer and healthy regions of interest (ROIs) in 11 rats; (ii) anti-angiogenic therapy monitoring by comparing parameter values in responders, non-responders, and control mice at 5 time points during anti-angiogenic treatment. Results and Discussion Table I shows the differences in USMI parameters in healthy and tumor ROIs, obtained in 11 prostate-tumor bearing rats. Figure 1 shows the changes in USMI parameters during antiangiogenic treatment, obtained in 17 colorectal cancer-bearing mice. The results suggest the microbubble binding rate Kb to be a promising quantitative biomarker of angiogenesis, enabling the distinction between healthy and cancer tissue, and the early prediction of the response to anti-antiangiogenic therapy.

Resonantly driven monodisperse phospholipid-coated microbubbles are expected to substantially increase the sensitivity and efficiency in contrast-enhanced ultrasound imaging and therapy. They can be produced in a microfluidic flow-focusing device, but questions remain as to the role of the device geometry, the liquid and gas flow, and the phospholipid formulation on bubble stability. Here, we develop a model based on simple continuum mechanics equations that reveals the scaling of the coalescence probability with the key physical parameters. It is used to characterize short-term coalescence behavior and long-term size stability as a function of flow-focusing geometry, bulk viscosity, lipid cosolvent mass fraction, lipid concentration, lipopolymer molecular weight, and lipopolymer molar fraction. All collected data collapse on two master curves given by universal equations for the coalescence probability and the long-term size stability. This work is therefore a route to a more fundamental understanding of the physicochemical monolayer properties of microfluidically formed bubbles and their coalescence behavior in a flow-focusing device.

OBJECTIVES:To compare physicochemical characteristics and in vitro and in vivo contrast-enhanced ultrasound imaging performance of 3 commercially available ultrasound contrast agents: SonoVue (Bracco Imaging SpA, Colleretto Giacosa, Italy; also marketed as Lumason in the USA), Definity (Lantheus Medical Imaging, North Billerica, MA) and Optison (GE Healthcare AS, Oslo, Norway).METHODS:Physicochemical characteristics were measured with a Multisizer Coulter Counter (Beckman Coulter, Fullerton, CA). Two ultrasound systems (Aplio 500; Toshiba Medical Systems Corp, Tochigi-ken, Japan; and Logiq E9; GE Healthcare, Little Chalfont, England) were used with different transducers. Contrast enhancement was measured in vitro by dose-ranging measurements using a custom-built beaker setup; in vivo imaging performances were compared in pigs (heart and liver) and rabbits (liver). Quantitative analyses were performed with VueBox quantification software (Bracco Suisse SA, Plan-les-Ouates, Switzerland).RESULTS:Measured physicochemical characteristics were in agreement with those provided by the manufacturers. In vitro data demonstrated that the performance of SonoVue was similar to or better than that of Definity but superior to Optison (normalized scattered power 2- to 10-fold higher with SonoVue). Similar results were obtained in vivo, although the duration of enhancement in the pig heart was longer for SonoVue compared to Definity, and quantitative analysis revealed higher enhancement for SonoVue (1.5-fold increase). For liver imaging, SonoVue and Definity showed similar contrast enhancement and duration of enhancement, but compared to Optison, both peak enhancement and duration of enhancement were superior for SonoVue (up to 2-fold increase).CONCLUSIONS:Imaging performance of SonoVue was similar to or slightly better than that of Definity, but it was superior to Optison for the conditions used in this study.

OBJECTIVE:To compare the proportion of clinically significant prostate cancers (PCa) found in lesions detected by multiparametric MRI (mpMRI) with that found in lesions detected by multiparametric ultrasound (mpUSS), in men at risk. PATIENTS AND METHODS:CADMUS (Cancer Detection by Multiparametric Ultrasound of the prostate) is a prospective, multi-centre paired cohort diagnostic utility study with built-in randomisation of order of biopsies. The trial is registered ISRCTN38541912. All patients will undergo the index test under evaluation (mpUSS±biopsies), as well as the standard test (mpMRI±biopsies). Eligible men will be those at risk of harbouring prostate cancer usually recommended for prostate biopsy, either for the first time or as a repeat, who have not had any prior treatment for prostate cancer. Men in need of repeat biopsy will include those with prior negative results but ongoing suspicion, and those with an existing prostate cancer diagnosis but a need for accurate risk stratification. Both scans will be reported blind to the results of the other and the order in which the targeted biopsies derived from the two different imaging modalities are taken will be randomised. Comparison will be drawn between biopsy results of lesions detected by mpUSS with those lesions detected by mpMRI. Agreement over position between the two imaging modalities will be studied. DISCUSSION:CADMUS will provide level one evidence on the performance of mpUSS derived targeted biopsies in the identification of clinically significant prostate cancer in comparison to mpMRI targeted biopsies. Recruitment is underway and expected to complete in 2018.

