Nonlinear Ultrasound Imaging Research Articles

Impact of wavefront shape on nonlinear ultrasound imaging of monodisperse microbubbles

Impact of wavefront shape on nonlinear ultrasound imaging of monodisperse microbubbles

Nonlinear ultrasound imaging of the microcirculation

Blood flow at and near the tissue level is a physiological parameter of significant experimental and clinical importance, as it reflects the adaptive response of organs to their normal biological environment, to disease, trauma, and the malignant progression of cancer. The use of microbubbles gives ultrasound imaging access to microvascular hemodynamics, which are beyond the ability of Doppler ultrasound methods. However, expanded clinical use of microbubbles has been limited due in part to a lack of quantification and providing only relative differences in microvascular flow. Past and more recent approaches to the quantification of microvascular blood flow will be presented. Past approaches included the use of focused nonlinear pulsing sequences to isolate microbubble from tissue signals followed by analysis of bolus kinetics or flash replenishment dynamics. Although successfull to some extent, their clinical limitations prohibited extended use and the overall application of contrast enhanced ultrasound (CEUS). Newer approaches include higher frame-rate plane wave acquisitions combined with nonlinear pulsing schemes, which enable the tracking of microbubble flow through microvascular networks. Different approaches of these elevated frame-rate acquisitions will be presented, including use of ultrasound localized microscopy.

Geometric effects in gas vesicle buckling under ultrasound

Acoustic reporter genes based on gas vesicles (GVs) have enabled the use of ultrasound to noninvasively visualize cellular function in vivo. The specific detection of GV signals relative to background acoustic scattering in tissues is facilitated by nonlinear ultrasound imaging techniques taking advantage of the sonomechanical buckling of GVs. However, the effect of geometry on the buckling behavior of GVs under exposure to ultrasound has not been studied. To understand such geometric effects, we developed computational models of GVs of various lengths and diameters and used finite element simulations to predict their threshold buckling pressures and postbuckling deformations. We demonstrated that the GV diameter has an inverse cubic relation to the threshold buckling pressure, whereas length has no substantial effect. To complement these simulations, we experimentally probed the effect of geometry on the mechanical properties of GVs and the corresponding nonlinear ultrasound signals. The results of these experiments corroborate our computational predictions. This study provides fundamental insights into how geometry affects the sonomechanical properties of GVs, which, in turn, can inform further engineering of these nanostructures for high-contrast, nonlinear ultrasound imaging.

Power Modulation Echocardiography to Detect and Quantify Myocardial Scar

Myocardial scar correlates with clinical outcomes. Traditionally, late gadolinium enhancement (LGE) cardiovascular magnetic resonance (CMR) is used to detect and quantify scar. In this prospective study using LGE CMR as reference, the authors hypothesized that nonlinear ultrasound imaging, namely, power modulation, can detect and quantify myocardial scar in selected patients with previous myocardial infarction. In addition, given the different histopathology between ischemic and nonischemic scar, a further aim was to test the diagnostic performance of this echocardiographic technique in unselected consecutive individuals with ischemic and nonischemic LGE or no LGE on CMR. Seventy-one patients with previous myocardial infarction underwent power modulation echocardiography following CMR imaging (group A). Subsequently, 101 consecutive patients with or without LGE on CMR, including individuals with nonischemic LGE, were scanned using power modulation echocardiography (group B). In group A, echocardiography detected myocardial scar in all 71 patients, with good scar volume agreement with CMR (bias = -1.9 cm3; limits of agreement [LOA], -8.0 to 4.2 cm3). On a per-segment basis, sensitivity was 82%, specificity 97%, and accuracy 92%. Sensitivity was higher in the inferior and posterior segments and lower in the anterior and lateral walls. In group B, on a per-subject basis, the sensitivity of echocardiography was 62% (91% for ischemic and 30% for nonischemic LGE), with specificity and accuracy of 89% and 72%, respectively. The bias for scar volume between modalities was 5.9 cm3, with LOA of 34.6 to 22.9 cm3 (bias = -1.9 cm3 [LOA, -11.4 to 7.6 cm3] for ischemic LGE, and bias = 18.9 cm3 [LOA, -67.4 to 29.7.6 cm3] for nonischemic LGE). Power modulation echocardiography can detect myocardial scar in both selected and unselected individuals with previous myocardial infarction and has good agreement for scar volume quantification with CMR. In an unselected cohort with nonischemic LGE, sensitivity is low.

