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Ultrasound contrast agents (UCAs) have tremendous potential for in vivo molecular imaging because of their high sensitivity. However, the diagnostic potential of UCAs has been difficult to exploit because current UCAs are based on pre-formed microbubbles, which can only detect cell surface receptors. Here, we demonstrate that chemical reactions that generate gas forming molecules can be used to perform molecular imaging by ultrasound in vivo. This new approach was demonstrated by imaging reactive oxygen species in vivo with allylhydrazine, a liquid compound that is converted into nitrogen and propylene gas after reacting with radical oxidants. We demonstrate that allylhydrazine encapsulated within liposomes can detect a 10 micromolar concentration of radical oxidants by ultrasound, and can image oxidative stress in mice, induced by lipopolysaccharide, using a clinical ultrasound system. We anticipate numerous applications of chemically-generated microbubbles for molecular imaging by ultrasound, given ultrasound's ability to detect small increments above the gas saturation limit, its spatial resolution and widespread clinical use.

Contrast agent-enhanced ultrasound can facilitate personalized therapeutic strategies by providing the technology to measure local blood flow rate, to selectively image receptors on the vascular endothelium, and to enhance localized drug delivery. Ultrasound contrast agents are micron-diameter encapsulated bubbles that circulate within the vascular compartment and can be selectively imaged with ultrasound. Microbubble transport-based estimates of local blood flow can quantify changes resulting from anti-angiogenic therapies and facilitate differentiation of angiogenic mechanisms. Microbubbles that are conjugated with targeting ligands attach to endothelial surface receptors that are upregulated in disease, providing high signal-to-noise ratio images of pathological vasculature. In addition to imaging applications, microbubbles can be used to enhance localized gene and drug delivery, either by changing membrane and vascular permeability or by carrying and locally releasing cargo. Our goal in this review is to provide an overview of the use of contrast-enhanced ultrasound methodologies in the design and evaluation of therapeutic strategies with emphases on quantitative blood flow mapping, molecular imaging, and enhanced drug delivery.

The use of ultrasound radiation force to manipulate microbubbles in blood vessels has attracted recent interest as a method to increase the efficiency of ultrasonic molecular imaging and drug delivery. However, recent studies indicate that microbubble oscillation is diminished within small blood vessels, and therefore we investigate microbubble oscillation and translation within 12 μ m vessels using high-speed photography. With each 0.1- to 1-MPa ultrasound pulse, microbubbles (radius of 1, 1.5 and 2 μ m) within 12 μ m tubes translate 5 to 10 times less than those within 200 μ m tubes. Application of a pulse train with a high pulse repetition frequency displaces bubbles to the wall of 12- and 200- μ m tubes within an interval (∼1 s) that is reasonable for clinical translation. Modeling of coupled oscillation and translation for unconstrained microbubbles, based on a modified Rayleigh-Plesset (RP) and the trajectory equations, is compared with experimental observations and demonstrates agreement for the larger displacements observed within the 200 μ m tubes. This study has implications for contrast-assisted ultrasound applications, aiding the manipulation of targeted microbubbles and for further theoretical understanding of the complex bubble dynamics within constrained vessel. (E-mail: kwferrara@ucdavis.edu )

Many thousands of contrast ultrasound studies have been conducted in clinics around the world. In addition, the microbubbles employed in these examinations are being widely investigated to deliver drugs and genes. Here, for the first time, the oscillation of these microbubbles in small vessels is directly observed and shown to be substantially different than that predicted by previous models and imaged within large fluid volumes. Using pulsed ultrasound with a center frequency of 1 MHz and peak rarefactional pressure of 0.8 or 2.0 MPa , microbubble expansion was significantly reduced when microbubbles were constrained within small vessels in the rat cecum ( p < 0.05 ) . A model for microbubble oscillation within compliant vessels is presented that accurately predicts oscillation and vessel displacement within small vessels. As a result of the decreased oscillation in small vessels, a large resting microbubble diameter resulting from agent fusion or a high mechanical index was required to bring the agent shell into contact with the endothelium. Also, contact with the endothelium was observed during asymmetrical collapse, not during expansion. These results will be used to improve the design of drug delivery techniques using microbubbles.

