Ryan C. Gessner

High-resolution, high-contrast ultrasound imaging using a prototype dual-frequency transducer: In vitro and in vivo studies

With recent advances in animal models of disease, there has been great interest in capabilities for highresolution contrast-enhanced ultrasound imaging. Microbubble contrast agents are unique in that they scatter broadband ultrasound energy because of their nonlinear behavior. For optimal response, it is desirable to excite the microbubbles near their resonant frequency. To date, this has been challenging with high-frequency imaging systems because most contrast agents are resonant at frequencies in the order of several megahertz. Our team has developed a unique dual-frequency confocal transducer which enables low-frequency excitation of bubbles near their resonance with one element, and detection of their emitted high-frequency content with the second element. Using this imaging approach, we have attained an average 12.3 dB improvement in contrast-to-tissue ratios over fundamental mode imaging, with spatial resolution near that of the high-frequency element. Because this detection method does not rely on signal decorrelation, it is not susceptible to corruption by tissue motion. This probe demonstrates contrast imaging capability with significant tissue suppression, enabling high-resolution contrast-enhanced images of microvascular blood flow. Additionally, this probe can readily produce radiation force on flowing contrast agents, which may be beneficial for targeted imaging or therapy.

Quantitative Volumetric Perfusion Mapping of the Microvasculature Using Contrast Ultrasound

Objectives:Contrast-enhanced ultrasound imaging has demonstrated significant potential as a noninvasive technology for monitoring blood flow in the microvasculature. With the application of nondestructive contrast imaging pulse sequences combined with a clearance-refill approach, it is possible to create quantitative time-to-refill maps of tissue correlating to blood perfusion rate. One limitation to standard two-dimensional (2D) perfusion imaging is that the narrow elevational beamwidth of 1- or 1.5-D ultrasound transducers provides information in only a single slice of tissue, and thus it is difficult to image exactly the same plane from study to study. We hypothesize that inhomogeneity in vascularization, such as that common in many types of tumors, makes serial perfusion estimates inconsistent unless the same region can be imaged repeatedly. Our objective was to evaluate error in 2D quantitative perfusion estimation in an in vivo sample volume because of differences in transducer positioning. To mitigate observed errors due to imaging plane misalignment, we propose and demonstrate the application of quantitative 3-dimensional (3D) perfusion imaging. We also evaluate the effect of contrast agent concentration and infusion rate on perfusion estimates. Materials and Methods:Contrast-enhanced destruction-reperfusion imaging was performed using parametric mapping of refill times and custom software for image alignment to compensate for tissue motion. Imaging was performed in rats using a Siemens Sequoia 512 imaging system with a 15L8 transducer. A custom 3D perfusion mapping system was designed by incorporating a computer-controlled positioning system to move the transducer in the elevational direction, and the Sequoia was interfaced to the motion system for timing of the destruction-reperfusion sequence and data acquisition. Perfusion estimates were acquired from rat kidneys as a function of imaging plane and in response to the vasoactive drug dopamine. Results:Our results indicate that perfusion estimates generated by 2D imaging in the rat kidney have mean standard deviations on the order of 10%, and as high as 22%, because of differences in initial transducer position. This difference was larger than changes in kidney perfusion induced by dopamine. With application of 3D perfusion mapping, repeatability in perfusion estimated in the kidney is reduced to a standard deviation of less than 3%, despite random initial transducer positioning. Varying contrast agent administration rate was also observed to bias measured perfusion time, especially at low concentrations; however, we observed that contrast administration rates between 2.7 × 108 and 3.9 × 108 bubbles/min provided results that were consistent within 3% for the contrast agent type evaluated. Conclusions:Three-dimensional perfusion imaging allows a significant reduction in the error caused by transducer positioning, and significantly improves the reliability of quantitative perfusion time estimates in a rat kidney model. When performing perfusion imaging, it is important to use appropriate and consistent contrast agent infusion rates to avoid bias.

Mapping Microvasculature with Acoustic Angiography Yields Quantifiable Differences between Healthy and Tumor-bearing Tissue Volumes in a Rodent Model

PURPOSE To determine if the morphologies of microvessels could be extracted from contrast material-enhanced acoustic angiographic ultrasonographic (US) images and used as a quantitative basis for distinguishing healthy from diseased tissue. MATERIALS AND METHODS All studies were institutional animal care and use committee approved. Three-dimensional contrast-enhanced acoustic angiographic images were acquired in both healthy (n = 7) and tumor-bearing (n = 10) rats. High-spatial-resolution and high signal-to-noise acquisition was enabled by using a prototype dual-frequency US transducer (transmit at 4 MHz, receive at 30 MHz). A segmentation algorithm was utilized to extract microvessel structure from image data, and the distance metric (DM) and the sum of angles metric (SOAM), designed to distinguish different types of tortuosity, were applied to image data. The vessel populations extracted from tumor-bearing tissue volumes were compared against vessels extracted from tissue volumes in the same anatomic location within healthy control animals by using the two-sided Student t test. RESULTS Metrics of microvascular tortuosity were significantly higher in the tumor population. The average DM of the tumor population (1.34 ± 0.40 [standard deviation]) was 23.76% higher than that of the control population (1.08 ± 0.08) (P < .0001), while the average SOAM (22.53 ± 7.82) was 50.73% higher than that of the control population (14.95 ± 4.83) (P < .0001). The DM and SOAM metrics for the control and tumor populations were significantly different when all vessels were pooled between the two animal populations. In addition, each animal in the tumor population had significantly different DM and SOAM metrics relative to the control population (P < .05 for all; P value ranges for DM, 3.89 × 10(-)(7) to 5.63 × 10(-)(3); and those for SOAM, 2.42 × 10(-)(12) to 1.57 × 10(-)(3)). CONCLUSION Vascular network quantification by using high-spatial-resolution acoustic angiographic images is feasible. Data suggest that the angiogenic processes associated with tumor development in the models studied result in higher instances of vessel tortuosity near the tumor site.

