Acoustic Angiography Research Articles

L’utilisation de la caméra thermique smartphone compatible (FLIR One) pour le repérage préopératoire des perforantes de DIEP

The success of surgeries involving free flaps largely depends on accurate preoperative mapping of perforator arteries. Various imaging techniques, such as Doppler ultrasound and CT angiography, are typically used, each having its advantages and disadvantages in terms of cost, accuracy, and patient risk. The main objective of our study is to compare the effectiveness of the FLIR One device for detecting these arteries compared with traditional methods. Thermal imaging appears to be a simpler, less expensive, and less invasive alternative for surgical planning. The study, conducted at the Regional Hospital Center of Nancy, included 25 free flaps (DIEP) on an exclusively female cohort of 22 patients, with follow-up from 2022 to 2023. Before the procedure, an abdominal-pelvic CT angiography was performed, followed by additional evaluation with thermal imaging using FLIR One and acoustic Doppler examination on the eve of the operation. This approach aimed to provide precise mapping of vascular perforators for each patient. Three different operators performed these markings, and the results were then compared with intraoperative observations. The imaging protocol also included a Doppler examination to validate the results of the thermal imaging. Statistical analyses with intraclass correlation coefficients (ICC) were performed to evaluate the correlation between different preoperative localization methods of perforating vessels. In 22 patients undergoing 25 DIEP flaps for breast reconstructions, three imaging techniques were used to identify vascular perforators: thermal imaging with FLIR, acoustic Doppler, and CT angiography. FLIR identified the most perforators (n=137), followed by acoustic Doppler (n=128) and CT angiography (n=126). Comparing these with intraoperative results, 66% of perforators identified by FLIR were confirmed, 70% for acoustic Doppler, and 95% for CT angiography. The ICCs shows a significant correlation between these imaging techniques and intraoperative results. FLIR demonstrated a strong correlation with intraoperative observations (ICC of 0.74, P<0.001), followed by a moderate correlation with acoustic Doppler (ICC of 0.56, P<0.03) and CT angiography (ICC of 0.52, P<0.006). The study concludes that thermal imaging with FLIR is a reliable and effective tool for locating vascular perforators. Although the study and FLIR have their own limitations, the tool presents several advantages such as ease of use, speed, and affordability. These characteristics make FLIR particularly attractive as a complement to traditional detection methods, such as acoustic Doppler and CT angiography.

Abstract 4159: Noninvasive measurement of murine glioblastoma in vivo with a benchtop ultrasound instrument

Abstract The purpose of this study was to evaluate a new in vivo approach for rapidly measuring tumor volumes in rodent models for glioblastoma (GBM). GBM is the most common primary brain tumor with &amp;lt;5% of patients surviving &amp;gt;5 years post diagnosis. These poor outcomes underscore the importance of researching new treatment options, and murine models play a vital role in this. Noninvasive measurements of murine GBM tumors are commonly performed using CT and MRI but these tools are costly and time-consuming to use. BLI can quickly detect the presence of GBM cells in vivo, but image data can be challenging to correlate to tumor volume. In this study we demonstrate the feasibility of imaging GBM in vivo through the intact mouse skull using a benchtop robotic ultrasound scanner. This approach could offer researchers a means to quickly size tumors in the murine brain noninvasively. To test feasibility of this approach, a female athymic nude mouse aged 16 weeks was implanted with 2e5 U87 GBM cells in the brain. The tumor was given 25 days to develop prior to ultrasound imaging. After imaging, the brain was harvested for ex vivo validation of tumor presence and volume. Imaging was performed using a Vega ultrasound system (Revvity, Waltham, MA, USA) with the mouse in supine position and fur removed from head with depilatory cream. In vivo imaging consisted of a contrast-enhanced acoustic angiography (AA) scan to assess tumor perfusion. An ex vivo B-mode scan of the brain was collected after sacrificing the animal to validate the location and size of the tumor without interference from the skull. The tumor volumes were measured in each image and compared. 3D segmentations in the ex vivo and in vivo images yielded volumes of 43.1 mm3 and 44.6 mm3, respectively, a 3.5% variance. In the in vivo AA image, the borders of the tumor were adequately well-defined for volume estimation, despite some shadowing from the sagittal suture of the skull. In the B-mode image of the excised brain, the tumor appeared hyperechoic compared to the contralateral hemisphere. Images were acquired in &amp;lt; 2 min. This pilot study demonstrates a potential alternative to CT and MRI for monitoring of GBM volumes in vivo. Citation Format: Thomas M. Kierski, Juan D. Rojas, Morrent Thang, Shawn D. Hingtgen, Phillip G. Durham, Paul A. Dayton, Tomek J. Czernuszewicz, Ryan C. Gessner. Noninvasive measurement of murine glioblastoma in vivo with a benchtop ultrasound instrument [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4159.