Automated software improves the accuracy and reliability of blood velocity, vessel diameter, blood flow, and shear rate ultrasound measurements, but existing software offers limited flexibility to customize and validate analyses. We developed FloWave.US —open-source software to automate ultrasound blood flow analysis—and demonstrated the validity of its blood velocity (aggregate relative error, 4.32%) and vessel diameter (0.31%) measures with a skeletal muscle ultrasound flow phantom. Compared with a commercial, manual analysis software program, FloWave.US produced equivalent in vivo cardiac cycle time-averaged mean (TAMean) velocities at rest and following a 10-s muscle contraction (mean bias

FUS pressure field mapping is important for dosimetry, quality assurance, and other uses. Hydrophone measurements are the current standard, and are accurate but costly and slow. As a simple low-cost alternative, background-oriented schlieren (BOS) imaging of ultrasound fields has been proposed.[1] In that technique, a predetermined image (usually a grid of lines or a random dot pattern) is placed on one side of a water tank and viewed from the other side, through the water and FUS pressure field. When the FUS is on, spatial variations in the water’s index of refraction are created that blur the image. Subtracting images with and without the FUS field provides a rapid visualization of it. The method has also been used to tomographically reconstruct air flow density.[2] The overall goal of the present work is to develop a low-cost BOS hardware system and BOS tomography acquisitions and reconstructions to enable rapid and cheap volumetric measurements of continuous-wave FUS fields. Here we present our current progress towards that goal.

Ultrasound thermometry provides noninvasive 2-D temperature monitoring, and in this paper, we have investigated the use of computed tomography (CT) radiodensity to characterize tissues to improve the accuracy of ultrasound thermometry. Agarose-based tissue-mimicking phantoms were created with glyceryl trioleate (a fat-mimicking material) concentration of 0%, 10%, 20%, 30%, 40%, and 50%. The speed of sound (SOS) of the phantoms was measured over a temperature range of 22.1-41.1 °C. CT images of the phantoms were acquired by a clinical dedicated breast CT scanner, followed by calculation of the Hounsfield units (HU). The phantom was heated with a therapeutic acoustic pulse (1.54 MHz), while RF data were acquired with a 10-MHz linear-array transducer. Two-dimensional speckle tracking was used to calculate the thermal strain offline. The tissue-dependent thermal strain parameter required for ultrasound thermometry was analyzed and correlated with CT radiodensity, followed by the validation of the temperature prediction. Results showed that the change in SOS with the temperature increase was opposite in sign between the 0%-10% and 20%-50% trioleate phantoms. The inverse of the tissue-dependent thermal strain parameter of the phantoms was correlated with the CT radiodensity (R(2) = 0.99). A blinded ultrasound thermometry study on phantoms with a trioleate range of 5%-35% demonstrated the capability to estimate the tissue-dependent thermal strain parameter and estimate temperature with error less than ~1 °C. In conclusion, CT radiodensity may provide a method for improving ultrasound thermometry in heterogeneous tissues.

Ultrasound can selectively and specifically visualize upregulated vascular receptors through the detection of bound microbubbles. However, most current ultrasound molecular imaging methods incur delays that result in longer acquisition times and reduced frame rates. These delays occur for two main reasons: 1) multi-pulse imaging techniques are used to differentiate microbubbles from tissue and 2) acquisition occurs after free bubble clearance (>6 minutes) in order to differentiate bound from freely circulating microbubbles. In this paper, we validate tumor imaging with a broadband single pulse molecular imaging method that is faster than the multi-pulse methods typically implemented on commercial scanners. We also combine the single pulse method with interframe filtering to selectively image targeted microbubbles without waiting for unbound bubble clearance, thereby reducing acquisition time from 10 to 2 minutes. The single pulse imaging method leverages non-linear bubble behavior by transmitting at low and receiving at high frequencies (TLRH). We implemented TLRH imaging and visualized the accumulation of intravenously administrated integrin-targeted microbubbles in a phantom and a Met-1 mouse tumor model. We found that the TLRH contrast imaging has a ~2-fold resolution improvement over standard contrast pulse sequencing (CPS) imaging. By using interframe filtering, the tumor contrast was 24.8±1.6 dB higher after the injection of integrin-targeted microbubbles than non-targeted control MBs, while echoes from regions lacking the target integrin were suppressed by 26.2±2.1 dB as compared with tumor echoes. Since real-time three-dimensional (3D) molecular imaging provides a more comprehensive view of receptor distribution, we generated 3D images of tumors to estimate their volume, and these measurements correlated well with expected tumor sizes. We conclude that TLRH combined with interframe filtering is a feasible method for 3D targeted ultrasound imaging that is faster than current multi-pulse strategies.