You have accessJournal of UrologyProstate Cancer: Detection and Screening I1 Apr 2015MP48-03 CONTRAST ENHANCED ULTRASOUND WITH PARAMETRIC MAPS FOR THE DETECTION OF PROSTATE CANCER Arnoud Postema, Peter Frinking, Martijn Smeenge, Theo De Reijke, Jean De la Rosette, Francois Tranquart, and Hessel Wijkstra Arnoud PostemaArnoud Postema More articles by this author , Peter FrinkingPeter Frinking More articles by this author , Martijn SmeengeMartijn Smeenge More articles by this author , Theo De ReijkeTheo De Reijke More articles by this author , Jean De la RosetteJean De la Rosette More articles by this author , Francois TranquartFrancois Tranquart More articles by this author , and Hessel WijkstraHessel Wijkstra More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.1677AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Reliable imaging is needed to improve prostate cancer(PCa) diagnostic pathways. The altered microvascularity of malignant tissue is targeted by dynamic contrast enhanced ultrasound (DCE-US). Parametric maps generated by software that extracts perfusion parameters from DCE-US recordings can aid interpretation, increasing accuracy and decreasing user-dependency. This study aims to investigate the value of DCE-US, the added value of parametric maps and their potential to reduce the number of negative biopsy cores. METHODS For 651 biopsy locations in 82 consecutive patients that underwent DCE-US imaging, we correlated DCE-US interpretation with and without parametric maps with biopsy results. We used SonoVue® (Bracco, Milan, Italy) as contrast agent and parametric imaging software developed by Bracco Suisse SA (Geneva, Switzerland). We performed a stringent analysis including all positive cores and a clinical analysis including only cores with ≥10% of Gleason ≥7. We determined the potential reduction in biopsies (negative on imaging) and resulting missed PCa (false negatives). We calculated sensitivity, specificty, Positive Predictive Value (PPV) and Negative Predictive Value (NPV) on the per-patient level. RESULTS DCE-US alone classified 470/651 (72.2%) of the biopsies as benign. In the stringent analysis 71 (15.1%) of these were false negatives and in the clinical analysis 40(8.5%). Including parametric maps 411/651 (63.1%) biopsies were classified as benign. Assessed stringently 50 (12.1%) were false negative, clinically 23 (5.6%) were false negative. The clinical per-patient analysis produced our most interesting results: DCE-US classified 36/82 patients as not needing biopsies, missing 8 diagnoses. Including parametric maps, 29/82 patients were classified as benign resulting in 3 missed diagnoses. Sensitivity, specificty, PPV and NPV were 73%, 58%, 50% and 79% for DCE-US alone and 91% (p=0.0588), 56%, 57% and 90% with parametric maps. The figure shows a parametric map with the red zone indicating a high chance of PCa. The corresponding biopsy showed Gleason 3+4 PCa. CONCLUSIONS In our study of 651 biopsy locations a good prediction of biopsy outcome could be made using DCE-US with parametric maps. In our data almost two-thirds of the biopsy cores could be omitted with a modest decrease in PCa detection. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e595 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Arnoud Postema More articles by this author Peter Frinking More articles by this author Martijn Smeenge More articles by this author Theo De Reijke More articles by this author Jean De la Rosette More articles by this author Francois Tranquart More articles by this author Hessel Wijkstra More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Purpose of review An imaging tool providing reliable prostate cancer (PCa) detection and localization is necessary to improve the diagnostic pathway with imaging targeted biopsies. This review presents the latest developments in existing and novel ultrasound modalities for the detection and localization of PCa. Recent findings The ultrasound modalities that were very promising on introduction (HistoScanning and Doppler) have shown a wane in performance when tested in larger patient populations. In the meantime, novel ultrasound modalities have emerged in the field of PCa detection. Modalities, such as shear wave elastography (SWE) and contrast-enhanced ultrasound (CEUS) show very promising results. SWE produces an absolute elasticity measure and removes the need for manual compression of the tissue. The former allows comparison between scans and patients, the latter reduces the interoperator variability. Quantification of CEUS enables easily interpretable and accurate imaging of the microvascular changes associated with clinically significant prostate tumors. Summary The novel ultrasound modalities of SWE and CEUS imaging open the door for taking targeted biopsies based on the detection and localization of PCa by these novel modalities. This potentially improves PCa detection wherein significantly reducing the number of biopsy cores.

