Search

Transcranial focused ultrasound (FUS) stimulation under MRI guidance, coupled with functional MRI (fMRI) monitoring of effects, offers a precise, noninvasive technology to dissect functional brain circuits and to modulate altered brain functional networks in neurological and psychiatric disorders. Here we show that ultrasound at moderate intensities modulated neural activity bi-directionally. Concurrent sonication of somatosensory areas 3a/3b with 250 kHz FUS suppressed the fMRI signals produced there by peripheral tactile stimulation, while at the same time eliciting fMRI activation at inter-connected, off-target brain regions. Direct FUS stimulation of the cortex resulted in different degrees of BOLD signal changes across all five off-target regions, indicating that its modulatory effects on active and resting neurons differed. This is the first demonstration of the dual suppressive and excitative modulations of FUS on a specific functional circuit and of ability of concurrent FUS and MRI to evaluate causal interactions between functional circuits with neuron-class selectivity.

Focused ultrasound (FUS) can concentrate energy noninvasively through the skull to elicit neuromodulation. Although the natural response of neurons to ultrasound has been explored, acoustically active drug loaded nanoparticles have the benefit of yielding a pharmacological response that may be more predictable than the effects of FUS alone. Researchers have developed drug delivery vehicles that release small molecule neuromodulatory drugs without opening blood brain barrier. It is desirable to increase drug release, and drug release typically increases with increased peak negative pressure (PNP). However, the likelihood of undesirable bioeffects, such as cavitation-induced focal hemorrhages increases with increasing PNP. Here, we examined echoes from propofol-containing nanoparticles during ultrasound exposures known to uncage propofol from the vehicle. Our goals were to 1) increase drug release without generating cavitation and 2) identify echoes that may be related to propofol release.

The aim of this study was to improve the sensitivity of magnetic resonance-acoustic radiation force imaging (MR-ARFI) to minimize pressures required to localize focused ultrasound (FUS) beams, and to establish safe FUS localization parameters for ongoing ultrasound neuromodulation experiments in living non-human primates. We developed an optical tracking method to ensure that the MR-ARFI motion-encoding gradients (MEGs) were aligned with a single-element FUS transducer and that the imaged slice was prescribed at the optically tracked location of the acoustic focus. This method was validated in phantoms, which showed that MR-ARFI-derived displacement sensitivity is maximized when the MR-ARFI MEGs were maximally aligned with the FUS propagation direction. The method was then applied in vivo to acquire displacement images in two healthy macaque monkeys (M fascicularis) which showed the FUS beam within the brain. Temperature images were acquired using MR thermometry to provide an estimate of in vivo brain temperature changes during MR-ARFI, and pressure and thermal simulations of the acoustic pulses were performed using the k-Wave package which showed no significant heating at the focus of the FUS beam. The methods presented here will benefit the multitude of transcranial FUS applications as well as future human applications.

High intensity focused ultrasound (FUS) is a noninvasive technique for treatment of tissues that can lie deep within the body. There is a need for methods to rapidly and quantitatively map FUS pressure beams for quality assurance and accelerate development of FUS systems and techniques. However, conventional ultrasound pressure beam mapping instruments, including hydrophones and optical techniques, are slow, not portable, and expensive, and most cannot map beams at actual therapeutic pressure levels. Here, a rapid projection imaging method to quantitatively map FUS pressure beams based on continuous-wave background-oriented schlieren (CW-BOS) imaging is reported. The method requires only a water tank, a background pattern, and a camera and uses a multi-layer deep neural network to reconstruct two-dimensional root-mean-square (RMS) projected pressure maps that resolve the ultrasound propagation dimension and one lateral dimension. In this work, the method was applied to collect beam maps over a 3 × 1 cm(2) field-of-view with 0.425 mm resolution for focal pressures up to 9 MPa. Results at two frequencies and comparisons to hydrophone measurements show that CW-BOS imaging produces high-resolution quantitative RMS projected FUS pressure maps in under 10 s, the technique is linear and robust to beam rotations and translations, and it can map aberrated beams.

Ultrasound is gaining traction as a neuromodulation method due to its ability to remotely and non-invasively modulate neuronal activity with millimeter precision. However, there is little consensus about optimal ultrasound parameters required to elicit neuromodulation and how specific parameters drive mechanisms that underlie ultrasound neuromodulation. We address these questions in this work by performing a study to determine effective ultrasound parameters in a transgenic mouse brain slice model that enables calcium imaging as a quantitative readout of neuronal activity for ultrasound neuromodulation. We report that (1) calcium signaling increases with the application of ultrasound; (2) the neuronal response rate to ultrasound is dependent on pulse repetition frequency (PRF); and (3) ultrasound can reversibly alter the inhibitory effects of tetrodotoxin (TTX) in pharmacological studies. This study offers mechanistic insight into the PRF dependence of ultrasound neuromodulation and the nature of ultrasound/ion channel interaction.