Characterization of therapeutic response in primary and metastatic tumors is limited by endpoint studies and tissue collection. We have successfully developed a unique double-transgenic mouse model, which combines the polyoma virus middle T oncogene (PyVT), a model of human breast cancer, with a nuclear factor-kappaB (NF-kB) reporter model (NGL). PyVT mice spontaneously form primary mammary fat pad tumors, with 80% of mice developing metastatic lesions in the lungs. The reporter is luciferase, driven by NF-kB activation, to provide quantitative bioluminescent measurements of spatially-specific NF-kB activity. The resulting double transgenic (PyNGL) mice were monitored using in vivo imaging systems (IVIS) multiple times, before and after treatment, to quantitatively and spatially characterize NF-kB therapeutic response, both locally and systemically. High intensity focused ultrasound (HIFU) was guided by magnetic resonance (MR) thermometry for tumor-localized hyperthermia which was dosage-controlled by the thermal feedback MR guided HIFU (MRgHIFU) system. A single primary mammary tumor in each PyNGL was treated with sub-ablative hyperthermia of 42⁰ for up to 25 minutes. Pre-treatment IVIS measurements were collected to determine baseline NF-kB activity within each mouse, followed by IVIS imaging every twelve hours after treatment until tissue collection. Treated tumor, contralateral (untreated) tumors, and spleens were collected for immunophenotyping and cytokine analysis using flow cytometry. PyNGL mice exhibited decreased NF-kB activity, spatially correlated with HIFU treatment, as early as 12 hours. In contrast, NF-kB increased in contralateral tumors as early as 24 hours following treatment. Immunophenotyping reveals changes in tumor infiltrating leukocyte populations in HIFU-treated and contralateral tumors, including quantity and phenotype of T cells. The unique murine model developed allowed quantitative, temporal characterization of local and distant tumor response to MFgHIFU. The spontaneous, metastatic nature of PyVT tumor formation in concert with the non-destructive, spatiotemporal imaging capabilities of the NF-kB reporter transgene make the PyNGL model an unparalleled tool for research in metastatic disease. Citation Format: Mary Dockery, Megan Poorman, Stephanie Dudzinski, Whitney Barham, Vandiver Chaplin, Ryan Spears, Jiro Kusunose, Fiona Yull, Charles Caskey, William Grissom, Todd Giorgio. Novel NF-kB reporter murine model of spontaneous, metastatic breast cancer for spatiotemporal monitoring of local and systemic therapeutic response. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A06.

While Magnetic Resonance Thermometry (MRT) has been extensively utilized for non-invasive temperature measurement, there is limited data on the use of high field (≥7T) scanners for this purpose. MR-guided Focused Ultrasound (MRgFUS) is a promising non-invasive method for localized hyperthermia and drug delivery. MRT based on the temperature sensitivity of the proton resonance frequency (PRF) has been implemented in both a tissue phantom and in vivo in a mouse Met-1 tumor model, using partial parallel imaging (PPI) to speed acquisition. An MRgFUS system capable of delivering a controlled 3D acoustic dose during real time MRT with proportional, integral, and derivative (PID) feedback control was developed and validated. Real-time MRT was validated in a tofu phantom with fluoroptic temperature measurements, and acoustic heating simulations were in good agreement with MR temperature maps. In an in vivo Met-1 mouse tumor, the real-time PID feedback control is capable of maintaining the desired temperature with high accuracy. We found that real time MR control of hyperthermia is feasible at high field, and k-space based PPI techniques may be implemented for increasing temporal resolution while maintaining temperature accuracy on the order of 1°C.

