Acoustic Angiography: Superharmonic Contrast-Enhanced Ultrasound Imaging for Noninvasive Visualization of Microvasculature.

Acoustic angiography is a contrast-enhanced ultrasound technique that relies on superharmonic imaging to form high-resolution, three-dimensional maps of the microvasculature. In order to obtain signal separation between tissue and contrast, acoustic angiography has been performed with dual-frequency transducers with nonoverlapping bandwidths. This enables a high contrast-to-tissue ratio, and the choice of a high frequency receiving element provides high resolution. In this chapter, we describe the technology behind acoustic angiography as well as the step-by-step implementation of this contrast enhanced microvascular imaging technique.

A Handheld Imaging Probe for Acoustic Angiography With an Ultrawideband Capacitive Micromachined Ultrasonic Transducer (CMUT) Array.

This article presents an imaging probe with a 256-element ultrawideband (UWB) 1-D capacitive micromachined ultrasonic transducer (CMUT) array designed for acoustic angiography (AA). This array was fabricated on a borosilicate glass wafer with a reduced bottom electrode and an additional central plate mass to achieve the broad bandwidth. A custom 256-channel handheld probe was designed and implemented with integrated low-noise amplifiers and supporting power circuitry. This probe was used to characterize the UWB CMUT, which has a functional 3-dB frequency band from 3.5 to 23.5 MHz. A mechanical index (MI) of 0.33 was achieved at 3.5 MHz at a depth of 11 mm. These promising measurements are then combined to demonstrate AA. The use of alternate amplitude modulation (aAM) combined with a frequency analysis of the measured transmit signal demonstrates the suitability of the UWB CMUT for AA. This is achieved by measuring only a low level of unwanted high-frequency harmonics in both the transmit signal and the reconstructed image in the areas other than the contrast bubbles.

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.

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.

Characterization of an Array-Based Dual-Frequency Transducer for Superharmonic Contrast Imaging.

Superharmonic imaging with dual-frequency imaging systems uses conventional low-frequency ultrasound transducers on transmit, and high-frequency transducers on receive to detect higher order harmonic signals from microbubble contrast agents, enabling high-contrast imaging while suppressing clutter from background tissues. Current dual-frequency imaging systems for superharmonic imaging have been used for visualizing tumor microvasculature, with single-element transducers for each of the low- and high-frequency components. However, the useful field of view is limited by the fixed focus of single-element transducers, while image frame rates are limited by the mechanical translation of the transducers. In this article, we introduce an array-based dual-frequency transducer, with low-frequency and high-frequency arrays integrated within the probe head, to overcome the limitations of single-channel dual-frequency probes. The purpose of this study is to evaluate the line-by-line high-frequency imaging and superharmonic imaging capabilities of the array-based dual-frequency probe for acoustic angiography applications in vitro and in vivo. We report center frequencies of 1.86 MHz and 20.3 MHz with -6 dB bandwidths of 1.2 MHz (1.2-2.4 MHz) and 14.5 MHz (13.3-27.8 MHz) for the low- and high-frequency arrays, respectively. With the proposed beamforming schemes, excitation pressure was found to range from 336 to 458 kPa at its azimuthal foci. This was sufficient to induce nonlinear scattering from microbubble contrast agents. Specifically, in vitro contrast channel phantom imaging and in vivo xenograft mouse tumor imaging by this probe with superharmonic imaging showed contrast-to-tissue ratio improvements of 17.7 and 16.2 dB, respectively, compared to line-by-line micro-ultrasound B-mode imaging.

Visualization of Microvascular Angiogenesis Using Dual-Frequency Contrast-Enhanced Acoustic Angiography: A Review.

Cancerous tumor growth is associated with the development of tortuous, chaotic microvasculature, and this aberrant microvascular morphology can act as a biomarker of malignant disease. Acoustic angiography is a contrast-enhanced ultrasound technique that relies on superharmonic imaging to form high-resolution 3-D maps of the microvasculature. To date, acoustic angiography has been performed with dual-element transducers that can achieve high contrast-to-tissue ratio and resolution in pre-clinical small animal models. In this review, we first describe the development of acoustic angiography, including the principle, transducer design, and optimization of superharmonic imaging techniques. We then detail several preclinical applications of this microvascular imaging method, as well as the current and future development of acoustic angiography as a pre-clinical and clinical diagnostic tool.

An Improved CMUT Structure Enabling Release and Collapse of the Plate in the Same Tx/Rx Cycle for Dual-Frequency Acoustic Angiography.

This study demonstrates, in detail, the potential of using capacitive micromachined ultrasonic transducers (CMUTs) for acoustic angiography of the microvasculature. It is known that when ultrasound contrast agents (microbubbles) are excited with moderate acoustic pressure around their resonance (2-4 MHz), they produce higher order harmonics (greater than third harmonic) due to their nonlinear behavior. To date, the fundamental challenge has been the availability of a transducer that can generate the transmit signals to excite the microbubbles at low frequencies and, in the same cycle, confocally detect harmonics in the higher frequencies. We present a novel device structure and dual-mode operation of a CMUT that operates with a center frequency of 4.3 MHz and 150% bandwidth in the conventional mode for transmitting and a center frequency of 9.8 MHz and a 125.5% bandwidth in collapse mode for receiving. Output pressure of 1.7 MPapp is achieved on the surface of a single unfocused transducer. The mechanical index at the transducer surface is 0.56. FEM simulations are performed first to show the functionality of the proposed device, and then, the device fabrication is described in detail. Finally, we experimentally demonstrate the ability to detect the microbubble signals with good contrast, and the background reflection is adequately suppressed, indicating the feasibility of the presented approach for acoustic angiography.

