Development of a Small-Footprint 50 MHz Linear Array: Fabrication and Micro-Ultrasound Imaging Demonstration.

Compact high-frequency arrays are of interest for clinical and preclinical applications in which a small-footprint or endoscopic device is needed to reach the target anatomy. However, the fabrication of compact arrays entails the connection of several dozens of small elements to the imaging system through a combination of flexible printed circuit boards at the array end and micro-coaxial cabling to the imaging system. The methods currently used, such as wire bonding, conductive adhesives, or a dry connection to a flexible circuit, considerably increase the array footprint. Here, we propose an interconnection method that uses vacuum-deposited metals, laser patterning, and electroplating to achieve a right-angle, compact, reliable connection between array elements and flexible-circuit traces. The array elements are thickened at the edges using patterned copper traces, which increases their cross-sectional area and facilitates the connection. We fabricated a 2.3 mm by 1.7 mm, 64-element linear array with elements at a 36 µm pitch connected to a 4 cm long flexible circuit, where the interconnect adds only 100 µm to each side of the array. Pulse-echo measurements yielded an average center frequency of 55 MHz and a -6 dB bandwidth of 41%. We measured an imaging resolution of 35 µm in the axial direction and 114 µm in the lateral direction and demonstrated the ex vivo imaging of porcine esophageal tissue and the in vivo imaging of avian embryonic vasculature.

Real-time visualisation of peripheral nerve trauma during subepineural injection in pig brachial plexus using micro-ultrasound.

BACKGROUND: Nerve damage is consistently demonstrated after subepineural injection in animal studies, but not after purposeful injection in patients participating in clinical studies. There is a need to better visualise nerves in order to understand the structural changes that occur during subepineural injection. METHODS: We scanned the brachial plexuses of three anaesthetised pigs using micro-ultrasound imaging (55-22 MHz probe), inserted 21 gauge block needles into the radial, median, and axillary nerves, and injected two 0.5 ml boluses of saline into nerves at a rate of 12 ml min-1. Our objectives were to measure the area and diameter of nerves and fascicles, and to describe changes in nerve anatomy, comparing our findings with histology. RESULTS: Images were acquired at 42 sites across 18 nerves in three pigs and compared dimensions (geometric ratio; 95% confidence interval; P value). As expected, the nerve cross-sectional area was greater in the proximal brachial plexus compared with the mid-plexus (2.10; 1.07-4.11; P<0.001) and the distal plexus (2.64; 1.42-4.87; P<0.001). Nerve area expanded after 0.5 ml injection (2.13; 1.48-3.08; P<0.001). Using microultrasound, subepineural injection was characterised by nerve and fascicle rotation, uniform, or localised swelling and epineural rupture. Micro-ultrasound revealed a unique pattern suggestive of subperineural injection after a median nerve injection, and good face validity with histology. Histology showed epineural trauma and inflammation to the perineurium. CONCLUSION: We accurately identified fascicles and real-time structural changes to peripheral nerves using micro-ultrasound. This is the first study to visualise in vivo and in real-time the motion of nerves and fascicles in response to anaesthetic needle insertion and fluid injection.

Tetrazine-Derived Near-Infrared Dye as a Facile Reagent for Developing Targeted Photoacoustic Imaging Agents.

A new photoacoustic (PA) dye was developed as a simple-to-use reagent for creating targeted PA imaging agents. The lead molecule was prepared via an efficient two-step synthesis from an inexpensive commercially available starting material. With the dye's innate albumin-binding properties, the resulting tetrazine-derived dye is capable of localizing to tumor and exhibits a biological half-life of a few hours, allowing for an optimized distribution profile. The presence of tetrazine in turn makes it possible to link the albumin-binding optoacoustic signaling agent to a wide range of targeting molecules. To demonstrate the utility and ease of use of the platform, a novel PA probe for imaging calcium accretion was generated using a single-step bioorthogonal coupling reaction where high-resolution PA images of the knee joint in mice were obtained as early as 1 h post injection. Whole-body distribution was subsequently determined by labeling the probe with 99mTc and performing tissue counting following necropsy. These studies, along with tumor imaging and in vitro albumin binding studies, revealed that the core PA contrast agent can be imaged in vivo and can be easily linked to targeting molecules for organ-specific uptake.

Piezoelectric micromachined ultrasound transducer (PMUT) arrays for integrated sensing, actuation and imaging.