Therapeutic ultrasound (US) can be noninvasively focused to activate drugs, ablate tumors and deliver drugs beyond the blood brain barrier. However, well-controlled guidance of US therapy requires fusion with a navigational modality, such as magnetic resonance imaging (MRI) or X-ray computed tomography (CT). Here, we developed and validated tissue characterization using a fusion between US and CT. The performance of the CT/US fusion was quantified by the calibration error, target registration error and fiducial registration error. Met-1 tumors in the fat pads of 12 female FVB mice provided a model of developing breast cancer with which to evaluate CT-based tissue segmentation. Hounsfield units (HU) within the tumor and surrounding fat pad were quantified, validated with histology and segmented for parametric analysis (fat: -300 to 0 HU, protein-rich: 1 to 300 HU, and bone: HU>300). Our open source CT/US fusion system differentiated soft tissue, bone and fat with a spatial accuracy of ∼1 mm. Region of interest (ROI) analysis of the tumor and surrounding fat pad using a 1 mm(2) ROI resulted in mean HU of 68±44 within the tumor and -97±52 within the fat pad adjacent to the tumor (p150 mm(3), respectively. Further, CT mapped bone-soft tissue interfaces near the acoustic beam during real-time imaging. Combined CT/US is a feasible method for guiding interventions by tracking the acoustic focus within a pre-acquired CT image volume and characterizing tissues proximal to and surrounding the acoustic focus.

Mild hyperthermia is increasingly important for the activation of temperature-sensitive drug delivery vehicles. Noninvasive ultrasound thermometry based on a 2-D speckle tracking algorithm was examined in this study. Here, a commercial ultrasound scanner, a customized co-linear array transducer, and a controlling PC system were used to generate mild hyperthermia. Because the co-linear array transducer is capable of both therapy and imaging at widely separated frequencies, RF image frames were acquired during therapeutic insonation and then exported for off-line analysis. For in vivo studies in a mouse model, before temperature estimation, motion correction was applied between a reference RF frame and subsequent RF frames. Both in vitro and in vivo experiments were examined; in the in vitro and in vivo studies, the average temperature error had a standard deviation of 0.7°C and 0.8°C, respectively. The application of motion correction improved the accuracy of temperature estimation, where the error range was -1.9 to 4.5°C without correction compared with -1.1 to 1.0°C following correction. This study demonstrates the feasibility of combining therapy and monitoring using a commercial system. In the future, real-time temperature estimation will be incorporated into this system.

The fusion of computed tomography (CT) and ultrasound (US) imaging has recently been examined for guiding interventional procedures such as biopsy, local drug delivery and radiofrequency ablation. During combined CT/US procedures, US facilitates real time visualization of blood flow and tissue structure, while CT provides a three-dimensional view of an extended region and is capable of differentiating tissue types based on Hounsfield units (HU). Our laboratory is working to extend this fusion to include therapeutic US co-registered with CT. Here, we demonstrate the ability to differentiate fat, protein-rich regions, and bone with CT by comparing segmented CT images to histology. Then we present methods for semi-automated registration between tracked US and CT with the objective of imaging and guiding acoustic therapy in small animals. A calibration phantom and fiducial markers were used to generate accurate transformation between the real-time US and pre-acquired CT image stack. The mean error in transducer calibration was 0.7±0.5mm with a maximum error of 1.75mm. Target registration error (TRE) of 1.98+/−1.12mm was observed between US and CT images of a dual-modality phantom. The combined CT/US system provides CT-based guidance with enough accuracy to plan and guide US-induced hyperthermia.

When microbubble contrast agents are driven by ultrasound, the transport of drugs and particles across cell membranes and blood vessel walls is enhanced. While a wide range of acoustic parameters enhance delivery, the acoustic parameters that maximize delivery while simultaneously minimizing biological effects have not been fully characterized. Here, we use a gel phantom with a Young's modulus similar to that of tissue to directly observe bubble interaction with the gel surface during insonation. Using parameters relevant to diagnostic imaging and drug delivery, we observe fluid jets that impinge on the surface and tunnels that follow the sound beam axis.

A commercial ultrasound scanner, a customized co-linear array ultrasound transducer and a real-time temperature feedback controller have been combined to generate controlled mild hyperthermia. Previous studies have shown that single beam-based image-guided local drug delivery can improve the treatment of small tumors. In this study, a scanned-beam based insonation mode was developed to achieve uniform therapy over a larger region. A color-region of interest (ROI) was used to define the scanned area, while periodic B-mode imaging was used to visualize the tumor. In this study, a thermal strain algorithm was also examined for temperature estimation and this algorithm will be applied for temperature control in the future.