Nonlinear Ultrasound Imaging Modeled by a Westervelt Equation

We consider the ultrasound imaging problem governed by a nonlinear wave equation of Westervelt type with variable wave speed. We show that the coefficient of nonlinearity can be recovered uniquely from knowledge of the Dirichlet-to-Neumann map. Our proof is based on a second order linearization and the use of Gaussian beam solutions to reduce the problem to the inversion of a weighted geodesic ray transform. We propose an inversion algorithm and report the results of a numerical implementation to solve the nonlinear ultrasound imaging problem in a transmission setting in the frequency domain.

Online nonlinear ultrasound imaging of crack closure during thermal fatigue loading

A thermal fatigue surface crack (6 mm deep × 23 mm wide) in a thick austenitic stainless steel plate is opened and closed with thermal heating and cooling cycles. This closing and opening of the crack is recorded with using fundamental wave amplitude difference (FAD) technique with 5 MHz 64 element phased array probe. In addition, the contact pressure and crack opening state at different time intervals were estimated using finite element (FE) simulation. In combination with the nonlinear output from the FAD and FE simulation gave insight at which level the contact pressure or the distance between crack faces prevents the contact acoustic nonlinearity (CAN) from happening. Predictably, no clear nonlinear response was recorded at crack face locations experiencing high contact pressure and on the locations where the crack could be considered fully open, as predicted by the FE simulation. Therefore, the FAD technique can be utilized within the constraints quantified in the paper.

Multi-tonal subwavelength metamaterial for absorption and amplification of acoustic and ultrasonic waves

In this work, an acousto-ultrasound metamaterial-based concept is proposed to achieve high multi-tonal sound absorption at specific design frequencies and their multiple harmonics, which generally requires large and complex systems. This structure can be deployed to improve the performance of air-coupled nonlinear acoustic/ultrasound imaging by filtering unwanted fundamental ultrasound responses while amplifying high order harmonics, since nonlinear ultrasonic experiments generally necessitate advanced signal processing tools digital and pass-band filters to highlight nonlinear features. The structure proposed is an Archimedean inspired spiral cavity metamaterial with a thickness of 1/62 wavelength to achieve high multi-tonal sound absorption performances at a design frequency and the multiple harmonics. The same geometrical configuration can also be used to filter a fundamental design excitation frequency f0 and amplify second harmonic of the desired excitation frequencies, 2f0. An analytical model was developed to optimise the sound absorption and amplification frequencies of the structure with a design frequency of 690 Hz, by matching the geometrical parameters with the resonance and antiresonance mechanisms of the system. Additionally, a parallel arrangement of two Archimedean-inspired spirals is also analytically and experimentally proposed, in order to achieve harmonic absorptions at the resonant frequency of each subsystem (i.e. f01 = 850 Hz, f02 = 950 Hz). Furthermore, acoustic impedance analyses have been analytically conducted in order to physically explain all the resonance and antiresonance mechanism occurring with the proposed structures. Experimental investigations show that the proposed 3D printed metamaterial-based structures are capable to achieve multi-tonal high absorption peaks (above 90%) at the fundamental frequency f0 and odd harmonics (3f0, 5f0, etc) and sound amplification of the even harmonics (2f0 and 4f0). The results show good correlation between the predicted model and experimental results, and thus the sub-wavelength metamaterial provides promising potential for controlling and achieving high level sound absorption at low frequencies and enhancing accuracy of nonlinear ultrasound imaging applications.

On the Development of a Novel Contrast Pulse Sequence for Polymer-Shelled Microbubbles.

Contrast agents are routinely used in ultrasound examinations. Nonlinear ultrasound imaging techniques have been developed over decades to enhance the contrast between the tissue and the blood pool after the injection of ultrasound contrast agents (UCAs). In this study, we introduce a new contrast pulse sequence, CPS4. The CPS4 combines pulse inversion (PI), subharmonic (SH), and ultraharmonic (UH) techniques to remove propagation distortion while capturing the unique SH and UH responses from UCAs. The novel CPS4 and conventional PI, SH, and UH techniques were used to detect the presence of a research-grade, thick-shell, polymer microbubble in a tissue-mimicking flow phantom. The contrast-to-tissue ratios (CTRs) obtained from the applications of all techniques were compared. The results show that the highest CTR of approximately 16 dB was obtained using CPS4, which was superior to the individual reference techniques: PI, SH, and UH techniques, in all scenarios considered in this study.