Microbubble contrast agents are widely used in ultrasound imaging and therapy, typically with transmission center frequencies in the MHz range. Currently, an ultrasound center frequency near 250 kHz is proposed for clinical trials in which ultrasound combined with microbubble contrast agents is applied to open the blood brain barrier, since at this low frequency focusing through the human skull to a predetermined location can be performed with reduced distortion and attenuation compared to higher frequencies. However, the microbubble vibrational response has not yet been carefully evaluated at this low frequency (an order of magnitude below the resonance frequency of these contrast agents). In the past, it was assumed that encapsulated microbubble expansion is maximized near the resonance frequency and monotonically decreases with decreasing frequency. Our results indicated that microbubble expansion was enhanced for 250 kHz transmission as compared with the 1 MHz center frequency. Following 250 kHz insonation, microbubble expansion increased nonlinearly with increasing ultrasonic pressure, and was accurately predicted by either the modified Rayleigh–Plesset equation for a clean bubble or the Marmottant model of a lipid-shelled microbubble. The expansion ratio reached 30-fold with 250 kHz at a peak negative pressure of 400 kPa, as compared to a measured expansion ratio of 1.6 fold for 1 MHz transmission at a similar peak negative pressure. Further, the range of peak negative pressure yielding stable cavitation in vitro was narrow (~100 kPa) for the 250 kHz transmission frequency. Blood brain barrier opening using in vivo transcranial ultrasound in mice followed the same trend as the in vitro experiments, and the pressure range for safe and effective treatment was 75–150 kPa. For pressures above 150 kPa, inertial cavitation and hemorrhage occurred. Therefore, we conclude that (1) at this low frequency, and for the large oscillations, lipid-shelled microbubbles can be approximately modeled as clean gas microbubbles and (2) the development of safe and successful protocols for therapeutic delivery to the brain utilizing 250 kHz or a similar center frequency requires consideration of the narrow pressure window between stable and inertial cavitation.

Plane-wave ultrasound contrast imaging offers a faster, less destructive means for imaging microbubbles compared with traditional ultrasound imaging. Even though many of the most acoustically responsive microbubbles have resonant frequencies in the lower-megahertz range, higher frequencies (>3 MHz) have typically been employed to achieve high spatial resolution. In this work we implement and optimize low-frequency (1.5-4 MHz) plane-wave pulse inversion imaging on a commercial, phased-array imaging transducer in vitro and illustrate its use in vivo by imaging a mouse xenograft model. We found that the 1.8-MHz contrast signal was about four times that acquired at 3.1 MHz on matched probes and nine times greater than echoes received on a higher-frequency probe. Low-frequency imaging was also much more resilient to motion. In vivo, we could identify sub-millimeter vasculature inside a xenograft tumor model and easily assess microbubble half-life. Our results indicate that low-frequency imaging can provide better signal-to-noise because it generates stronger non-linear responses. Combined with high-speed plane-wave imaging, this method could open the door to super-resolution imaging at depth, while high power pulses could be used for image-guided therapeutics.

Thermal ablation by ultrasound is being explored as a local therapy for cancers of soft tissue, such as the liver or breast. One challenge for these treatments are off-target effects, including heating outside of the intended region or skin burns. Improvements in heating efficiency can mitigate these undesired outcomes, and here, we describe methods for using phase-shift nanodroplets (PSNDs) with multi-focus sonications to enhance volumetric ablation and ablation efficiency at constant powers while increasing the pre-focal temperature by less than 6 [Formula: see text]C. Multi-focus ablation with 4 foci performed the best and achieved a mean ablation volume of 120.2 ± 22.4 mm3 and ablation efficiency of 0.04 mm3 J-1 versus an ablation volume of 61.2 ± 21.16 mm3 and ablation efficiency of 0.02 mm3 J-1 in single focus case. The combined use of PSNDs with multi-focal ultrasound presented here is a new approach to increasing ablation efficiency while reducing off-target effects and could be generally applied in various focused ultrasound thermal ablation treatments.

Focused ultrasound is a promising method for non-invasive neural stimulation in the brain. Early studies have shown that neurological conditions such as essential tremors can be effectively treated with ultrasound ablation, while sub-thermal stimulation is being explored to probe functional connections in the brain and may have therapeutic benefits in a variety of neurological diseases. Although systems for transcranial ablation exist for human subjects, they are not optimized for cortical stimulation or studies in non-human primates. Here we report on the design and characterization of a new spherically-focused randomized sparse array transducer.