Insonified microbubbles were observed in vessels within a gel with a Young’s modulus similar to that of tissue, demonstrating shape instabilities, liquid jets, and the formation of small tunnels. In this study, tunnel formulation occurred in the direction of the propagating ultrasound wave, where radiation pressure directed the contact of the bubble and gel, facilitating the activity of the liquid jets. Combinations of ultrasonic parameters and microbubble concentrations that are relevant for diagnostic imaging and drug delivery and that lead to tunnel formation were applied and the resulting tunnel formation was quantified.

Microbubble contrast agents and the associated imaging systems have developed over the past twenty-five years, originating with manually-agitated fluids introduced for intra-coronary injection. Over this period, stabilizing shells and low diffusivity gas materials have been incorporated in microbubbles, extending stability in vitro and in vivo. Simultaneously, the interaction of these small gas bubbles with ultrasonic waves has been extensively studied, resulting in models for oscillation and increasingly sophisticated imaging strategies. Early studies recognized that echoes from microbubbles contained frequencies that are multiples of the microbubble resonance frequency. Although individual microbubble contrast agents cannot be resolved—given that their diameter is on the order of microns—nonlinear echoes from these agents are used to map regions of perfused tissue and to estimate the local microvascular flow rate. Such strategies overcome a fundamental limitation of previous ultrasound blood flow strategies; the previous Doppler-based strategies are insensitive to capillary flow. Further, the insonation of resonant bubbles results in interesting physical phenomena that have been widely studied for use in drug and gene delivery. Ultrasound pressure can enhance gas diffusion, rapidly fragment the agent into a set of smaller bubbles or displace the microbubble to a blood vessel wall. Insonation of a microbubble can also produce liquid jets and local shear stress that alter biological membranes and facilitate transport. In this review, we focus on the physical aspects of these agents, exploring microbubble imaging modes, models for microbubble oscillation and the interaction of the microbubble with the endothelium.

Mechanisms for permeability enhancement using contrast ultrasound are not fully understood, although the successful use of contrast agents for drug and gene delivery has been demonstrated. Here, we aim to elucidate mechanisms for vascular permeability enhancement by directly observing microbubble contrast agents in a tissue-like gel phantom. The phantom used in these experiments allows us to flow microbubbles through a small channel (230 mum in diameter) and observe microbubble interaction with a compliant wall during an acoustic pulse at center frequencies ranging from 1 MHz to 2.25 MHz using laser-based strobe microscopy. The phantom is made of agarose gel that has similar rigidity to in vivo tissue. During a 1-MHz pulse with peak rarefactional pressure of 1.2 MPa and 10 msec pulse duration, fluid jets were observed as the bubbles disrupted the gel and created tunnels beyond the lumen of the flow vessel. The degree of gel disruption was greatly reduced using short pulse length (10 musec) and a pulse repetition frequency relevant to diagnostic imaging (10 kHz), despite matched duty cycle. Using a transmission frequency of 2.25 MHz (pulse duration of 10 msec), the amount of damage increased as bubble concentration increased from no gel disruption at diagnostic concentrations (1 times 105 bubble/mL) to significant disruption at high concentrations (1 times 107 bubbles/mL). Observations of microbubbles and their effects on a gel phantom help identify the mechanisms and important parameters for optimizing drug delivery with contrast agents.

Objectives Low-power multipulse contrast ultrasound imaging provides a promising tool to quantify angiogenesis noninvasively when used with contrast agents targeted to vascular markers expressed by the angiogenic endothelium. Targeted ultrasound contrast agents, with a diameter on the order of micrometers, cannot extravasate and therefore are targeted solely to receptors expressed by the vascular endothelium. The aim of this study was to evaluate the potential of a low-power multipulse imaging sequence, CadenceTM contrast pulse sequencing (CPS), combined with targeted contrast agents to quantify angiogenesis. Material and Methods Targeted microbubbles were prepared by conjugating echistatin via biotin–avidin linkage to the surface of a phospholipid microbubble shell. The density of echistatin present on the shell was confirmed with flow-cytometry and quantified by total fluorescence. The binding of targeted microbubbles was evaluated in vitro by quantifying the adherence of targeted microbubbles to rat aortic endothelial cells, compared with control (nontargeted) microbubbles. The circulation time and adherence of targeted microbubbles was evaluated in vivo in a Matrigel model in rats and compared with control microbubbles using CPS in addition to a destructive ultrasound pulse. Results Using only the low-power CPS pulse, the echo intensity produced in the neovasculature of the Matrigel pellet was significantly greater with targeted microbubbles than with the control contrast agent (p