Progress in the Use of Echocardiography in Patients with Tumors.

Advances in cancer treatment have increased patient survival rates, shifting clinical focus towards minimizing treatment-related morbidity, including cardiovascular issues. Since echocardiography allows for a comprehensive non-invasive assessment at all cancer stages, it is well suited to monitor cardiovascular disease secondary to oncology treatment. This has earned it significant attention in the study of cardiac tumors and treatment-induced cardiac alterations. Ultrasound methods-ranging from transthoracic and transesophageal echocardiography to ultrasound diagnostic techniques including myocardial strain imaging, myocardial work indices, three-dimensional cardiac imaging-offer a holistic view of both the tumor and its treatment impact cardiac function. Stress echocardiography, myocardial contrast echocardiography, and myocardial acoustic angiography further augment this capability. Together, these echocardiographic techniques provide clinicians with early detection opportunities for cardiac damage, enabling timely interventions. As such, echocardiography continues to be instrumental in monitoring and managing the cardiovascular health of oncology patients, complementing efforts to optimize their overall treatment and survival outcomes.

Design and Fabrication of 1-D CMUT Arrays for Dual-Mode Dual-Frequency Acoustic Angiography Applications.

When microbubble contrast agents are excited at low frequencies (less than 5 MHz), they resonate and produce higher-order harmonics due to their nonlinear behavior. We propose a novel scheme with a capacitive micromachined ultrasonic transducer (CMUT) array to receive high-frequency microbubble harmonics in collapse mode and to transmit a low-frequency high-pressure pulse by releasing the CMUT plate from collapse and pull it back to collapse again in the same transmit-receive cycle. By patterning and etching the substrate to create glass spacers in the device cavity we can reliably operate the CMUT in collapse mode and receive high-frequency signals. Previously, we demonstrated a single-element CMUT with spacers operating in the described fashion. In this article, we present the design and fabrication of a dual-mode, dual-frequency 1-D CMUT array with 256 elements. We present two different insulating glass spacer designs in rectangular cells for the collapse mode. For the device with torus-shaped spacers, the 3 dB receive bandwidth is from 8 to 17 MHz, and the transmitted maximum peak-to-peak pressure from 32 elements at 4 mm focal depth was 2.12 MPa with a 1.21 MPa peak negative pressure, which corresponds to a mechanical index (MI) of 0.58 at 4.3 MHz. For the device with line-shaped spacers, the 3-dB receive bandwidth at 150 V dc bias extends from 10.9 to 19.2 MHz. By increasing the bias voltage to 180 V, the 3 dB bandwidth shifts, and extends from 11.7 to 20.4 MHz. The transmitting maximum peak-to-peak pressure with 32 elements at 4 mm was 2.06 MPa with a peak negative pressure of 1.19 MPa, which corresponds to an MI of 0.62 at 3.7 MHz.

Integrated Photoacoustic, Ultrasound, and Angiographic Tomography (PAUSAT) for NonInvasive Whole-Brain Imaging of Ischemic Stroke.