Contrast-enhanced ultrasound imaging using pulse inversion spectral deconvolution.

A contrast-enhanced ultrasound (CEUS) imaging approach, termed pulse inversion spectral deconvolution (PISD), is introduced. The approach uses two Gaussian-weighted Hermite polynomials to form two inverted pulse sequences. The two inversed pulses are then used to filter ultrasound (US) backscattered data and discrimination of the linear and nonlinear signal components. A research US scanner equipped with a linear array transducer was used for data acquisition. The receive data from all channels are shaped using plane wave imaging beamforming with angular compounding (from one to nine angles). In vitro data was collected with a tissue mimicking flow phantom perfused with an US contrast agent using PISD and traditional nonlinear (NLI) US imaging as comparison. The role of imaging frequency (between 4.5 and 6.25 MHz) and mechanical index (from 0.1 to 0.3) were evaluated. Preliminary in vivo data was collected in the hindlimb of three healthy mice. Preliminary experimental findings indicate that the PISD contrast-to-tissue ratio was improved nearly ten times compared to the NLI US imaging approach. Also, the spatial resolution was improved due to the effect of deconvolution and spatial angular compounding. Overall, PISD is a promising postprocessing technique for real-time CEUS imaging.

A combined linear and nonlinear ultrasound time-domain approach for impact damage detection in composite structures using a constructive nonlinear array technique

Discovery and evaluation concerns of barely visible impact damage in composite materials is a well-known issue in industries using these materials. This work proposes a frequency sweep method where damage assessment is conducted with respect to the time domain. Firstly, a combined linear and nonlinear ultrasound imaging technique is proposed, which focuses on the excitation of damage/defect regions using a frequency sweep methodology from multiple transducer locations. Secondly, the method deconstructs time domain signals, which allows for the visualisation of linear and nonlinear ultrasound components independently. While, a filtering and frequency band separation method was used to exploit defect responses over different frequency ranges and provide time domain visualisation at the damage region. Finally, image segmentation was employed to automate the damage sizing procedure, while a binary imaging method was used to remove false positive damage regions produced by material vibration mode excitation (fundamental frequency responses) by using the nonlinear responses as a baseline-free tool.The results showed that the combined linear and nonlinear results provided more accurate results than a purely linear or nonlinear approach, furthermore the results were shown to be equivalent to those of a standard phased array system. The ability of the method to visualise nonlinear outputs in time can improve the understanding of nonlinear ultrasound mechanisms while provides a clear argument that a complete approach, incorporating both linear and nonlinear methods should be regarded as the future of NDT/E systems.

Nonlinear elastic imaging of barely visible impact damage in composite structures using a constructive nonlinear array sweep technique

Linear and nonlinear ultrasound imaging methods highlight different damage features: the linear method detects large stiffness changes, while the nonlinear technique identifies small impedance mismatches, such as microcracks or closed delaminations. Typically, nonlinear ultrasound techniques detect damage/defects in materials by measuring higher order harmonics. These harmonics can be difficult to measure due to low magnitude and signal to noise ratios (SNR): hence large excitation amplitudes are needed, which can further complicate the reliability of these methods as equipment nonlinearities can be generated. To overcome these issues, exciting at specific frequencies, known as local defect resonances (LDR), produce a much larger displacements at the damaged regions. However, estimation of LDR is time-consuming, complex and not an easily automated process.A coupled baseline-free linear and nonlinear ultrasonic imaging approach is proposed, using a Constructive Nonlinear Array Sweep excitation and an image subtraction method for identifying damage in layered materials. The signal sweep method uses a narrow band frequency excitation to increase the probability of detection of a LDR frequency. The novel imaging approach was employed using laser vibrometry measurements in various complex composite structures to assess barely visible impact damage, critical for the aircraft industry. The results showed better estimation of impact damage when compared to classical linear or nonlinear ultrasonic methods leading to improved reliability of aircraft inspections.

