3D Super-Resolution US Imaging of Rabbit Lymph Node Vasculature in Vivo by Using Microbubbles.

Background Variations in lymph node (LN) microcirculation can be indicative of metastasis. The identification and quantification of metastatic LNs remains essential for prognosis and treatment planning, but a reliable noninvasive imaging technique is lacking. Three-dimensional super-resolution (SR) US has shown potential to noninvasively visualize microvascular networks in vivo. Purpose To study the feasibility of three-dimensional SR US imaging of rabbit LN microvascular structure and blood flow by using microbubbles. Materials and Methods In vivo studies were carried out to image popliteal LNs of two healthy male New Zealand white rabbits aged 6-8 weeks. Three-dimensional, high-frame-rate, contrast material-enhanced US was achieved by mechanically scanning with a linear imaging probe. Individual microbubbles were identified, localized, and tracked to form three-dimensional SR images and super-resolved velocity maps. Acoustic subaperture processing was used to improve image contrast and to generate enhanced power Doppler and color Doppler images. Vessel size and blood flow velocity distributions were evaluated and assessed by using Student paired t test. Results SR images revealed microvessels in the rabbit LN, with branches clearly resolved when separated by 30 µm, which is less than half of the acoustic wavelength and not resolvable by using power or color Doppler. The apparent size distribution of most vessels in the SR images was below 80 µm and agrees with micro-CT data, whereas most of those detected with Doppler techniques were larger than 80 µm in the images. The blood flow velocity distribution indicated that most of the blood flow in rabbit popliteal LN was at velocities lower than 5 mm/sec. Conclusion Three-dimensional super-resolution US imaging using microbubbles allows noninvasive nonionizing visualization and quantification of lymph node microvascular structures and blood flow dynamics with resolution below the wave diffraction limit. This technology has potential for studying the physiologic functions of the lymph system and for clinical detection of lymph node metastasis. Published under a CC BY 4.0 license. Online supplemental material is available for this article.

Contrast Agent-Free Assessment of Blood Flow and Wall Shear Stress in the Rabbit Aorta using Ultrasound Image Velocimetry.

Blood flow velocity and wall shear stress (WSS) influence and are influenced by vascular disease. Their measurement is consequently useful in the laboratory and clinic. Contrast-enhanced ultrasound image velocimetry (UIV) can estimate them accurately but the need to inject contrast agents limits utility. Singular value decomposition and high-frame-rate imaging may render contrast agents dispensable. Here we determined whether contrast agent-free UIV can measure flow and WSS. In simulation, accurate measurements were achieved with a signal-to-noise ratio of 13.5 dB or higher. Signal intensity in the rabbit aorta increased monotonically with mechanical index; it was lowest during stagnant flow and uneven across the vessel. In vivo measurements with contrast-free and contrast-enhanced UIV differed by 4.4% and 1.9% for velocity magnitude and angle and by 9.47% for WSS. Bland-Altman analysis of waveforms revealed good agreement between contrast-free and contrast-enhanced UIV. In five rabbits, the root-mean-square errors were as low as 0.022 m/s (0.81%) and 0.11 Pa (1.7%). This study indicates that with an optimised protocol, UIV can assess flow and WSS without contrast agents. Unlike contrast-enhanced UIV, contrast-free UIV could be routinely employed.

Investigating CXCR4 expression of tumor cells and the vascular compartment: A multimodal approach.