Functional ultrasound imaging for assessment of extracellular matrix scaffolds used for liver organoid formation.

A method of 3D functional ultrasound imaging has been developed to enable non-destructive assessment of extracellular matrix scaffolds that have been prepared by decellularization protocols and are intended for recellularization to create organoids. A major challenge in organ decellularization is retaining patent micro-vascular structures crucial for nutrient access and functionality of organoids. The imaging method described here provides statistical distributions of flow rates throughout the tissue volumes, 3D vessel network architecture visualization, characterization of microvessel volumes and sizes, and delineation of matrix from vascular circuits. The imaging protocol was tested on matrix scaffolds that are tissue-specific, but not species-specific, matrix extracts, prepared by a process that preserved >98% of the collagens, collagen-associated matrix components, and matrix-bound growth factors and cytokines. Image-derived data are discussed with respect to assessment of scaffolds followed by proof-of-concept studies in organoid establishment using Hep3B, a human hepatoblast-like cell line. Histology showed that the cells attached to scaffolds with patent vasculature within minutes, achieved engraftment at near 100%, expressed liver-specific functions within 24 h, and yielded evidence of proliferation and increasing differentiation of cells throughout the two weeks of culture studies. This imaging method should prove valuable in analyses of such matrix scaffolds.

Therapeutic ultrasound as a potential male contraceptive: power, frequency and temperature required to deplete rat testes of meiotic cells and epididymides of sperm determined using a commercially available system

BackgroundStudies published in the 1970s by Mostafa S. Fahim and colleagues showed that a short treatment with ultrasound caused the depletion of germ cells and infertility. The goal of the current study was to determine if a commercially available therapeutic ultrasound generator and transducer could be used as the basis for a male contraceptive.MethodsSprague-Dawley rats were anesthetized and their testes were treated with 1 MHz or 3 MHz ultrasound while varying power, duration and temperature of treatment.ResultsWe found that 3 MHz ultrasound delivered with 2.2 Watt per square cm power for fifteen minutes was necessary to deplete spermatocytes and spermatids from the testis and that this treatment significantly reduced epididymal sperm reserves. 3 MHz ultrasound treatment reduced total epididymal sperm count 10-fold lower than the wet-heat control and decreased motile sperm counts 1,000-fold lower than wet-heat alone. The current treatment regimen provided nominally more energy to the treatment chamber than Fahims originally reported conditions of 1 MHz ultrasound delivered at 1 Watt per square cm for ten minutes. However, the true spatial average intensity, effective radiating area and power output of the transducers used by Fahim were not reported, making a direct comparison impossible. We found that germ cell depletion was most uniform and effective when we rotated the therapeutic transducer to mitigate non-uniformity of the beam field. The lowest sperm count was achieved when the coupling medium (3% saline) was held at 37 degrees C and two consecutive 15-minute treatments of 3 MHz ultrasound at 2.2 Watt per square cm were separated by 2 days.ConclusionsThe non-invasive nature of ultrasound and its efficacy in reducing sperm count make therapeutic ultrasound a promising candidate for a male contraceptive. However, further studies must be conducted to confirm its efficacy in providing a contraceptive effect, to test the result of repeated use, to verify that the contraceptive effect is reversible and to demonstrate that there are no detrimental, long-term effects from using ultrasound as a method of male contraception.

Microbubbles in imaging: Applications beyond ultrasound

Since their introduction as ultrasound contrast agents, microbubbles have demonstrated the potential to revolutionise the use of ultrasound at the bedside. Aside from clinical application, where microbubbles are used to enhance ultrasonic assessment of myocardial perfusion, they have demonstrated potential in an exciting host of pre-clinical ultrasound imaging and therapeutic applications. These include the ability to target specific cellular markers of disease, provide dynamic blood flow estimation, deliver localised chemotherapy, potentiate the mechanisms of gene therapy, enhance lesion ablation through cavitation, and spatiotemporally permeabilise the blood-brain barrier. The unique and flexible construction of microbubbles not only enables a variety of ultrasound applications, but also opens the door to detection of microbubbles with modalities other than ultrasound. In this review, non-ultrasound imaging applications utilizing microbubbles are discussed, including MRI, PET, and DEI. These various imaging approaches illustrate novel applications of microbubbles, and may provide the groundwork for future multi-modality imaging or image-guided therapeutics.