Detailed temporal characterization of in vivo response to anti-tumor immunotherapies are limited by the frequency and volume of tissue that can be practically collected. We have successfully generated a novel double-transgenic murine model, which incorporates a nuclear factor-kappaB (NF-kB) reporter transgene (NGL) into the polyoma virus middle T oncogene (PyVT) model, a widely accepted transgenic mouse with spontaneous tumor formation and disease progression comparable to that of human breast cancer. The reporter transgene uses luciferase to quantitatively indicate NF-kB activation, which is central to immunomodulatory inflammation. In vivo imaging systems (IVIS) were used for luminescent imaging of luciferase activity as a spatial reporter of NF-kB activation that was carried out repeatedly on individual double transgenic (Py-NGL) mice. Magnetic resonance thermometry (MRT) was used to guide therapeutic, high intensity focused ultrasound (HIFU) for quantitatively repeatable, sub-ablative thermal dosing as an inflammatory stimulus. Py-NGL mice were treated with the MRT-HIFU system at a sub-ablative level of 42 °C for up to 25 minutes. Mice were imaged with IVIS before treatment and every twelve hours after treatment until the collection of tissues. The untreated tumors, treated tumors, and spleens were collected, digested, and quantitatively analyzed with flow cytometry for immunophenotyping and cytokine expression. Py-NGL mice have a peak in NF-kB activation, spatially correlated with the administration of MRT-guided HIFU, between 48-96 hours post treatment. Preliminary measures of inflammation by NF-kB activity demonstrate up to a 20-fold increase from baseline in the HIFU treated tissue. Following treatment, NF-kB activity increased from baseline as early as 24 hours and reached a peak between 48 and 96 hours, consistent with recruitment and activation of inflammatory cell phenotypes as a consequence of MRT-HIFU treatment. Elevated NF-kB activity remained localized in the HIFU-treated tissue throughout the measurement period, and smoothly declined towards, but remained above, baseline levels following the activation peak. Immunophenotyping data reveals changes in immune cell infiltrates following treatment, including the number and phenotypes of T cells and macrophages. This novel murine model enabled real-time, quantitative live-animal imaging of spatially specific, time-dependent NF-kB activation. Measurement of NF-kB response to controlled-dose HIFU provides the first opportunity to correlate induced inflammation with immune cell and cytokine characterization of the tumors. The objective is to quantitatively elucidate the spatiotemporal immunologic response to inflammatory stimulus in tumors. In this way, therapeutic methods based on elevating inflammation in tumors can be assessed for reversal of the immunosuppressive microenvironment and the generation of functional anti-tumor immunotherapy. Citation Format: Mary D. Dockery, Megan E. Poorman, Vandiver L. Chaplin, Ryan A. Spears, Charles F. Caskey, William A. Grissom, Todd D. Giorgio. Real-time in vivo characterization of spatiotemporal immunotherapeutic response to high intensity focused ultrasound with a novel NF-kB reporter model of human breast cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A086.

The use of ultrasound contrast agents for gene and drug delivery has shown much promise in many recent studies, while simultaneously raising questions regarding the safety of methods used. There is currently no consensus on the optimal safe operating regime, since the mechanisms for contrast- enhanced drug delivery are not well-understood. Here, the mechanisms for increased permeability are investigated by using high-speed microscopy to directly observe microbubbles in a vessel phantom and ex vivo preparation. The vessel phantom was a small block (30 mm times 20 mm times 3 mm) of 0.75% agarose gel with a 200-mum diameter cylindrical channel along the length of the block. For ex vivo work, the preparation used was an excised rat cecum with a cannulated ileocolic vein to allow for microbubble perfusion. Images of microbubble oscillation were acquired using a strobe imaging system with a 30 nsec flash provided by a copper vapor laser. Images of vessel wall disruption and tunnels formed in the wall were acquired with 1, 2.25, and 5 MHz center frequencies using microbubble concentration, duty cycle, and rarefactional pressures that were similar to those used for in vivo drug delivery experiments. Observations from in vitro and ex vivo studies indicate that expansion of microbubbles within small vessels is smaller than in an infinite fluid and that bubbles persist over many transmitted cycles and pulses. In vitro studies over a long pulse train indicate that microbubbles create pores within the tissue phantom and travel long distances along the beam axis. The width and depth of tunnels created by microbubbles at varied frequencies indicate a stronger dependence on insonation frequency than suggested by mechanical index.