The C-X-C chemokine receptor 4 (CXCR4) is G protein-coupled receptor that upon binding to its cognate ligand, can lead to tumor progression. Several CXCR4-targeted therapies are currently under investigation, and with it comes the need for imaging agents capable of accurate depiction of CXCR4 for therapeutic stratification and monitoring. PET agents enjoy the most success, but more cost-effective and radiation-free approaches such as ultrasound (US) imaging could represent an attractive alternative. In this work, we developed a targeted microbubble (MB) for imaging of vascular CXCR4 expression in cancer. A CXCR4-targeted MB was developed through incorporation of the T140 peptide into the MB shell. Binding properties of the T140-MB and control, non-targeted MB (NT-MB) were evaluated in MDA-MB-231 cells where CXCR4 expression was knocked-down (via shRNA) through optical imaging, and in the lymphoma tumor models U2932 and SuDHL8 (high and low CXCR4 expression, respectively) by US imaging. PET imaging of [18F]MCFB, a tumor-penetrating CXCR4-targeted small molecule, was used to provide whole-tumor CXCR4 readouts. CXCR4 expression and microvessel density were performed by immunohistochemistry analysis and western blot. T140-MB were formed with similar properties to NT-MB and accumulated sensitively and specifically in cells according to their CXCR4 expression. In NOD SCID mice, T140-MB persisted longer in tumors than NT-MB, indicative of target interaction, but showed no difference between U2932 and SuDHL8. In contrast, PET imaging with [18F]MCFB showed a marked difference in tumor uptake at 40-60 min post-injection between the two tumor models (p<0.05). Ex vivo analysis revealed that the large differences in CXCR4 expression between the two models are not reflected in the vascular compartment, where the MB are restricted; in fact, microvessel density and CXCR4 expression in the vasculature was comparable between U2932 and SuDHL8 tumors. In conclusion, we successfully developed a T140-MB that can be used for imaging CXCR4 expression in the tumor vasculature.

3-D Super-Resolution Ultrasound Imaging With a 2-D Sparse Array.

High-frame-rate 3-D ultrasound imaging technology combined with super-resolution processing method can visualize 3-D microvascular structures by overcoming the diffraction-limited resolution in every spatial direction. However, 3-D super-resolution ultrasound imaging using a full 2-D array requires a system with a large number of independent channels, the design of which might be impractical due to the high cost, complexity, and volume of data produced. In this study, a 2-D sparse array was designed and fabricated with 512 elements chosen from a density-tapered 2-D spiral layout. High-frame-rate volumetric imaging was performed using two synchronized ULA-OP 256 research scanners. Volumetric images were constructed by coherently compounding nine-angle plane waves acquired at a pulse repetition frequency of 4500 Hz. Localization-based 3-D super-resolution images of two touching subwavelength tubes were generated from 6000 volumes acquired in 12 s. Finally, this work demonstrates the feasibility of 3-D super-resolution imaging and super-resolved velocity mapping using a customized 2-D sparse array transducer.

Contrast-Enhanced High-Frame-Rate Ultrasound Imaging of Flow Patterns in Cardiac Chambers and Deep Vessels.

Cardiac function and vascular function are closely related to the flow of blood within. The flow velocities in these larger cavities easily reach 1 m/s, and generally complex spatiotemporal flow patterns are involved, especially in a non-physiologic state. Visualization of such flow patterns using ultrasound can be greatly enhanced by administration of contrast agents. Tracking the high-velocity complex flows is challenging with current clinical echographic tools, mostly because of limitations in signal-to-noise ratio; estimation of lateral velocities; and/or frame rate of the contrast-enhanced imaging mode. This review addresses the state of the art in 2-D high-frame-rate contrast-enhanced echography of ventricular and deep-vessel flow, from both technological and clinical perspectives. It concludes that current advanced ultrasound equipment is technologically ready for use in human contrast-enhanced studies, thus potentially leading to identification of the most clinically relevant flow parameters for quantifying cardiac and vascular function.

Measurement of Flow Volume in the Presence of Reverse Flow with Ultrasound Speckle Decorrelation.

Direct measurement of volumetric flow rate in the cardiovascular system with ultrasound is valuable but has been a challenge because most current 2-D flow imaging techniques are only able to estimate the flow velocity in the scanning plane (in-plane). Our recent study demonstrated that high frame rate contrast ultrasound and speckle decorrelation (SDC) can be used to accurately measure the speed of flow going through the scanning plane (through-plane). The volumetric flow could then be calculated by integrating over the luminal area, when the blood vessel was scanned from the transverse view. However, a key disadvantage of this SDC method is that it cannot distinguish the direction of the through-plane flow, which limited its applications to blood vessels with unidirectional flow. Physiologic flow in the cardiovascular system could be bidirectional due to its pulsatility, geometric features, or under pathologic situations. In this study, we proposed a method to distinguish the through-plane flow direction by inspecting the flow within the scanning plane from a tilted transverse view. This method was tested on computer simulations and experimental flow phantoms. It was found that the proposed method could detect flow direction and improved the estimation of the flow volume, reducing the overestimation from over 100% to less than 15% when there was flow reversal. This method showed significant improvement over the current SDC method in volume flow estimation and can be applied to a wider range of clinical applications where bidirectional flow exists.