Blood vessel structural morphology derived from 3D dual-frequency ultrasound images

The method of dual-frequency contrast ultrasound imaging can produce high resolution, high contrast 3D maps of the microvasculature. In this manuscript, we demonstrate an extension of the technique, showing the potential to segment individual blood vessels from 3D datasets in order to quantify their structural morphologies. There is a known relationship between abnormal blood vessel structure and the presence of cancer, with diseased vasculature generally showing a high degree of tortuosity and irregularity. These irregular vessel paths have been shown to normalize with successful anti-cancer treatment, effects which generally precede gross tumor volume changes. Thus, we hypothesize that our ultra-broadband imaging technique could be used in conjunction with vessel segmentation and morphological analyses to assess in-vivo therapeutic response with greater sensitivity than previously possible. In this paper, we demonstrate the consistency of our segmentation algorithm, and estimate the limitations of detectable tortuosity levels.

3-D microvessel-mimicking ultrasound phantoms produced with a scanning motion system.

Ultrasound techniques are currently being developed that can assess the vascularization of tissue as a marker for therapeutic response. Some of these ultrasound imaging techniques seek to extract quantitative features about vessel networks, whereas high-frequency imaging also allows individual vessels to be resolved. The development of these new techniques, and subsequent imaging analysis strategies, necessitates an understanding of their sensitivities to vessel and vessel network structural abnormalities. Constructing in-vitro flow phantoms for this purpose can be prohibitively challenging, because simulating precise flow environments with nontrivial structures is often impossible using conventional methods of construction for flow phantoms. Presented in this manuscript is a method to create predefined structures with <10 μm precision using a three-axis motion system. The application of this technique is demonstrated for the creation of individual vessel and vessel networks, which can easily be made to simulate the development of structural abnormalities typical of diseased vasculature in vivo. In addition, beyond facilitating the creation of phantoms that would otherwise be very challenging to construct, the method presented herein enables one to precisely simulate very slow blood flow and respiration artifacts, and to measure imaging resolution.

Early assessment of tumor response to radiation therapy using high-resolution quantitative microvascular ultrasound imaging

Measuring changes in tumor volume using anatomical imaging weeks to months post radiation therapy (RT) is currently the clinical standard for indicating treatment response to RT. For patients whose tumors do not respond successfully to treatment, this approach is suboptimal as timely modification of the treatment approach may lead to better clinical outcomes. We propose to use tumor microvasculature as a biomarker for early assessment of tumor response to RT. Acoustic angiography is a novel contrast ultrasound imaging technique that enables high-resolution microvascular imaging and has been shown to detect changes in microvascular structure due to cancer growth. Data suggest that acoustic angiography can detect longitudinal changes in the tumor microvascular environment that correlate with RT response. Methods: Three cohorts of Fisher 344 rats were implanted with rat fibrosarcoma tumors and were treated with a single fraction of RT at three dose levels (15 Gy, 20 Gy, and 25 Gy) at a dose rate of 300 MU/min. A simple treatment condition was chosen for testing the feasibility of our imaging technique. All tumors were longitudinally imaged immediately prior to and after treatment and then every 3 days after treatment for a total of 30 days. Both acoustic angiography (using in-house produced microbubble contrast agents) and standard b-mode imaging was performed at each imaging time point using a pre-clinical Vevo770 scanner and a custom modified dual-frequency transducer. Results: Results show that all treated tumors in each dose group initially responded to treatment between days 3-15 as indicated by decreased tumor growth accompanied with decreased vascular density. Untreated tumors continued to increase in both volume and vascular density until they reached the maximum allowable size of 2 cm in diameter. Tumors that displayed complete control (no tumor recurrence) continued to decrease in size and vascular density, while tumors that progressed after the initial response presented an increase in tumor volume and volumetric vascular density. The increase in tumor volumetric vascular density in recurring tumors can be detected 10.25 ± 1.5 days, 6 ± 0 days, and 4 ± 1.4 days earlier than the measurable increase in tumor volume in the 15, 20, and 25 Gy dose groups, respectively. A dose-dependent growth rate for tumor recurrence was also observed. Conclusions: In this feasibility study we have demonstrated the ability of acoustic angiography to detect longitudinal changes in vascular density, which was shown to be a potential biomarker for tumor response to RT.

Acoustic angiography: a new high frequency contrast ultrasound technique for biomedical imaging

Acoustic Angiography is a new approach to high-resolution contrast enhanced ultrasound imaging enabled by ultra-broadband transducer designs. The high frequency imaging technique provides signal separation from tissue which does not produce significant harmonics in the same frequency range, as well as high resolution. This approach enables imaging of microvasculature in-vivo with high resolution and signal to noise, producing images that resemble x-ray angiography. Data shows that acoustic angiography can provide important information about the presence of disease based on vascular patterns, and may enable a new paradigm in medical imaging.