Presented here is an experimental ischemic stroke study using our newly developed noninvasive imaging system that integrates three acoustic-based imaging technologies: photoacoustic, ultrasound, and angiographic tomography (PAUSAT). Combining these three modalities helps acquire multi-spectral photoacoustic tomography (PAT) of the brain blood oxygenation, high-frequency ultrasound imaging of the brain tissue, and acoustic angiography of the cerebral blood perfusion. The multi-modal imaging platform allows the study of cerebral perfusion and oxygenation changes in the whole mouse brain after stroke. Two commonly used ischemic stroke models were evaluated: the permanent middle cerebral artery occlusion (pMCAO) model and the photothrombotic (PT) model. PAUSAT was used to image the same mouse brains before and after a stroke and quantitatively analyze both stroke models. This imaging system was able to clearly show the brain vascular changes after ischemic stroke, including significantly reduced blood perfusion and oxygenation in the stroke infarct region (ipsilateral) compared to the uninjured tissue (contralateral). The results were confirmed by both laser speckle contrast imaging and triphenyltetrazolium chloride (TTC) staining. Furthermore, stroke infarct volume in both stroke models was measured and validated by TTC staining as the ground truth. Through this study, we have demonstrated that PAUSAT can be a powerful tool in noninvasive and longitudinal preclinical studies of ischemic stroke.

Three-Dimensional Deep-Tissue Functional and Molecular Imaging by Integrated Photoacoustic, Ultrasound, and Angiographic Tomography (PAUSAT).

Non-invasive small-animal imaging technologies, such as optical imaging, magnetic resonance imaging and x -ray computed tomography, have enabled researchers to study normal biological phenomena or disease progression in their native conditions. However, existing small-animal imaging technologies often lack either the penetration capability for interrogating deep tissues (e.g., optical microscopy), or the functional and molecular sensitivity for tracking specific activities (e.g., magnetic resonance imaging). To achieve functional and molecular imaging in deep tissues, we have developed an integrated photoacoustic, ultrasound and acoustic angiographic tomography (PAUSAT) system by seamlessly combining light and ultrasound. PAUSAT can perform three imaging modes simultaneously with complementary contrast: high-frequency B-mode ultrasound imaging of tissue morphology, microbubble-enabled acoustic angiography of tissue vasculature, and multi-spectral photoacoustic imaging of molecular probes. PAUSAT can provide three-dimensional (3D) multi-contrast images that are co-registered, with high spatial resolutions at large depths. Using PAUSAT, we performed proof-of-concept in vivo experiments on various small animal models: monitoring longitudinal development of placenta and embryo during mouse pregnancy, tracking biodistribution and metabolism of near-infrared organic dye on the whole-body scale, and detecting breast tumor expressing genetically-encoded photoswitchable phytochromes. These results have collectively demonstrated that PAUSAT has broad applicability in biomedical research, providing comprehensive structural, functional, and molecular imaging of small animal models.

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 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.

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.

Assessing Polycystic Kidney Disease in Rodents: Comparison of Robotic 3D Ultrasound and Magnetic Resonance Imaging.

Polycystic kidney disease (PKD) is an inherited disorder characterized by renal cyst formation and enlargement of the kidney. PKD severity can be staged noninvasively by measuring total kidney volume (TKV), a promising biomarker that has recently received regulatory qualification. In preclinical mouse models, where the disease is studied and potential therapeutics are evaluated, the most popular noninvasive method of measuring TKV is magnetic resonance imaging (MRI). Although MRI provides excellent 3D resolution and contrast, these systems are expensive to operate, have long acquisition times, and, consequently, are not heavily used in preclinical PKD research. In this study, a new imaging instrument, based on robotic ultrasound (US), was evaluated as a complementary approach for assessing PKD in rodent models. The objective was to determine the extent to which TKV measurements on the robotic US scanner correlated with both in vivo and ex vivo reference standards (MRI and Vernier calipers, respectively). A cross-sectional study design was implemented that included both PKD-affected mice and healthy wild types, spanning sex and age for a wide range of kidney volumes. It was found that US-derived TKV measurements and kidney lengths were strongly associated with both in vivo MRI and ex vivo Vernier caliper measurements (R 2=0.94 and 0.90, respectively). In addition to measuring TKV, renal vascular density was assessed using acoustic angiography (AA), a novel contrast-enhanced US methodology. AA image intensity, indicative of volumetric vascularity, was seen to have a strong negative correlation with TKV (R 2=0.82), suggesting impaired renal vascular function in mice with larger kidneys. These studies demonstrate that robotic US can provide a rapid and accurate approach for noninvasively evaluating PKD in rodent models.