Optimization of 3-D Divergence-Free Flow Field Reconstruction Using 2-D Ultrasound Vector Flow Imaging.

3-D blood vector flow imaging is of great value in understanding and detecting cardiovascular diseases. Currently, 3-D ultrasound vector flow imaging requires 2-D matrix probes, which are expensive and suffer from suboptimal image quality. Our recent study proposed an interpolation algorithm to obtain a divergence-free reconstruction of the 3-D flow field from 2-D velocities obtained by high-frame-rate ultrasound particle imaging velocimetry (High Frame Rate echo-Particle Imaging Velocimetry, also known as HFR Ultrasound Imaging Velocimetry (UIV)), using a 1-D array transducer. The aim of this work was to significantly improve the accuracy and reduce the time-to-solution of our previous approach, thereby paving the way for clinical translation. More specifically, accuracy was improved by optimising the divergence-free basis to reduce Runge phenomena near domain boundaries, and time-to-solution was reduced by demonstrating that under certain conditions, the resulting system could be solved using widely available and highly optimised generalised minimum residual algorithms. To initially illustrate the utility of the approach, coarse 2-D subsamplings of an analytical unsteady Womersely flow solution and a steady helical flow solution obtained using computational fluid dynamics were used successfully to reconstruct full flow solutions, with 0.82% and 4.8% average relative errors in the velocity field, respectively. Subsequently, multiplane 2-D velocity fields were obtained through HFR UIV for a straight-tube phantom and a carotid bifurcation phantom, from which full 3-D flow fields were reconstructed. These were then compared with flow fields obtained via computational fluid dynamics in each of the two configurations, and average relative errors of 6.01% and 12.8% in the velocity field were obtained. These results reflect 15%-75% improvements in accuracy and 53- to 874-fold acceleration of reconstruction speeds for the four cases, compared with the previous divergence-free flow reconstruction method. In conclusion, the proposed method provides an effective and fast method to reconstruct 3-D flow in arteries using a 1-D array transducer.

3-D Flow Reconstruction Using Divergence-Free Interpolation of Multiple 2-D Contrast-Enhanced Ultrasound Particle Imaging Velocimetry Measurements.

Quantification of 3-D intravascular flow is valuable for studying arterial wall diseases but currently there is a lack of effective clinical tools for this purpose. Divergence-free interpolation (DFI) using radial basis function (RBF) is an emerging approach for full-field flow reconstruction using experimental sparse flow field samples. Previous DFI reconstructs full-field flow from scattered 3-D velocity input obtained using phase-contrast magnetic resonance imaging with low temporal resolution. In this study, a new DFI algorithm is proposed to reconstruct full-field flow from scattered 2-D in-plane velocity vectors obtained using ultrafast contrast-enhanced ultrasound (>1000 fps) and particle imaging velocimetry. The full 3-D flow field is represented by a sum of weighted divergence-free RBFs in space. Because the acquired velocity vectors are only in 2-D and hence the problem is ill-conditioned, a regularized solution of the RBF weighting is achieved through singular value decomposition (SVD) and the L-curve method. The effectiveness of the algorithm is determined via numerical experiments for Poiseuille flow and helical flow with added noise, and it is found that an accuracy as high as 95.6% can be achieved for Poiseuille flow (with 5% input noise). Experimental feasibility is also determined by reconstructing full-field 3-D flow from experimental 2-D ultrasound image velocimetry measurements in a carotid bifurcation phantom. The method is typically faster for a range of problems compared with computational fluid dynamics, and has been found to be effective for the three flow cases.

High Frame Rate Contrast-Enhanced Ultrasound Imaging for Slow Lymphatic Flow: Influence of Ultrasound Pressure and Flow Rate on Bubble Disruption and Image Persistence.