Cancer growth requires angiogenesis; imaging of angiogenesis holds thus great potential for improved cancer detection and treatment. In this context, ultrasound molecular imaging permits the visualization of cancer angiogenesis by use of novel targeted contrast agents (tUCA). These consist of ligand-bearing microbubbles designed to specifically bind molecular angiogenic expressions, thus providing selective enhancement especially in the late phase after injection. Discrimination between bound and free microbubbles is crucial to assess the degree of binding and thus to quantify angiogenesis. Currently, binding is mainly assessed by the differential targeted enhancement, i.e., the difference in signal intensity in the late phase before and after the application of a high-pressure destructive pulse. However, this method is not quantitative, and it requires long acquisitions and a high-pressure pulse, which may damage the endothelial tissue. To overcome these limitations, here we propose a new method for quantification of the microbubble binding kinetics by fitting a dedicated compartmental model to the tUCA first-pass. We investigated the feasibility of the method in three prostate tumor-bearing rats. The novel information provided by the proposed method may be combined with the late enhancement analysis to gain deeper insight into tumor angionesis, and thus potentially improve cancer diagnosis and management.

To investigate the value of dynamic contrast‐enhanced (DCE)‐ultrasonography (US) and software‐generated parametric maps in predicting biopsy outcome and their potential to reduce the amount of negative biopsy cores.

Purpose of review An imaging tool providing reliable prostate cancer (PCa) detection and localization is necessary to improve the diagnostic pathway with imaging targeted biopsies. This review presents the latest developments in existing and novel ultrasound modalities for the detection and localization of PCa. Recent findings The ultrasound modalities that were very promising on introduction (HistoScanning and Doppler) have shown a wane in performance when tested in larger patient populations. In the meantime, novel ultrasound modalities have emerged in the field of PCa detection. Modalities, such as shear wave elastography (SWE) and contrast-enhanced ultrasound (CEUS) show very promising results. SWE produces an absolute elasticity measure and removes the need for manual compression of the tissue. The former allows comparison between scans and patients, the latter reduces the interoperator variability. Quantification of CEUS enables easily interpretable and accurate imaging of the microvascular changes associated with clinically significant prostate tumors. Summary The novel ultrasound modalities of SWE and CEUS imaging open the door for taking targeted biopsies based on the detection and localization of PCa by these novel modalities. This potentially improves PCa detection wherein significantly reducing the number of biopsy cores.

This work presents a new approach for automatic motion compensation in the context of ultrasound contrast imaging. Image registration is obtained by performing a rigid transformation on the basis of a similarity criterion between a reference image and the images to be realigned. Mutual information was found to be the most suitable and robust similarity criterion in the presence of changes in contrast occurring during the ultrasound contrast agent wash-in and wash-out phases. A series of optimization strategies is also presented in order to maximize processing speed, such as a predictive-motion model and adaptive spatial sub-sampling, taking into account the image content. Parametric perfusion imaging computed on motion-compensated sequences shows substantial improvements, both in accuracy and in spatial resolution of the parameter estimates, compared to the results obtained on original image sequences. Examples using in-vivo sequences demonstrate the potential of this approach, which will undoubtedly play an increasing role in the future of ultrasound contrast imaging in radiological applications.