Superharmonic Ultrasound for Motion-Independent Localization Microscopy: Applications to Microvascular Imaging From Low to High Flow Rates.

Recent advances in high frame rate biomedical ultrasound have led to the development of ultrasound localization microscopy (ULM), a method of imaging microbubble (MB) contrast agents beyond the diffraction limit of conventional coherent imaging techniques. By localizing and tracking the positions of thousands of individual MBs, ultrahigh resolution vascular maps are generated which can be further analyzed to study disease. Isolating bubble echoes from tissue signal is a key requirement for super-resolution imaging which relies on the spatiotemporal separability and localization of the bubble signals. To date, this has been accomplished either during acquisition using contrast imaging sequences or post-beamforming by applying a spatiotemporal filter to the B-mode images. Superharmonic imaging (SHI) is another contrast imaging method that separates bubbles from tissue based on their strongly nonlinear acoustic properties. This approach is highly sensitive, and, unlike spatiotemporal filters, it does not require decorrelation of contrast agent signals. Since this superharmonic method does not rely on bubble velocity, it can detect completely stationary and moving bubbles alike. In this work, we apply SHI to ULM and demonstrate an average improvement in SNR of 10.3-dB in vitro when compared with the standard singular value decomposition filter approach and an increase in SNR at low flow ( [Formula: see text]/frame) from 5 to 16.5 dB. Additionally, we apply this method to imaging a rodent kidney in vivo and measure vessels as small as [Formula: see text] in diameter after motion correction.

Assessment of the Superharmonic Response of Microbubble Contrast Agents for Acoustic Angiography as a Function of Microbubble Parameters.

Acoustic angiography is a superharmonic contrast-enhanced ultrasound imaging technique that enables 3-D high-resolution microvascular visualization. This technique utilizes a dual-frequency imaging strategy, transmitting at a low frequency and receiving at a higher frequency, to detect high-frequency contrast agent signatures and separate them from tissue background. Prior studies have illustrated differences in microbubble scatter dependent on microbubble size and composition; however, most previously reported data have utilized a relatively narrow frequency bandwidth centered around the excitation frequency. To date, a comprehensive study of isolated microbubble superharmonic responses with a broadband dual-frequency system has not been performed. Here, the superharmonic signal production of 14 contrast agents with various gas cores, shell compositions, and bubble diameters at mechanical indices of 0.2 to 1.2 was evaluated using a transmit 4 MHz, receive 25 MHz configuration. Results indicate that perfluorocarbon cores or lipid shells with 18- or 20-carbon acyl chains produce more superharmonic signal than sulfur hexafluoride cores or lipid shells with 16-carbon acyl chains, respectively. As microbubble diameter increases from 1 to 4 µm, superharmonic generation decreases. In a comparison of two clinical agents, Definity and Optison, and one preclinical agent, Micromarker, Optison produced the least superharmonic signal. Overall, this work suggests that microbubbles around 1 µm in diameter with perfluorocarbon cores and longer-chained lipid shells perform best for superharmonic imaging at 4 MHz. Studies have found that microbubble superharmonic response follows trends different from those described in prior studies using a narrower frequency bandwidth centered around the excitation frequency. Future work will apply these results in vivo to optimize the sensitivity of acoustic angiography.

Ultrasound multiple scattering with microbubbles can differentiate between tumor and healthy tissue in vivo.

Most solid tumors are characterized by highly dense, isotropic vessel networks. Characterization of such features has shown promise for early cancer diagnosis. Ultrasound diffusion has been used to characterize the micro-architecture of complex media, such as bone and the lungs. In this work, we examine a non-invasive diffusion-based ultrasound technique to assess neo-vascularization. Because the diffusion constant reflects the density of scatterers in heterogeneous media, we hypothesize that by injecting microbubbles into the vasculature, ultrasound diffusivity can reflect vascular density (VD), thus differentiating the microvascular patterns between tumors and healthy tissue. The diffusion constant and its anisotropy are shown to be significantly different between fibrosarcoma tumors (n = 16) and control tissue (n = 18) in a rat animal model in vivo. The diffusion constant values for control and tumor were found to be 1.38 ± 0.51 mm2 µs-1 and 0.65 ± 0.27 mm2 µs-1, respectively. These results are corroborated with VD from acoustic angiography (AA) data, confirming increased vessel density in tumors compared to controls. The diffusion constant offers a promising way to quantitatively assess vascular networks when combined with contrast agents, which may allow early tumor detection and characterization.

Precision mouse models with expanded tropism for human pathogens.

A major limitation of current humanized mouse models is that they primarily enable the analysis of human-specific pathogens that infect hematopoietic cells. However, most human pathogens target other cell types, including epithelial, endothelial and mesenchymal cells. Here, we show that implantation of human lung tissue, which contains up to 40 cell types, including nonhematopoietic cells, into immunodeficient mice (lung-only mice) resulted in the development of a highly vascularized lung implant. We demonstrate that emerging and clinically relevant human pathogens such as Middle East respiratory syndrome coronavirus, Zika virus, respiratory syncytial virus and cytomegalovirus replicate in vivo in these lung implants. When incorporated into bone marrow/liver/thymus humanized mice, lung implants are repopulated with autologous human hematopoietic cells. We show robust antigen-specific humoral and T-cell responses following cytomegalovirus infection that control virus replication. Lung-only mice and bone marrow/liver/thymus-lung humanized mice substantially increase the number of human pathogens that can be studied in vivo, facilitating the in vivo testing of therapeutics.