Phase and Amplitude Modulation Methods for Nonlinear Ultrasound Imaging With CMUTs.

Conventional amplitude and phase modulated pulse sequences for selective imaging of nonlinear tissue and ultrasound contrast agents are designed for piezoelectric transducers that behave linearly. Inherent nonlinearity of capacitive micromachined ultrasonic transducers (CMUTs), especially during large-signal operation, renders these methods inapplicable. In this paper, we present different pulse sequences for nonlinear imaging that are valid for small- and large-signal CMUT operations. For small-signal operation, two-pulse amplitude and phase modulation methods for microbubble and tissue harmonic imaging are presented, where CMUT nonlinearity is compensated via subharmonic excitation. In the large-signal regime, using a nonlinear model, we first show that there is a simple linear relationship between the phases of each harmonic distortion component generated and the input drive signal. Based on this observation, we demonstrate a pulse sequence using N+1 consecutive phase modulated transmit events to extract N harmonics of the nonlinear contrast agent echo content uncorrupted by CMUT nonlinearity. The proposed methods assume no apriori information about the transducer and, therefore, are applicable to any CMUT. The phase modulation method is also valid for piezoelectric transducers and systems with nonlinearities described by Taylor series where the same phase relationship between the input signal and the harmonic content is valid. The proof of principle experiments using a commercial contrast agent validates the phase modulated pulse sequences for CMUTs, operating in a highly nonlinear collapse-snapback mode and for piezoelectric transducers.

Multiparameter evaluation of in vivo gene delivery using ultrasound-guided, microbubble-enhanced sonoporation

More than 1800 gene therapy clinical trials worldwide have targeted a wide range of conditions including cancer, cardiovascular diseases, and monogenic diseases. Biological (i.e. viral), chemical, and physical approaches have been developed to deliver nucleic acids into cells. Although viral vectors offer the greatest efficiency, they also raise major safety concerns including carcinogenesis and immunogenicity. The goal of microbubble-mediated sonoporation is to enhance the uptake of drugs and nucleic acids. Insonation of microbubbles is thought to facilitate two mechanisms for enhanced uptake: first, deflection of the cell membrane inducing endocytotic uptake, and second, microbubble jetting inducing the formation of pores in the cell membrane. We hypothesized that ultrasound could be used to guide local microbubble-enhanced sonoporation of plasmid DNA. With the aim of optimizing delivery efficiency, we used nonlinear ultrasound and bioluminescence imaging to optimize the acoustic pressure, microbubble concentration, treatment duration, DNA dosage, and number of treatments required for in vivo Luciferase gene expression in a mouse thigh muscle model. We found that mice injected with 50μg luciferase plasmid DNA and 5×105 microbubbles followed by ultrasound treatment at 1.4MHz, 200kPa, 100-cycle pulse length, and 540 Hz pulse repetition frequency (PRF) for 2min exhibited superior transgene expression compared to all other treatment groups. The bioluminescent signal measured for these mice on Day 4 post-treatment was 100-fold higher (p<0.0001, n=5 or 6) than the signals for controls treated with DNA injection alone, DNA and microbubble injection, or DNA injection and ultrasound treatment. Our results indicate that these conditions result in efficient gene delivery and prolonged gene expression (up to 21days) with no evidence of tissue damage or off-target delivery. We believe that these promising results bear great promise for the development of microbubble-enhanced sonoporation–induced gene therapies.

Abstract 1496: Quantification of tumor blood perfusion of an orthotopic mouse model of neuroblastoma using nonlinear contrast-enhanced ultrasound imaging