A theoretical model was established to study interaction between ultrasound contrast agents (UCAs, called microbubbles) and compliant microvessels. The predicted microbubble expansion and vessel wall displacement are in good agreement with the ex vivo experimental results using the rat cecum vessels for ultrasound with a center frequency of 1 MHz and a PRP (peak rarefactional pressure) of 800 kPa. We demonstrate that compared to the bubble oscillation within an infinite liquid or within a 200-micron tube, bubble maximum expansion within the small vessels is substantially reduced. With two-cycle 1 MHz, 0.8 MPa PRP ultrasound, the relative expansion of the shelled microbubbles with a diameter of 4 and 7.2-mum estimated by the Rayleigh-Plesset equation is 4.1 and 3.0, respectively, while within a 14-mum vessel, we observed that the bubble expansion was 2.6 and 1.3-fold, respectively. Our model predicted that the corresponding relative expansion is 2.6 and 1.8, respectively. With 0.8 MPa PRP, 1 MHZ ultrasound, a 4 mum bubble within a 25 mum vessel can displace the vessel wall up to 2 microns and induce a circumferential stress near 36 kPa.

To characterize the effect of low-frequency contrast material-enhanced ultrasound on the vascular endothelium and to determine the parameters and techniques required to deliver a therapeutic agent by using the chorioallantoic membrane (CAM) model.All in vivo animal procedures were conducted with institutional Animal Care and Use Committee approval. Extravasation of 8.5-nm-diameter fluorescein isothiocyanate-labeled dextran was evaluated in the vasculature of a chick CAM model. Intravital microscopy was performed during contrast-enhanced ultrasound exposure (1.00 or 2.25 MHz); results were compared with results of electron microscopy of the insonated regions. Data acquired after insonation with greater mechanical stress (n = 30 animals) (mechanical index [MI]1.3) and with lower mechanical stress (n = 86 animals) (MI1.13) were compared with measurements in control conditions (n = 46 animals). The diameter of affected vessels; number of extravasation sites; extravasation rate, area, and location; and changes in endothelial cells and basement membrane were evaluated. Differences were tested with analysis of variance or the Student t test.After ultrasound application, convective transport of the model drug was observed through micron-sized openings with a mean fluid velocity of 188.6 microm/sec in the low-stress class and 362.5 microm/sec in the high-stress class. Electron microscopy revealed micron-sized focal endothelial gaps and disseminated blebs, vacuoles, and filopodia extending across tens of microns. The threshold pressure for extravasation was 0.5 MPa for a transmitted center frequency of 1.00 MHz (MI = 0.5) and 1.6 MPa for a frequency of 2.25 MHz (MI = 1.06); thus, the frequency dependence of the threshold was not predicted simply by the MI.Low-frequency contrast-enhanced ultrasound can increase vascular permeability and result in convective extravasation of an 8.5-nm-diameter model drug.

A novel form of acoustic modulation of light from an incoherent source has been detected in water as well as in turbid media. We demonstrate that patterns of modulated light intensity appear to propagate as the optical shadow of the density variations caused by ultrasound within an illuminated ultrasonic focal zone. This pattern differs from previous reports of acousto-optical interactions that produce diffraction effects that rely on phase shifts and changes in light directions caused by the acoustic modulation. Moreover, previous studies of acousto-optic interactions have mainly reported the effects of sound on coherent light sources via photon tagging, and/or the production of diffraction phenomena from phase effects that give rise to discrete sidebands. We aimed to assess whether the effects of ultrasound modulation of the intensity of light from an incoherent light source could be detected directly, and how the acoustically modulated (AOM) light signal depended on experimental parameters. Our observations suggest that ultrasound at moderate intensities can induce sufficiently large density variations within a uniform medium to cause measurable modulation of the intensity of an incoherent light source by absorption. Light passing through a region of high intensity ultrasound then produces a pattern that is the projection of the density variations within the region of their interaction. The patterns exhibit distinct maxima and minima that are observed at locations much different from those predicted by Raman-Nath, Bragg, or other diffraction theory. The observed patterns scaled appropriately with the geometrical magnification and sound wavelength. We conclude that these observed patterns are simple projections of the ultrasound induced density changes which cause spatial and temporal variations of the optical absorption within the illuminated sound field. These effects potentially provide a novel method for visualizing sound fields and may assist the interpretation of other hybrid imaging methods.