Many applications of ultrasound for sensing, actuation and imaging require miniaturized and low power transducers and transducer arrays integrated with electronic systems. Piezoelectric micromachined ultrasound transducers (PMUTs), diaphragm-like thin film flexural transducers typically formed on silicon substrates, are a potential solution for integrated transducer arrays. This paper presents an overview of the current development status of PMUTs and a discussion of their suitability for miniaturized and integrated devices. The thin film piezoelectric materials required to functionalize these devices are discussed, followed by the microfabrication techniques used to create PMUT elements and the constraints the fabrication imposes on device design. Approaches for electrical interconnection and integration with on-chip electronics are discussed. Electrical and acoustic measurements from fabricated PMUT arrays with up to 320 diaphragm elements are presented. The PMUTs are shown to be broadband devices with an operating frequency which is tunable by tailoring the lateral dimensions of the flexural membrane or the thicknesses of the constituent layers. Finally, the outlook for future development of PMUT technology and the potential applications made feasible by integrated PMUT devices are discussed.

Acoustic tractor beam.

Negative radiation forces act opposite to the direction of propagation, or net momentum, of a beam but have previously been challenging to definitively demonstrate. We report an experimental acoustic tractor beam generated by an ultrasonic array operating on macroscopic targets (>1 cm) to demonstrate the negative radiation forces and to map out regimes over which they dominate, which we compare to simulations. The result and the geometrically simple configuration show that the effect is due to nonconservative forces, produced by redirection of a momentum flux from the angled sides of a target and not by conservative forces from a potential energy gradient. Use of a simple acoustic setup provides an easily understood illustration of the negative radiation pressure concept for tractor beams and demonstrates continuous attraction towards the source, against a net momentum flux in the system.

Acoustic devices for particle and cell manipulation and sensing.

An emerging demand for the precise manipulation of cells and particles for applications in cell biology and analytical chemistry has driven rapid development of ultrasonic manipulation technology. Compared to the other manipulation technologies, such as magnetic tweezing, dielectrophoresis and optical tweezing, ultrasonic manipulation has shown potential in a variety of applications, with its advantages of versatile, inexpensive and easy integration into microfluidic systems, maintenance of cell viability, and generation of sufficient forces to handle particles, cells and their agglomerates. This article briefly reviews current practice and reports our development of various ultrasonic standing wave manipulation devices, including simple devices integrated with high frequency (>20 MHz) ultrasonic transducers for the investigation of biological cells and complex ultrasonic transducer array systems to explore the feasibility of electronically controlled 2-D and 3-D manipulation. Piezoelectric and passive materials, fabrication techniques, characterization methods and possible applications are discussed. The behavior and performance of the devices have been investigated and predicted with computer simulations, and verified experimentally. Issues met during development are highlighted and discussed. To assist long term practical adoption, approaches to low-cost, wafer level batch-production and commercialization potential are also addressed.

Micro-ultrasound based characterization of cerebrovasculature following focal ischemic stroke and upon short-term rehabilitation.

Notwithstanding recanalization treatments in the acute stage of stroke, many survivors suffer long-term impairments. Physical rehabilitation is the only widely available strategy for chronic-stage recovery, but its optimization is hindered by limited understanding of its effects on brain structure and function. Using micro-ultrasound, behavioral testing, and electrophysiology, we investigated the impact of skilled reaching rehabilitation on cerebral hemodynamics, motor function, and neuronal activity in a rat model of focal ischemic stroke. A 50 MHz micro-ultrasound transducer and intracortical electrophysiology were utilized to characterize neurovascular changes three weeks following focal ischemia elicited by endothelin-1 injection into the sensorimotor cortex. Sprague-Dawley rats were rehabilitated through tray reaching, and their fine skilled reaching was assessed via the Montoya staircase. Focal ischemia led to a sustained deficit in forelimb reaching; and increased tortuosity of the penetrating vessels in the perilesional cortex; with no lateralization of spontaneous neuronal activity. Rehabilitation improved skilled reaching; decreased cortical vascularity; was associated with elevated peri- vs. contralesional hypercapnia-induced flow homogenization and increased perilesional spontaneous cortical neuronal activity. Our study demonstrated neurovascular plasticity accompanying rehabilitation-elicited functional recovery in the subacute stage following stroke, and multiple micro-ultrasound-based markers of cerebrovascular structure and function modified in recovery from ischemia and upon rehabilitation.