Contrast-enhanced ultrasound (CEUS) utilising microbubbles shows great potential for visualising lymphatic vessels and identifying sentinel lymph nodes (SLNs) which are valuable for axillary staging in breast cancer patients. However, current CEUS imaging techniques have limitations that affect the accurate visualisation and tracking of lymphatic vessels and SLN. (i) Tissue artefacts and bubble disruption can reduce the image contrast. (ii) Limited spatial and temporal resolution diminishes the amount of information that can be captured by CEUS. (iii) The slow lymph flow makes Doppler-based approaches less effective. This work evaluates on a lymphatic vessel phantom the use of high frame rate (HFR) CEUS for the detection of lymphatic vessels where flow is slow. Specifically, the work particularly investigates the impact of key factors in lymphatic imaging, including ultrasound pressure and flow velocity as well as probe motion during vessel tracking, on bubble disruption and image contrast. Experiments were also conducted to apply HFR CEUS imaging on vasculature in a rabbit popliteal lymph node (LN). Our results show that (i) HFR imaging and singular value decomposition (SVD) filtering can significantly reduce tissue artefacts in the phantom at high clinical frequencies; (ii) the slow flow rate within the phantom makes image contrast and signal persistence more susceptible to changes in ultrasound amplitude or mechanical index (MI), and an MI value can be chosen to reach a compromise between images contrast and bubble disruption under slow flow condition; (iii) probe motion significantly decreases image contrast of the vessel, which can be improved by applying motion correction before SVD filtering; (iv) the optical observation of the impact of ultrasound pressure on HFR CEUS further confirms the importance of optimising ultrasound amplitude and (v) vessels inside rabbit LN with blood flow less than 3 mm/s are clearly visualised.

3-D Microvascular Imaging Using High Frame Rate Ultrasound and ASAP Without Contrast Agents: Development and Initial In Vivo Evaluation on Nontumor and Tumor Models.

Three-dimensional imaging is valuable to noninvasively assess angiogenesis given the complex 3-D architecture of vascular networks. The emergence of high frame rate (HFR) ultrasound, which can produce thousands of images per second, has inspired novel signal processing techniques and their applications in structural and functional imaging of blood vessels. Although highly sensitive vascular mapping has been demonstrated using ultrafast Doppler, the detectability of microvasculature from the background noise may be hindered by the low signal-to-noise ratio (SNR) particularly in the deeper region and without the use of contrast agents. We have recently demonstrated a coherence-based technique, acoustic subaperture imaging (ASAP), for super-contrast vascular imaging and illustrated the contrast improvement using HFR contrast-enhanced ultrasound. In this work, we provide a feasibility study for microvascular imaging using ASAP without contrast agents, and extend its capability from 2-D to volumetric vascular mapping. Using an ultrasound research system and a preclinical probe, we demonstrated the improved visibility of microvascular mapping using ASAP in comparison to ultrafast power Doppler (PD) on a mouse kidney, liver, and tumor without contrast agent injection. The SNR of ASAP images improves in average by 10 dB when compared to PD. In addition, directional velocity mappings were also demonstrated by combining ASAP with the phase information extracted from lag-1 autocorrelation. The 3-D vascular and velocity mapping of the mouse kidney, liver, and tumor were demonstrated by stacking the ASAP images acquired using 2-D ultrasound imaging and a trigger-controlled linear translation stage. The 3-D results depicted clear microvasculature morphologies and functional information in terms of flow direction and velocity in two nontumor models and a tumor model. In conclusion, we have demonstrated a new 3-D in vivo ultrasound microvascular imaging technique with significantly improved SNR over existing ultrafast Doppler.

Fast Acoustic Wave Sparsely Activated Localization Microscopy (fast-AWSALM): Ultrasound Super-Resolution using Plane-Wave Activation of Nanodroplets.

Localization-based ultrasound super-resolution imaging using microbubble contrast agents and phase-change nano-droplets has been developed to visualize microvascular structures beyond the diffraction limit. However, the long data acquisition time makes the clinical translation more challenging. In this study, fast acoustic wave sparsely activated localization microscopy (fast-AWSALM) was developed to achieve super-resolved frames with sub-second temporal resolution, by using low-boiling-point octafluoropropane nanodroplets and high frame rate plane waves for activation, destruction, as well as imaging. Fast-AWSALM was demonstrated on an in vitro microvascular phantom to super-resolve structures that could not be resolved by conventional B-mode imaging. The effects of the temperature and mechanical index on fast-AWSALM was investigated. Experimental results show that sub-wavelength micro-structures as small as 190 lm were resolvable in 200 ms with plane-wave transmission at a center frequency of 3.5 MHz and a pulse repetition frequency of 5000 Hz. This is about a 3.5 fold reduction in point spread function full-width-half-maximum compared to that measured in conventional B-mode, and two orders of magnitude faster than the recently reported AWSALM under a non-flow/very slow flow situations and other localization based methods. Just as in AWSALM, fast-AWSALM does not require flow, as is required by current microbubble based ultrasound super resolution techniques. In conclusion, this study shows the promise of fast-AWSALM, a super-resolution ultrasound technique using nanodroplets, which can generate super-resolution images in milli-seconds and does not require flow.