Quantification of the wideband signal associated with bubble collapse is desirable to measure cavitation dosage during microbubble therapy. We have developed a cavitation detection algorithm based on time-frequency analysis with the Stockwell transform. This transform is ideal since it isolates cavitation transients in time and frequency at much higher resolution than other time-frequency methods. In this study, we acquired simultaneous high-speed images and acoustic signals from bubbles undergoing oscillation during acoustic pulses commonly used for permeability enhancement (Transmit: 1.5 MHz, 10-cycle, peak negative pressure range 150–500 kPa, Receive: 15 MHz cavitation detector). Above 200 kPa, linescan vs time images showed relative bubble expansion greater than 2 and echoes contained a wideband signal that coincided with bubble collapse. The Stockwell transform of detected echoes revealed wideband spectrums localized to the time of bubble collapse. After filtering below 4 MHz to reject linear echoes, the root mean square (RMS) of the spectral amplitude was obtained from the time-frequency decomposition, yielding peaks at cavitation transients. The magnitude of the Fourier transform of the RMS signal at 1.5 MHz provided a sensitive indicator for bubble collapse and differed significantly across pressures where cavitation occurred (p < 0.05,t-test). The proposed measurement provides a sensitive metric for cavitation activity during microbubble therapy.

Fusion of computed tomography (CT) and ultrasound (US) imaging is currently used for guiding interventional procedures, including biopsy, local drug delivery, and radiofrequency ablation. While US facilitates real‐time visualization of blood flow and tissue structure, CT provides a 3‐D view of an extended region of interest, can differentiate tissue types, and operate in a fluoroscopic mode for catheter placement. Our laboratory is working to extend this fusion to include US therapy and imaging co‐registered with CT. Here, we present methods for semi‐automated registration between tracked US and CT with the objective of imaging and guiding acoustic therapy in small animals. A calibration phantom and fiducial markers were used to generate accurate transformation between the real‐time US and pre‐acquired CT image stack. The mean error in the transducer calibration was 1.1 ± 0.76 mm with a maximum error of 3.1 mm. A target registration error of 1.7 ± 0.9 mm was observed in US and CT images of a dual‐modality...

A multi‐disciplinary approach is required to engineer ultrasound contrast agents for drug delivery and imaging. These micrometer‐sized bubbles oscillate in response to acoustic energy, emitting non‐linear echoes that can be differentiated from the tissue when imaged. If large enough, the oscillations generate perturbations capable of affecting the region surrounding the bubble. Here, microbubble activity is observed in scenarios that mimic those found in vivo to provide intuition about the mechanisms for contrast‐enhanced imaging and drug delivery. High‐speed imaging of microbubble oscillation at 1 MHz within ex vivo vessels reveals that microbubble expansion is significantly reduced, and the constrained oscillation imparts a force capable of displacing the surrounding vessel wall. [Caskey et al., 122, 1191‐1200.] Bubble oscillation near a tissue‐mimicking gel boundary shows fluid jets impinging on and disrupting the gel surface at 1, 2.25, and 5 MHz. [Caskey et al., 125, EL183–EL189.] The bubble concentr...

Ultrasound contrast agents are often used to measure flow rate in the microvasculature; however, the oscillation of these agents in capillary-sized tubes has not been directly observed. Here, oscillations of microbubbles are examined in microvessel phantoms with diameters similar to those of capillaries. High-speed camera images demonstrate the effects of ultrasonic pressure and tube diameter and length on microbubble expansion and fragmentation occurrence. Microbubble displacement due to radiation force is also demonstrated in a phantom microvessel.