Adaptation of a Clinical High-Frequency Transrectal Ultrasound System for Prostate Photoacoustic Imaging: Implementation and Pre-clinical Demonstration.

OBJECTIVE: High-frequency, high-resolution transrectal micro-ultrasound (micro-US: ≥15 MHz) imaging of the prostate is emerging as a beneficial tool for scoring disease risk and accurately targeting biopsies. Adding photoacoustic (PA) imaging to visualize abnormal vascularization and accumulation of contrast agents in tumors has potential for guiding focal therapies. In this work, we describe a new imaging platform that combines a transrectal micro-US system with transurethral light delivery for PA imaging. METHODS: A clinical transrectal micro-US system was adapted to acquire PA images synchronous to a tunable laser pulse. A transurethral side-firing optical fiber was developed for light delivery. A polyvinyl chloride (PVC)-plastisol phantom was developed and characterized to image PA contrast agents in wall-less channels. After resolution measurement in water, PA imaging was demonstrated in phantom channels with dyes and biodegradable nanoparticle contrast agents called porphysomes. In vivo imaging of a tumor model was performed, with porphysomes administered intravenously. RESULTS: Photoacoustic imaging data were acquired at 5 Hz, and image reconstruction was performed offline. PA image resolution at a 14-mm depth was 74 and 261 µm in the axial and lateral directions, respectively. The speed of sound in PVC-plastisol was 1383 m/s, and the attenuation was 4 dB/mm at 20 MHz. PA signal from porphysomes was spectrally unmixed from blood signals in the tumor, and a signal increase was observed 3 h after porphysome injection. CONCLUSION: A combined transrectal micro-US and PA imaging system was developed and characterized, and in vivo imaging demonstrated. High-resolution PA imaging may provide valuable additional information for diagnostic and therapeutic applications in the prostate.

The CathEye: A Forward-Looking Ultrasound Catheter for Image-Guided Cardiovascular Procedures.

Catheter based procedures are typically guided by X-Ray, which suffers from low soft tissue contrast and only provides 2D projection images of a 3D volume. Intravascular ultrasound (IVUS) can serve as a complementary imaging technique. Forward viewing catheters are useful for visualizing obstructions along the path of the catheter. The CathEye system mechanically steers a single-element transducer to generate a forward-looking surface reconstruction from an irregularly spaced 2-D scan pattern. The steerable catheter leverages an expandable frame with cables to manipulate the distal end independently of vessel tortuosity. The tip position is estimated by measuring the cable displacements and used to create surface reconstructions of the imaging workspace with the single-element transducer. CathEye's imaging capabilities were tested with an agar phantom and an ex vivo chronic total occlusion (CTO) sample while the catheter was confined to various tortuous paths. The CathEye maintained similar scan patterns regardless of path tortuosity and was able to recreate major features of the imaging targets, such as holes and extrusions. The feasibility of forward-looking IVUS with the CathEye is demonstrated in this study. The CathEye mechanism can be applied to other imaging modalities with field-of-view (FOV) limitations and represents the basis for an interventional device fully integrated with image guidance.

Spatially segmented SVD clutter filtering in cardiac blood flow imaging with diverging waves.

Ultrafast ultrasound imaging enables the visualization of rapidly changing blood flow dynamics in the chambers of the heart. Singular value decomposition (SVD) filters outperform conventional high pass clutter rejection filters for ultrafast blood flow imaging of small and shallow fields of view (e.g., functional imaging of brain activity). However, implementing SVD filters can be challenging in cardiac imaging due to the complex spatially and temporally varying tissue characteristics. To address this challenge, we describe a method that involves excluding the proximal portion of the image (near the chest wall) and divides the reduced field of view into overlapped segments, within which tissue signals are expected to be spatially and temporally coherent. SVD filtering with automatic selection of cut-off singular vector orders to remove tissue and noise signals is implemented for each segment. Auto-thresholding is based on the coherence of spatial singular vectors, delineating tissue, blood, and noise subspaces within a spatial similarity matrix calculated for each segment. Filtered blood flow signals from the segments are reconstructed and then combined and Doppler processing is used to form a set of blood flow images. Preliminary experimental results suggest that the spatially segmented approach improves the separation of the tissue and blood subsets in the spatial similarity matrix so that automatic thresholding is significantly improved, and tissue clutter can then be rejected more effectively in cardiac ultrafast imaging, compared to using the full field of view. In the case studied, spatially segmented SVD improved the rate of correct automatic selection of thresholds from 78% to 98.7% for the investigated cases and improved the post-filter power of blood signals by an average of more than 10 dB during a cardiac cycle.