Development of 68Ga-labelled ultrasound microbubbles for whole-body PET imaging.

Microbubble (MB) contrast agents have revolutionalised the way ultrasound (US) imaging can be used clinically and pre-clinically. Contrast-enhanced US offers improvements in soft-tissue contrast, as well as the ability to visualise disease processes at the molecular level. However, its inability to provide in vivo whole-body imaging can hamper the development of new MB formulations. Herein, we describe a fast and efficient method for achieving 68Ga-labelling of MBs after a direct comparison of two different strategies. The optimised approach produces 68Ga-labelled MBs in good yields through the bioorthogonal inverse-electron-demand Diel-Alder reaction between a trans-cyclooctene-modified phospholipid and a new tetrazine-bearing HBED-CC chelator. The ability to noninvasively study the whole-body distribution of 68Ga-labelled MBs was demonstrated in vivo using positron emission tomography (PET). This method could be broadly applicable to other phospholipid-based formulations, providing accessible solutions for in vivo tracking of MBs.

Spatio-Temporal Flow and Wall Shear Stress Mapping Based on Incoherent Ensemble-Correlation of Ultrafast Contrast Enhanced Ultrasound Images.

In this study, a technique for high-frame-rate ultrasound imaging velocimetry (UIV) is extended first to provide more robust quantitative flow velocity mapping using ensemble correlation of images without coherent compounding, and second to generate spatio-temporal wall shear stress (WSS) distribution. A simulation model, which couples the ultrasound simulator with analytical flow solution, was implemented to evaluate its accuracy. It is shown that the proposed approach can reduce errors in velocity estimation by up to 10-fold in comparison with the coherent correlation approach. Mean errors (ME) of 3.2% and 8.6% were estimated under a steady flow condition, while 3.0% and 10.6% were found under a pulsatile condition for the velocity and wall shear rate (WSR) measurement, respectively. Appropriate filter parameters were selected to constrain the velocity profiles before WSR estimations and the effects of incorrect wall tracking were quantified under a controlled environment. Although accurate wall tracking is found to be critical in WSR measurement (as a 200 µm deviation from the wall may yield up to a 60% error), this can be mitigated by HFR imaging (of up to 10 kHz) with contrast agents, which allow for improved differentiation of the wall-fluid boundaries. In vitro investigations on two carotid bifurcation phantoms, normal and diseased, were conducted, and their relative differences in terms of the flow patterns and WSR distribution were demonstrated. It is shown that high-frame-rate UIV technique can be a non-invasive tool to measure quantitatively the spatio-temporal velocity and WSS distribution.

ASAP: Super-Contrast Vasculature Imaging Using Coherence Analysis and High Frame-Rate Contrast Enhanced Ultrasound.

The very high frame rate afforded by ultrafast ultrasound, combined with microbubble contrast agents, opens new opportunities for imaging tissue microvasculature. However, new imaging paradigms are required to obtain superior image quality from the large amount of acquired data while allowing real-time implementation. In this paper, we report a technique-acoustic sub-aperture processing (ASAP)-capable of generating very high contrast/signal-to-noise ratio (SNR) images of macro-and microvessels, with similar computational complexity to classical power Doppler (PD) imaging. In ASAP, the received data are split into subgroups. The reconstructed data from each subgroup are temporally correlated over frames to generate the final image. As signals in subgroups are correlated but the noise is not, this substantially reduces the noise floor compared to PD. Using a clinical imaging probe, the method is shown to visualize vessels down to $200~\mu \text{m}$ with a SNR of 10 dB higher than PD and to resolve microvascular flow/perfusion information in rabbit kidneys noninvasively in vivo at multiple centimeter depths. With careful filter design, the technique also allows the estimation of flow direction and the separation of fast flow from tissue perfusion. ASAP can readily be implemented into hardware/firmware for real-time imaging and can be applied to contrast enhanced and potentially noncontrast imaging and 3-D imaging.