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.

Microvascular Ultrasonic Imaging of Angiogenesis Identifies Tumors in a Murine Spontaneous Breast Cancer Model.

The purpose of this study is to determine if microvascular tortuosity can be used as an imaging biomarker for the presence of tumor-associated angiogenesis and if imaging this biomarker can be used as a specific and sensitive method of locating solid tumors. Acoustic angiography, an ultrasound-based microvascular imaging technology, was used to visualize angiogenesis development of a spontaneous mouse model of breast cancer (n = 48). A reader study was used to assess visual discrimination between image types, and quantitative methods utilized metrics of tortuosity and spatial clustering for tumor detection. The reader study resulted in an area under the curve of 0.8, while the clustering approach resulted in the best classification with an area under the curve of 0.95. Both the qualitative and quantitative methods produced a correlation between sensitivity and tumor diameter. Imaging of vascular geometry with acoustic angiography provides a robust method for discriminating between tumor and healthy tissue in a mouse model of breast cancer. Multiple methods of analysis have been presented for a wide range of tumor sizes. Application of these techniques to clinical imaging could improve breast cancer diagnosis, as well as improve specificity in assessing cancer in other tissues. The clustering approach may be beneficial for other types of morphological analysis beyond vascular ultrasound images.

Use of 3-D Contrast-Enhanced Ultrasound to Evaluate Tumor Microvasculature After Nanoparticle-Mediated Modulation

A cost-effective method for serial in vivo imaging of tumor microvasculature has been developed. We evaluated acoustic angiography (AA) for visualizing and assessing non-small cell lung tumor (A549) microvasculature in mice before and after tumor vascular disruption by vascular-targeted gold nanoparticles and radiotherapy. Standard B-mode and microbubble-enhanced AA images were acquired at pre- and post-treatment time points. Using these modes, a new metric, 50% vessel penetration depth, was developed to characterize the 3-D spatial heterogeneity of microvascular networks. We observed an increase in tumor perfusion after radiation-induced vascular disruption, relative to control animals. This was also visualized in vessel morphology mode, which revealed a loss in vessel integrity. We found that tumors with poorly perfused vasculature at day 0 exhibited a reduced growth rate over time. This suggested a new method to reduce in-group treatment response variability using pre-treatment microvessel maps to objectively identify animals for study removal.

On Command Drug Delivery via Cell-Conveyed Phototherapeutics.

Herein, the use of red blood cells (RBCs) as carriers of cytoplasmically interned phototherapeutic agents is described. Photolysis promotes drug release from the RBC carrier thereby providing the means to target specific diseased sites. This strategy is realized with a vitamin B12-taxane conjugate (B12-TAX), in which the drug is linked to the vitamin via a photolabile CoC bond. The conjugate is introduced into mouse RBCs (mRBCs) via a pore-forming/pore-resealing procedure and is cytoplasmically retained due to the membrane impermeability of B12. Photolysis separates the taxane from the B12 cytoplasmic anchor, enabling the drug to exit the RBC carrier. A covalently appended Cy5 antenna sensitizes the conjugate (Cy5-B12-TAX) to far red light, thereby circumventing the intense light absorbing properties of hemoglobin (350-600 nm). Microscopy and imaging flow cytometry reveal that Cy5-B12-TAX-loaded mRBCs act as drug carriers. Furthermore, intravital imaging of mice furnish a real time assessment of circulating phototherapeutic-loaded mRBCs as well as evidence of the targeted photorelease of the taxane upon photolysis. Histopathology confirms that drug release occurs in a well resolved spatiotemporal fashion. Finally, acoustic angiography is employed to assess the consequences of taxane release at the tumor site in Nu/Nu-tumor-bearing mice.