Abstract This study quantifies tumor perfusion in individual tumors to estimate blood flow and blood volume parameters of an individualized tumor compartment of a comprehensive physiologically-based pharmacokinetic model of topotecan using an orthotopic xenograft model of pediatric neuroblastoma. We non-invasively imaged perfusion in orthotopic neuroblastoma (NB5) xenograft tumors (n = 3 CD1 nude mice/time point) using nonlinear contrast enhanced ultrasound technique (CEUS). Tumor tissue and organs from the mice were harvested at predefined time-points. We used a programmable syringe pump to inject MicroMarker® microbubbles via tail vein catheter and acquired images using VisualSonics VEVO 2100 imaging system. We used the burst-replenishment technique to image tumor perfusion, which requires a constant concentration of microbubbles in blood during acquisition. To maintain a steady concentration of microbubbles, we programmed the pump to inject a small bolus followed by constant infusion. Our preliminary analysis showed that healthy kidneys rapidly reach a steady state in less than 1 min, significantly shorter than the commonly used constant infusion without an initial bolus. The nonlinear CEUS signal intensities of kidney cortex showed less than 20% variation between mice. We used a custom program to acquire the CEUS perfusion images over a 3D volume that included the tumor and a kidney. We used the kidney as a reference organ to normalize whole tumor perfusion data. We fitted the log-normal perfusion model to estimate perfusion parameters for individual tumors. Our perfusion quantification over the entire tumor volume represents tumor perfusion more accurately than the commonly used methods based on a single 2D plane without a reference organ. Our approach provides population estimates of blood perfusion based on properly normalized estimates of individual blood perfusion parameters. Citation Format: Suresh Thiagarajan, Abbas Shirinifard, Megan O. Jacus, Abigail D. Davis, Yogesh T. Patel, Stacy L. Throm, Vinay Daryani, Clinton F. Stewart, András Sablauer. Quantification of tumor blood perfusion of an orthotopic mouse model of neuroblastoma using nonlinear contrast-enhanced ultrasound imaging. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1496. doi:10.1158/1538-7445.AM2015-1496

Abstract 1495: Quantification of blood perfusion in rhabdomyosarcoma xenograft in 3D using contrast-enhanced ultrasound imaging

Abstract This study aims to estimate individual 3D perfusion maps using nonlinear contrast-enhanced ultrasound imaging (CEUS) over the volume of individual rhabdomyosarcoma xenograft tumors in CD1 nude mice. We implanted the tumors subcutaneously close to the right kidney. We administered MicroMarker® microbubbles via tail vein catheter and acquired images using VisualSonics VEVO 2100 imaging system. We used the burst-replenishment technique to image tumor perfusion. To maintain a steady concentration of microbubbles, we used a programmable pump to inject a small bolus followed by constant infusion, achieving a steady state in less than 1 min, significantly shorter than the commonly used constant infusion without an initial bolus. Our preliminary analysis showed that the nonlinear CEUS signal intensities of kidney cortex have less than 20% variation between mice. We used a custom program to acquire the CEUS perfusion images over a 3D volume that included the tumor and the right kidney. We used the kidney as a reference organ to normalize whole tumor perfusion data. We applied temporal clustering methods to identify regions with similar perfusion profiles. We fitted three perfusion models to each region: a) mono-exponential, b) lognormal, c) beam-specific. We compared the performance of the models based on their overall quality of fit (across all the regions) and their error in estimated pre-burst signal level. Our preliminary analysis showed that log-normal and multi-vessel beam-specific models provide better performance than the mono-exponential model. We estimated individual 3D perfusion maps based on the model fits. These perfusion maps over the entire tumor volume represent tumor perfusion more accurately than the commonly used methods based on a single 2D plane over a region of interest and without including a reference organ. Our approach quantifies intratumoral blood perfusion heterogeneity and enables comparison of perfusion across individuals. Our method is extensible to other animal models and cancer types provided they can be imaged using ultrasound and a reference tissue can be imaged reliably in tandem on a subset of planes containing the tumor. Citation Format: Abbas Shirinifard, Suresh Thiagarajan, András Sablauer. Quantification of blood perfusion in rhabdomyosarcoma xenograft in 3D using contrast-enhanced ultrasound imaging. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1495. doi:10.1158/1538-7445.AM2015-1495

Multi-functional biomedical ultrasound: Imaging, manipulation, neuromodulation and therapy