Superharmonic and Microultrasound Imaging With Plane Wave Beamforming Techniques.

Superharmonic contrast imaging (SpHI) suppresses tissue clutter and allows high-contrast visualization of the vasculature. An array-based dual-frequency (DF) probe has been developed for SpHI, integrating a 21-MHz, 256-element microultrasound imaging array with a 2-MHz, 32-element array to take advantage of the broadband nonlinear responses from microbubble (MB) contrast agents. In this work, ultrafast imaging with plane waves was implemented for SpHI to increase the acquisition frame rate. Ultrafast imaging was also implemented for microultrasound B-mode imaging (HFPW B-mode) to enable high-resolution visualization of the tissue structure. Coherent compounding was demonstrated in vitro and in vivo in both imaging modes. Acquisition frame rates of 4.5 kHz and 187 Hz in HFPW B-mode imaging were achieved for imaging up to 21 mm with one and 25 angles, respectively, and 3.5 kHz and 396 Hz in the SpHI mode with one and nine coherently compounded angles, respectively. SpHI images showed suppression of tissue clutter prior to and after the introduction of MBs in vitro and in vivo. The nine-angle coherently compounded 2-D SpHI images of contrast-filled flow channel showed a contrast-to-tissue ratio (CTR) of 26.0 dB, a 2.5-dB improvement relative to images reconstructed from 0° steering. Consistent with in vitro imaging, the nine-angle compounded 2-D SpHI of a Lewis lung cancer tumor showed a 2.6-dB improvement in contrast enhancement, relative to 0° steering, and additionally revealed a region of nonviable tissue. The 3-D display of the volumetric SpHI data acquired from a xenograft mouse tumor using both 0° steering and nine-angle compounding allowed the visualization of the tumor vasculature. A small vessel visible in the compounded SpHI image, measuring around [Formula: see text], is not visualized in the 0° steering SpHI image, demonstrating the superiority of the latter in detecting fine structures within the tumor.

High-frequency quantitative ultrasound for the assessment of the acoustic properties of engineered tissues in vitro.

Acoustic properties of biomaterials and engineered tissues reflect their structure and cellularity. High-frequency ultrasound (US) can non-invasively characterize and monitor these properties with sub-millimetre resolution. We present an approach to estimate the speed of sound, acoustic impedance, and acoustic attenuation of cell-laden hydrogels that accounts for frequency-dependent effects of attenuation in coupling media, hydrogel thickness, and interfacial transmission/reflection coefficients of US waves, all of which can bias attenuation estimates. Cell-seeded fibrin hydrogel disks were raster-scanned using a 40 MHz US transducer. Thickness, speed of sound, acoustic impedance, and acoustic attenuation coefficients were determined from the difference in the time-of-flight and ratios of the magnitudes of US signals, interfacial transmission/reflection coefficients, and acoustic properties of the coupling media. With this approach, hydrogel thickness was accurately measured by US, with agreement to confocal microscopy (r2 = 0.97). Accurate thickness measurement enabled acoustic property measurements that were independent of hydrogel thickness, despite up to 60% reduction in thickness due to cell-mediated contraction. Notably, acoustic attenuation coefficients increased with increasing cell concentration (p < 0.001), reflecting hydrogel cellularity independent of contracted hydrogel thickness. This approach enables accurate measurement of the intrinsic acoustic properties of biomaterials and engineered tissues to provide new insights into their structure and cellularity. STATEMENT OF SIGNIFICANCE: High-frequency ultrasound can measure the acoustic properties of engineered tissues non-invasively and non-destructively with µm-scale resolution. Acoustic properties, including acoustic attenuation, are related to intrinsic material properties, such as scatterer density. We developed an analytical approach to estimate the acoustic properties of cell-laden hydrogels that accounts for the frequency-dependent effects of attenuation in coupling media, the reflection/transmission of ultrasound waves at the coupling interfaces, and the dependency of measurements on hydrogel thickness. Despite up to 60% reduction in hydrogel thickness due to cell-mediated contraction, our approach enabled measurements of acoustic properties that were substantially independent of thickness. Acoustic attenuation increased significantly with increasing cell concentration (p < 0.001), demonstrating the ability of acoustic attenuation to reflect intrinsic physical properties of engineered tissues.