Abstract 1935: Tracking angiogenesis induced microvascular changes in a lymphoma model via a new high throughput non-invasive dual modality imaging platform

Abstract This study presents a novel dual-modality imaging system for assessing cancer progression in rodents. The system incorporates bioluminescence imaging (BLI), used to assess tumor growth, and contrast-enhanced ultrasound (CEUS), used to assess anatomical information and map microvasculature. The combination of the two modalities has previously been shown to reduce inter-user variability of BLI quantification, and in this work, we demonstrate that a dual BLI/US system can provide a more holistic assessment of disease. NSG (NOD/scid/gamma) female mice were implanted with luc-tagged lymphoma cells (BCBL-1, RRID: CVCL_0165, 1x105 cells, intraperitoneal (IP) injection, N = 8 mice) and imaged using the US and BLI hybrid modality system (SonoVol, Inc.), and a BLI-alone system (Perkin Elmer, Inc.) for comparison to a widely available commercial BLI system. BLI sensitivity was evaluated using a weakly luminescent tritium phantom to find the shortest exposure required to detect signal. In vivo studies consisted of an IP injection of D-luciferin (250 µL at 15 mg/mL) and serial captures of images with exposure times of 60 s every 3 min. Acoustic Angiography (AA), a high-resolution CEUS technique, was used to acquire 3D volumes in the abdomen surrounding the tumor site to assess angiogenesis-induced vascular remodeling associated with tumor growth. In vitro BLI sensitivity experiments showed that the dual-modality system required an exposure of 3 sec to detect signal (p &amp;lt; 0.05) and the BLI-alone system required an exposure of 1 sec (p&amp;lt; 0.05). For in vivo studies, the change in luminescence occurring between week 2 and 3 post-cell implantation was calculated (a surrogate measurement for tumor growth), and the difference in signal was 17.15 ± 10.1 photons/sec and 16.04 ± 7.6 for the dual-modality and BLI-alone systems, respectively. Images of the vascular remodeling arising during the first two weeks of tumor growth were captured with AA and demonstrated an increase in perfusion in the vicinity of BLI signal by a factor of 1.4 ± 0.38, with vascular remodeling being evident even at the periphery of BLI signal. This work demonstrates that non-invasive measurements of in vivo microvascular remodeling can be precisely mapped to changes in tumor growth with a hybrid modality system. The system has comparable sensitivity to a BLI-alone system and provides similar assessments of longitudinal tumor growth. Adding quantitative metrics for vascular remodeling to the widely used luminescent imaging could provide a more comprehensive assessment for tumor functional status than either modality could individually. This should prove valuable when using antiangiogenic therapies because changes in vasculature will precede cell death, and the ability to monitor both the cells and their blood supply might help to elucidate underlying biological processes. Citation Format: Juan D. Rojas, Rajalekha Rajamahendiran, Tomasz J. Czernuszewicz, Brian Velasco, Jonathan Perdomo, Max Harlacher, Graeme O'Connell, James Butler, Blossom Damania, Paul A. Dayton, Ryan C. Gessner. Tracking angiogenesis induced microvascular changes in a lymphoma model via a new high throughput non-invasive dual modality imaging platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1935.