As a safe, low-cost, portable and fast imaging modality, ultrasound has been widely used in various clinical fields. Ultrasound imaging is used in every branch of medicine and can be used to visualize almost every organ. Recently, numerous new ultrasound technologies, such as microbubble-based nonlinear contrast imaging, ultrasound molecular imaging, ultrasound-assisted targeted drug delivery, ultrasonic neuro-modulation and theranostics-based integrative application, have been developed for early-stage diagnosis and treatment, based on the multi-disciplinary developments in physics, engineering, and biomedicine. The nature of biomedical ultrasound technology is also changing, from being an imaging technology into a multi-functional tool that allows both precise diagnosis and treatment. This review offers a critical analysis of the state-of-the-art biomedical ultrasound technology and its application in clinical diagnosis and drug delivery. In this paper, we first introduce some basic physical characteristics of biomedical ultrasound technology, including its wave and mechanical properties, the history of the development of ultrasound contrast agent microbubbles, and the biomedical effects of ultrasound, such as cavitation and heat deposition. Then, we review the recent progress and our research on the preparation and acoustic characterization of microbubbles, nonlinear ultrasound contrast imaging and ultrasound molecular imaging, and ultrasound-assisted drug delivery and treatment based on the large surface of a drug-loaded microbubble and on the surface modification-enabled elastic shell. In addition, we discuss the integrative applications of ultrasound diagnostics and therapeutics in clinical medicine. Moreover, we review the advances in ultrasound-based deep brain stimulation, which indicates that ultrasound technology might be used as a potential noninvasive tool in neuroscience and in the treatment of brain disorders. Finally, we review the challenges associated with the development of biomedical ultrasound technology, which include acoustic power thresholds in drug delivery and deep brain stimulation for the safety and precision control of acoustic fields in inhomogeneous media. In conclusion, besides assessing the conventional tissue anatomy and hemodynamics, we argue that biomedical ultrasound techniques can provide information on tissue elasticity, metabolic function, distribution of temperature, and even biochemical processes. Owing to its advantages such as acoustic radiation force, the use of ultrasound might open a new era in neuro-modulation in the neurosciences, in addition to its traditional areas of use such as imaging and thermal therapy.

Contrast Optimization by Metaheuristic for Inclusion Detection in Nonlinear Ultrasound Imaging

In ultrasound imaging, improvements have been made possible by taking into account the harmonic frequencies. However, the transmitted signal often consists of providing empirically pre-set transmit frequencies, even if the medium to be explored should be taken into account during the optimization process. To resolve this waveform optimization, transmission of stochastic sequences were proposed combined with a genetic algorithm. A medium with an inclusion was compared in term of contrast to a reference medium without defect. Two media were distinguished thanks an Euclidean distance. In simulation, the optimal distance could be multiplied by 4 in comparison with an usual excitation.

Contrast-enhanced molecular ultrasound differentiates endoglin genotypes in mouse embryos

Targeted ultrasound contrast imaging has the potential to become a reliable molecular imaging tool. A better understanding of the quantitative aspects of molecular ultrasound technology could facilitate the translation of this technique to the clinic for the purposes of assessing vascular pathology and detecting individual response to treatment. The objective of this study was to evaluate whether targeted ultrasound contrast-enhanced imaging can provide a quantitative measure of endogenous biomarkers. Endoglin, an endothelial biomarker involved in the processes of development, vascular homeostasis, and altered in diseases, including hereditary hemorrhagic telangiectasia type 1 and tumor angiogenesis, was the selected target. We used a parallel plate perfusion chamber in which endoglin-targeted (MBE), rat isotype IgG2 control and untargeted microbubbles were perfused across endoglin wild-type (Eng+/+), heterozygous (Eng+/-) and null (Eng-/-) embryonic mouse endothelial cells and their adhesion quantified. Microbubble binding was also assessed in late-gestation, isolated living transgenic Eng+/- and Eng+/+ embryos. Nonlinear contrast-specific ultrasound imaging performed at 21 MHz was used to collect contrast mean power ratios for all bubble types. Statistically significant differences in microbubble binding were found across genotypes for both in vitro (p<0.05) and embryonic studies (p<0.001); MBE binding was approximately twofold higher in Eng+/+ cells and embryos compared with their Eng+/- counterparts. These results suggest that molecular ultrasound is capable of reliably differentiating between molecular genotypes and relating receptor densities to quantifiable molecular ultrasound levels.