Imaging of vaporised sub-micron phase change contrast agents with high frame rate ultrasound and optics.

Phase-change ultrasound contrast agent (PCCA), or nanodroplet, shows promise as an alternative to the conventional microbubble agent over a wide range of diagnostic applications. Meanwhile, high-frame-rate (HFR) ultrasound imaging with microbubbles enables unprecedented temporal resolution compared to traditional contrast-enhanced ultrasound imaging. The combination of HFR ultrasound imaging and PCCAs can offer the opportunity to observe and better understand PCCA behaviour after vaporisation captures the fast phenomenon at a high temporal resolution. In this study, we utilised HFR ultrasound at frame rates in the kilohertz range (5-20 kHz) to image native and size-selected PCCA populations immediately after vaporisation in vitro within clinical acoustic parameters. The size-selected PCCAs through filtration are shown to preserve a sub-micron-sized (mean diameter < 200 nm) population without micron-sized outliers (>1 µm) that originate from native PCCA emulsion. The results demonstrate imaging signals with different amplitudes and temporal features compared to that of microbubbles. Compared with the microbubbles, both the B-mode and pulse-inversion (PI) signals from the vaporised PCCA populations were reduced significantly in the first tens of milliseconds, while only the B-mode signals from the PCCAs were recovered during the next 400 ms, suggesting significant changes to the size distribution of the PCCAs after vaporisation. It is also shown that such recovery in signal over time is not evident when using size-selective PCCAs. Furthermore, it was found that signals from the vaporised PCCA populations are affected by the amplitude and frame rate of the HFR ultrasound imaging. Using high-speed optical camera observation (30 kHz), we observed a change in particle size in the vaporised PCCA populations exposed to the HFR ultrasound imaging pulses. These findings can further the understanding of PCCA behaviour under HFR ultrasound imaging.

3-D Velocity and Volume Flow Measurement In Vivo Using Speckle Decorrelation and 2-D High-Frame-Rate Contrast-Enhanced Ultrasound.

Being able to measure 3-D flow velocity and volumetric flow rate effectively in the cardiovascular system is valuable but remains a significant challenge in both clinical practice and research. Currently, there has not been an effective and practical solution to the measurement of volume flow using ultrasound imaging systems due to challenges in existing 3-D imaging techniques and high system cost. In this study, a new technique for quantifying volumetric flow rate from the cross-sectional imaging plane of the blood vessel was developed by using speckle decorrelation (SDC), 2-D high-frame-rate imaging with a standard 1-D array transducer, microbubble contrast agents, and ultrasound imaging velocimetry (UIV). Through SDC analysis of microbubble signals acquired with a very high frame rate and by using UIV to estimate the two in-plane flow velocity components, the third and out-of-plane velocity component can be obtained over time and integrated to estimate volume flow. The proposed technique was evaluated on a wall-less flow phantom in both steady and pulsatile flow. UIV in the longitudinal direction was conducted as a reference. The influences of frame rate, mechanical index (MI), orientation of imaging plane, and compounding on velocity estimation were also studied. In addition, an in vivo trial on the abdominal aorta of a rabbit was conducted. The results show that the new system can estimate volume flow with an averaged error of 3.65% ± 2.37% at a flow rate of 360 mL/min and a peak velocity of 0.45 m/s, and an error of 5.03% ± 2.73% at a flow rate of 723 mL/min and a peak velocity of 0.8 m/s. The accuracy of the flow velocity and volumetric flow rate estimation directly depend on the imaging frame rate. With a frame rate of 6000 Hz, a velocity up to 0.8 m/s can be correctly estimated. A higher mechanical index (MI = 0.42) is shown to produce greater errors (up to 21.78±0.49%, compared to 3.65±2.37% at MI = 0.19). An in vivo trial, where velocities up to 1 m/s were correctly measured, demonstrated the potential of the technique in clinical applications.

Effects of microchannel confinement on acoustic vaporisation of ultrasound phase change contrast agents.