Abstract 1955: A preclinical ultrasound platform for widefield 3D imaging of rodent tumors

Abstract Background: Preclinical ultrasound (US) and contrast-enhanced ultrasound (CEUS) imaging have long been used in oncology to noninvasively measure tumor volume and vascularity. While the value of preclinical US has been repeatedly demonstrated, these modalities are not without several key limitations that make them unattractive to cancer researchers, including: high user-variability, low throughput, and limited imaging field-of-view (FOV). Herein, we present a novel robotic preclinical US/CEUS system that addresses these limitations and demonstrates its use in evaluating tumors in 3D in a rodent model. Methods: The imaging system was designed to allow seamless whole-body 3D imaging, which requires rodents to be imaged without physical contact between the US transducer and the animal. To achieve this, a custom dual-element transducer was mounted on a robotic carriage, submerged in a hydrocarbon fluid, and the reservoir sealed with an acoustically transmissive top platform. Eight NOD/scid/gamma (NSG) female mice were injected subcutaneously in the flank with 8×109 786-O human clear-cell renal cell carcinoma (ccRCC) cells. Weekly imaging commenced after tumors reached a size of 150 mm3 and continued until tumors reached a maximum size of 1 cm3 (∼4-5 weeks). An additional six nude athymic female mice were injected subcutaneously in the flank with 7 × 105 SVR angiosarcoma cells to perform an inter-operator variability study. Imaging consisted of 3D B-mode (conventional ultrasound) of the whole abdomen (&amp;lt; 1 min), as well as contrast-enhanced acoustic angiography (&amp;lt; 10 min) to measure blood vessel density (BVD). Tumors were manually segmented in 3D (&amp;lt; 2 min) and inter-operator and inter-reader reliability was assessed with Krippendorff’s alpha. Results: Wide-field US images reconstructed from 3D volumetric data showed superior FOV over conventional US. Several anatomical landmarks could be identified within each image surrounding the tumor, including the liver, small intestines, bladder, and inguinal lymph nodes. Tumor boundaries were clearly delineated in both B-mode and BVD images, with BVD images showing heterogeneous microvessel density at later timepoints suggesting tumor necrosis. Excellent agreement was measured for both inter-reader and inter-operator experiments, with alpha coefficients of 0.914 (95% CI: 0.824-0.948) and 0.959 (0.911-0.981), respectively. Conclusion: We have demonstrated a novel preclinical US imaging system that can accurately and consistently evaluate tumors in rodent models. The system leverages cost-effective robotic technology, and a new scanning paradigm that allows for easy and reproducible data acquisition to enable wide-field, 3D, multi-parametric ultrasound imaging. Note: This abstract was not presented at the meeting. Citation Format: Tomasz Czernuszewicz, Virginie Papadopoulou, Juan D. Rojas, Rajalekha Rajamahendiran, Jonathan Perdomo, James Butler, Max Harlacher, Graeme O'Connell, Dzenan Zukic, Paul A. Dayton, Stephen Aylward, Ryan C. Gessner. A preclinical ultrasound platform for widefield 3D imaging of rodent tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1955.

In Vitro Superharmonic Contrast Imaging Using a Hybrid Dual-Frequency Probe

Superharmonic imaging is an ultrasound contrast imaging technique that differentiates microbubble echoes from tissue through detection of higher-order bubble harmonics in a broad frequency range well above the excitation frequency. Application of superharmonic imaging in three dimensions allows specific visualization of the tissue microvasculature with high resolution and contrast, a technique referred to as acoustic angiography. Because of the need to transmit and receive across a bandwidth that spans up to the fifth harmonic of the fundamental and higher, this imaging approach requires imaging probes comprising dedicated transducers for transmit and receive. In this work, we report on a new dual-frequency probe including two 1.7-MHz rectangular transducers positioned one on each side of a 20-MHz 256-element array. Finite element modeling-based design, fabrication processes and assembly of the transducer are described, as is integration with a high-frequency ultrasound imaging platform. Dual-frequency single-plane-wave imaging was performed with a microbubble contrast agent in flow phantoms and compared with conventional high-frequency B-mode imaging, and resolution and contrast-to-tissue ratio were quantified. This work represents an intermediate but informative step toward the development of dual-frequency imaging probes based on array technology, specifically designed for clinical applications of acoustic angiography.

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.