Mechanical evidence of the orbital angular momentum to energy ratio of vortex beams.

We measure, in a single experiment, both the radiation pressure and the torque due to a wide variety of propagating acoustic vortex beams. The results validate, for the first time directly, the theoretically predicted ratio of the orbital angular momentum to linear momentum in a propagating beam. We experimentally determine this ratio using simultaneous measurements of both the levitation force and the torque on an acoustic absorber exerted by a broad range of helical ultrasonic beams produced by a 1000-element matrix transducer array. In general, beams with helical phase fronts have been shown to contain orbital angular momentum as the result of the azimuthal component of the Poynting vector around the propagation axis. Theory predicts that for both optical and acoustic helical beams the ratio of the angular momentum current of the beam to the power should be given by the ratio of the beam's topological charge to its angular frequency. This direct experimental observation that the ratio of the torque to power does convincingly match the expected value (given by the topological charge to angular frequency ratio of the beam) is a fundamental result.

Fine Pitch Flexible Printed Circuit Board Patterning for Miniaturized Endoscopic MicroUltrasound Arrays.

Microultrasound (micro-US) has become an invaluable tool for preclinical research and in emerging applications in clinical diagnosis and treatment guidance. Several such applications can benefit from arrays with a small footprint and endoscopic form factor. However, critical challenges arise in making electrical connections to array elements in such spatial constraints. In this work, we describe a method to pattern a high-density flexible circuit cabling on a copper-on polyimide film, using laser ablation of a polymer resist and wet etching, and then demonstrate a connection to a micro-US array. We investigate laser ablation process parameters and evaluate the ability to consistently pattern continuous copper traces. A minimum 30- [Formula: see text] pitch was achieved with 5- [Formula: see text]-wide electrode lines, and continuity of a 5-m-long trace is demonstrated. A flexible circuit with 30-mm-long traces with 30- [Formula: see text] line and 30- [Formula: see text] space before fan-out was fabricated to connect in an interleaved manner to a 32-element array with 30- [Formula: see text] element pitch. Metal deposition and laser ablation were used to connect and pattern the element electrodes to the copper traces of the flexible circuit. Electrical and acoustic measurements show good yield and consistent impedance across channels. Element pulse-echo tests demonstrated device functionality; the two-way pulse had 43-MHz center frequency and 40% fractional bandwidth (-6 dB). The proposed manufacturing methods facilitate the prototyping and fabrication of flexible endoscopic or small-footprint micro-US devices.

Acoustic Molecular Imaging Beyond the Diffraction Limit In Vivo.

Ultrasound molecular imaging (USMI) is a technique used to noninvasively estimate the distribution of molecular markers in vivo by imaging microbubble contrast agents (MCAs) that have been modified to target receptors of interest on the vascular endothelium. USMI is especially relevant for preclinical and clinical cancer research and has been used to predict tumor malignancy and response to treatment. In the last decade, methods that improve the resolution of contrast-enhanced ultrasound by an order of magnitude and allow researchers to noninvasively image individual capillaries have emerged. However, these approaches do not translate directly to molecular imaging. In this work, we demonstrate super-resolution visualization of biomarker expression in vivo using superharmonic ultrasound imaging (SpHI) with dual-frequency transducers, targeted contrast agents, and localization microscopy processing. We validate and optimize the proposed method in vitro using concurrent optical and ultrasound microscopy and a microvessel phantom. With the same technique, we perform a proof-of-concept experiment in vivo in a rat fibrosarcoma model and create maps of biomarker expression co-registered with images of microvasculature. From these images, we measure a resolution of 23 µm, a nearly fivefold improvement in resolution compared to previous diffraction-limited molecular imaging studies.

High frequency ultrasound nonlinear scattering from porphyrin nanobubbles.

Emerging contrast imaging studies have highlighted the potential of nanobubbles for both intravascular and extravascular applications. Reports to date on nanobubbles have generally utilized low frequencies (<12 MHz), high concentrations (>109 mL-1), and B-mode or contrast-mode on preclinical and clinical systems. However, none of these studies directly examined nanobubble acoustic signatures systematically to implement nonlinear imaging schemes in a methodical manner based on nanobubble behaviour. Here, nanobubble nonlinear behaviour is investigated at high frequencies (12.5, 25, 30 MHz) and low concentration (106 mL-1) in a channel phantom, with different pulse types in single- and multi-pulse sequences to examine behaviour under conditions relevant to high frequency imaging. Porphyrin nanobubbles are demonstrated to initiate nonlinear scattering at high frequencies in a pressure-threshold dependent manner, as previously observed at low frequencies. This threshold behaviour was then utilized to demonstrate enhanced nanobubble imaging with pulse inversion, amplitude modulation, and a combination of the two, progressing towards the improved sensitivity and expanded utility of these ultrasound contrast agents.