The sub-micron phase change contrast agent (PCCA) composed of a perfluorocarbon liquid core can be activated into gaseous state and form stable echogenic microbubbles for contrast-enhanced ultrasound imaging. It has shown great promise in imaging microvasculature, tumour microenvironment, and cancer cells. Although PCCAs have been extensively studied for different diagnostic and therapeutic applications, the effect of biologically geometrical confinement on the acoustic vaporisation of PCCAs is still not clear. We have investigated the difference in PCCA-produced ultrasound contrast enhancement after acoustic activation with and without a microvessel confinement on a microchannel phantom. The experimental results indicated more than one-order of magnitude less acoustic vaporisation in a microchannel than that in a free environment taking into account the attenuation effect of the vessel on the microbubble scattering. This may provide an improved understanding in the applications of PCCAs in vivo.

Microbubble Void Imaging: A Non-invasive Technique for Flow Visualisation and Quantification of Mixing in Large Vessels Using Plane Wave Ultrasound and Controlled Microbubble Contrast Agent Destruction.

There is increasing recognition of the influence of the flow field on the physiology of blood vessels and their development of pathology. Preliminary work is reported on a novel non-invasive technique, microbubble void imaging, which is based on ultrasound and controlled destruction of microbubble contrast agents, permitting flow visualisation and quantification of flow-induced mixing in large vessels. The generation of microbubble voids can be controlled both spatially and temporally using ultrasound parameters within the safety limits. Three different model vessel geometries-straight, planar-curved and helical-with known effects on the flow field and mixing were chosen to evaluate the technique. A high-frame-rate ultrasound system with plane wave transmission was used to acquire the contrast-enhanced ultrasound images, and an entropy measure was calculated to quantify mixing. The experimental results were cross-compared between the different geometries and with computational fluid dynamics. The results indicated that the technique is able to quantify the degree of mixing within the different configurations, with a helical geometry generating the greatest mixing, and a straight geometry, the lowest. There is a high level of concordance between the computational fluid dynamics and experimental results. The technique could also serve as a flow visualisation tool.

Flow Velocity Mapping Using Contrast Enhanced High-Frame-Rate Plane Wave Ultrasound and Image Tracking: Methods and Initial in Vitro and in Vivo Evaluation.

Ultrasound imaging is the most widely used method for visualising and quantifying blood flow in medical practice, but existing techniques have various limitations in terms of imaging sensitivity, field of view, flow angle dependence, and imaging depth. In this study, we developed an ultrasound imaging velocimetry approach capable of visualising and quantifying dynamic flow, by combining high-frame-rate plane wave ultrasound imaging, microbubble contrast agents, pulse inversion contrast imaging and speckle image tracking algorithms. The system was initially evaluated in vitro on both straight and carotid-mimicking vessels with steady and pulsatile flows and in vivo in the rabbit aorta. Colour and spectral Doppler measurements were also made. Initial flow mapping results were compared with theoretical prediction and reference Doppler measurements and indicate the potential of the new system as a highly sensitive, accurate, angle-independent and full field-of-view velocity mapping tool capable of tracking and quantifying fast and dynamic flows.

Surface Charge Measurement of SonoVue, Definity and Optison: A Comparison of Laser Doppler Electrophoresis and Micro-Electrophoresis.

Microbubble (MB) contrast-enhanced ultrasonography is a promising tool for targeted molecular imaging. It is important to determine the MB surface charge accurately as it affects the MB interactions with cell membranes. In this article, we report the surface charge measurement of SonoVue, Definity and Optison. We compare the performance of the widely used laser Doppler electrophoresis with an in-house micro-electrophoresis system. By optically tracking MB electrophoretic velocity in a microchannel, we determined the zeta potentials of MB samples. Using micro-electrophoresis, we obtained zeta potential values for SonoVue, Definity and Optison of -28.3, -4.2 and -9.5 mV, with relative standard deviations of 5%, 48% and 8%, respectively. In comparison, laser Doppler electrophoresis gave -8.7, +0.7 and +15.8 mV with relative standard deviations of 330%, 29,000% and 130%, respectively. We found that the reliability of laser Doppler electrophoresis is compromised by MB buoyancy. Micro-electrophoresis determined zeta potential values with a 10-fold improvement in relative standard deviation.