Abstract 2060: Quantifying vascular biomarkers with contrast-enhanced molecular ultrasound imaging

Abstract Introduction: Molecular imaging has the potential to dramatically impact all facets of patient care, from early disease detection to treatment monitoring and follow-up, as a tool for the characterization and measurement of key biomolecules in vivo. In ultrasound, functional and molecular imaging is possible through the use of microbubbles (MB), a contrast agent that can be transformed into targeting agents that bind to vascular biomarkers of interest. In this study, we evaluate whether targeted ultrasound contrast enhanced imaging can provide a quantitative measure of surface receptor expression in endothelial cell populations. Methods: The biomarker endoglin (Eng) was selected for targeting due to its involvement in the processes of development, vascular regulation and disease, including tumor angiogenesis. Endoglin wildtype (Eng+/+), heterozygous null (Eng+/-) and null (Eng-/-) mouse embryonic endothelial cells were cultured on glass slides and mounted in parallel plate flow chambers. MicroMarker microbubbles (endoglin targeted: MBE, isotype control: MBC or untargeted: MBU at 1x107 MB/mL in PBS) were perfused across the cells at 4 mL/min, corresponding to a shear stress of 2 dynes/cm2. Cell and bubble numbers were determined from bright field and phase images (Nikon, 40x), with adhesion quantified as the number of MB/cell. Binding of microbubbles was also assessed in late-gestational stage, isolated, living embryos (Eng+/+, Eng+/-). The highly regulated and controlled activities of normal angiogenesis and vasculogenesis in the mouse embryo make it an excellent surrogate for complex and heterogeneous tumor microenvironments, while genetic manipulation enables the generation of a variety of useful transgenic models. Nonlinear contrast-specific ultrasound imaging, performed at 21MHz with a Vevo-2100 scanner (VisualSonics Inc.), was used to collect contrast mean power ratios (CMPR, representative measure of MB binding) within the brains of each embryo 4 minutes after a bolus injection of MBE, MBC or MBU. Results: Expression levels in cells and embryos were significantly different across genotypes, with endoglin reduced by half in Eng+/- and totally absent in Eng-/- samples. In vitro, microbubble adhesion was found to vary significantly (p&amp;lt;0.05) across genotype populations, with minimal attachment of MBC and MBU compared to MBE. Endoglin-targeted binding was approximately two-fold higher (median = 0.96 MBE/cell) in Eng+/+ compared to Eng+/- (median = 0.42 MBE/cell) cells. In embryo studies, we observed minimal signal from MBC and MBU, while MBE binding was found to be significantly higher in Eng+/+ embryos (CMPR+/+ = 9.71 + 0.66, 95% CI) compared to Eng+/- embryos (CMPR+/- = 5.51 + 0.64, 95% CI). In conclusion, these results suggest that molecular ultrasound is capable of reliably differentiating between molecular genotypes and relating receptor densities to quantifiable molecular ultrasound levels. Citation Format: Janet M. Denbeigh, Brian A. Nixon, John J.Y. Lee, Mirjana Jerkic, Philip A. Marsden, Michelle Letarte, Mira C. Puri, F. Stuart Foster. Quantifying vascular biomarkers with contrast-enhanced molecular ultrasound imaging. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2060. doi:10.1158/1538-7445.AM2014-2060

Doxorubicin liposome-loaded microbubbles for contrast imaging and ultrasound-triggered drug delivery

Targeted drug delivery under image guidance is gaining more interest in the drug-delivery field. The use of microbubbles as contrast agents in diagnostic ultrasound provides new opportunities in noninvasive image-guided drug delivery. In the present study, the imaging and therapeutic properties of novel doxorubicin liposome-loaded microbubbles are evaluated. The results showed that at scanning settings (1.7 MHz and mechanical index 0.2), these microbubbles scatter sufficient signal for nonlinear ultrasound imaging and can thus be imaged in real time and be tracked in vivo. In vitro therapeutic evaluation showed that ultrasound at 1 MHz and pressures up to 600 kPa in combination with the doxorubicin liposomeloaded microbubbles induced 4-fold decrease of cell viability compared with treatment with free doxorubicin or doxorubicin liposome-loaded microbubbles alone. The therapeutic effectiveness is correlated to an ultrasound-triggered release of doxorubicin from the liposomes and an enhanced uptake of the free doxorubicin by glioblastoma cells. The results obtained demonstrate that the combination of ultrasound and the doxorubicin liposome-loaded microbubbles can provide a new method of noninvasive image-guided drug delivery.