Validation of the soft-embalmed Thiel cadaver as a high-fidelity simulator of pressure during targeted nerve injection.

INTRODUCTION: Although administration of regional anesthesia nerve blocks has increased during the COVID-19 pandemic, training opportunities in regional anesthesia have reduced. Simulation training may enhance skills, but simulators must be accurate enough for trainees to engage in a realistic way-for example, detection of excessive injection pressure. The soft-embalmed Thiel cadaver is a life-like, durable simulator that is used for dedicated practice and mastery learning training in regional anesthesia. We hypothesized that injection opening pressure in perineural tissue, at epineurium and in subepineurium were similar to opening pressures measured in experimental animals, fresh frozen cadavers, glycol soft-fix cadavers and patients. METHODS: We systematically reviewed historical data, then conducted three validation studies delivering a 0.5 mL hydrolocation bolus of embalming fluid and recording injection pressure. First, we delivered the bolus at 12 mL/min at epimysium, perineural tissue, epineurium and in subepineurium at 48 peripheral nerve sites on three cadavers. Second, we delivered the bolus at using three infusion rates: 1 mL/min, 6 mL/min and 12 mL/min on epineurium at 70 peripheral nerve sites on five cadavers. Third, we repeated three injections (12 mL/min) at 24 epineural sites over the median and sciatic nerves of three cadavers. RESULTS: Mean (95%) injection pressure was greater at epineurium compared with subepineurium (geometric ratio 1.2 (95% CI: 0.9 to 1.6)), p=0.04, and perineural tissue (geometric ratio 5.1 (95% CI: 3.7 to 7.0)), p<0.0001. Mean (95%) injection pressure was greater at 12 mL/min compared with 1 mL/min (geometric ratio 1.6 (95% CI: 1.2 to 2.1), p=0.005). Pressure measurements were similar in study 3 (p>0.05 for all comparisons). DISCUSSION: We conclude that the soft-embalmed Thiel cadaver is a realistic simulator of injection opening pressure.

High-Frequency Array-Based Nanobubble Nonlinear Imaging in a Phantom and In Vivo.

There has been growing interest in nanobubbles (NBs) for vascular and extravascular ultrasound contrast imaging and therapeutic applications. Studies to date have generally utilized low frequencies (<12 MHz), high concentrations (>109 mL-1), and uncalibrated B-mode or contrast-mode on commercial systems without reporting investigations on NB signatures upon which the imaging protocols should be based. We recently demonstrated that low concentrations (106 mL-1) of porphyrin-lipid-encapsulated NBs scatter nonlinearly at low (2.5, 8 MHz) and high (12.5, 25, 30 MHz) frequencies in a pressure threshold-dependent manner that is advantageous for amplitude modulation (AM) imaging. Here, we implement pressure-calibrated AM at high frequency on a commercial preclinical array system to enhance sensitivity to nonlinear scattering of three phospholipid-based NB formulations. With this approach, improvements in contrast to tissue ratio relative to B-mode between 12.4 and 22.8 dB are demonstrated in a tissue-mimicking phantom, and between 6.7 and 14.8 dB in vivo.

Implementation of a Novel 288-Element Dual-Frequency Array for Acoustic Angiography: In Vitro and In Vivo Characterization.

Acoustic angiography is a superharmonic contrast-enhanced ultrasound imaging method that produces high-resolution, 3-D maps of the microvasculature. Previous acoustic angiography studies have used twoelement, annular,mechanicallyactuated transducers(called "wobblers") to image microvasculature in preclinical tumor models with high contrast-to-tissue ratio and resolution, but these earlywobbler transducerscould not achieve the depth and sensitivity required for clinical acoustic angiography. In this work, we present a system for performing acoustic angiography with a novel dual-frequency(DF) transducer-a coaxially stacked DF array (DFA). We evaluate the DFA system bothin vitro andin vivo and demonstrate improvements in sensitivity and imaging depth up to 13.1 dB and 10 mm, respectively, compared with previous wobbler probes.