Non-invasive, quantitative methods to assess the properties of biological tissues are needed for many therapeutic and tissue engineering applications. Magnetic resonance elastography (MRE) has historically relied on external vibration to generate periodic shear waves. In order to focally assess a biomaterial or to monitor the response to ablative therapy, the interrogation of a specific region of interest by a focused beam is desirable and transient MRE (t-MRE) techniques have previously been developed to accomplish this goal. Also, strategies employing a series of discrete ultrasound pulses directed to increasing depths along a single line-of-sight have been designed to generate a quasi-planar shear wave. Such 'supersonic' excitations have been applied for ultrasound elasticity measurements. The resulting shear wave is higher in amplitude than that generated from a single excitation and the properties of the media are simply visualized and quantified due to the quasi-planar wave geometry and the opportunity to generate the wave at the site of interest. Here for the first time, we extend the application of supersonic methods by developing a protocol for supersonic transient magnetic resonance elastography (sst-MRE) using an MR-guided focused ultrasound system capable of therapeutic ablation. We apply the new protocol to quantify tissue elasticity in vitro using biologically-relevant inclusions and tissue-mimicking phantoms, compare the results with elasticity maps acquired with ultrasound shear wave elasticity imaging (US-SWEI), and validate both methods with mechanical testing. We found that a modified time-of-flight (TOF) method efficiently quantified shear modulus from sst-MRE data, and both the TOF and local inversion methods result in similar maps based on US-SWEI. With a three-pulse excitation, the proposed sst-MRE protocol was capable of visualizing quasi-planar shear waves propagating away from the excitation location and detecting differences in shear modulus of 1 kPa. The techniques demonstrated here have potential application in real-time in vivo lesion detection and monitoring, with particular significance for image-guided interventions.

Background Mild-hyperthermia therapy (40–45 °C) with high-intensity focused ultrasound (HIFU) is a technique being considered in a number of different treatments such as thermally activated drug delivery, immune-stimulation, and as a chemotherapy adjuvant. Mechanical damage and loss of cell viability associated with HIFU-induced acoustic cavitation may pose a risk during these treatments or may hinder their success. Here we present a method that achieves mild heating and reduces cavitation by using a multi-focused HIFU beam. We quantify cavitation level and temperature rise in multi-focal sonications and compare it to single-focus sonications at the transducer geometric focus. Methods Continuous wave sonications were performed with the Sonalleve V2 transducer in gel phantoms and pork at 5, 10, 20, 40, 60, 80 acoustic watts for 30 s. Cavitation activity was measured with two ultrasound (US) imaging probes, both by computing the raw channel variance and using passive acoustic mapping (PAM). Temperature rise was measured with MR thermometry at 3 T. Cavitation and heating were compared for single- and multi-focal sonication geometries. Multi-focal sonications used four points equally spaced on a ring of either 4 mm or 8 mm diameter. Single-focus sonications were not steered. Results Multi-focal sonication generated distinct foci that were visible in MRI thermal maps in both phantoms and pork, and visible in PAM images in phantoms only. Cavitation activity (measured by channel variance) and mean PAM image value were highly correlated (r > 0.9). In phantoms, cavitation exponentially decreased over the 30-second sonication, consistent with depletion of cavitation nuclei. In pork, sporadic spikes signaling cavitation were observed with single focusing only. In both materials, the widest beam reduced average and peak cavitation level by a factor of two or more at each power tested when compared to a single focus. The widest beam reduced peak temperature by at least 10 °C at powers above 5 W, and created heating that was more spatially diffuse than single focus, resulting in more voxels in the mild heating (3–8 °C) range. Conclusions Multi-focal HIFU can be used to achieve mild temperature elevation and reduce cavitation activity.

Ultrasound is a rapidly advancing field with many emerging diagnostic and therapeutic applications. For diagnostics, new vascular targets are routinely identified and mature technologies are being translated to humans, while other recent innovations may bring about the creation of acoustic reporter genes and micron-scale resolution with ultrasound. As a cancer therapy, ultrasound is being explored as an adjuvant to immune therapies and to deliver acoustically or thermally active drugs to tumor regions. Ultrasound-enhanced delivery across the blood brain barrier (BBB) could potentially be very impactful for brain cancers and neurodegenerative diseases where the BBB often impedes the delivery of therapeutic molecules. In this minireview, we provide an overview of these topics in the field of ultrasound that are especially relevant to the interests of World Molecular Imaging Society.

INTRODUCTION Low intensity focused ultrasound has recently been identified as a novel method of stimulating targeted neurons. We hypothesized that ultrasound stimulation of the posterior tibial nerve would inhibit bladder contractions in an established rat model of rhythmic bladder contractions. METHODS Cyclical detrusor contractions were initiated by placing a transurethral catheter in female rats and infusing saline into the bladder. Transcutaneous ultrasound pulses were then delivered to the lower extremity of a rat (overlying the posterior tibial nerve) using a single element spherically focused 250 kHz transducer. Sixty-three cycles were repeated at 2 kHz for 300 ms at peak negative pressure of 900 kPa pulsed at 0.5 Hz. RESULTS We report successful suppression of bladder contractions using ultrasound stimulation in 10 animals. The average latency between the initiation of ultrasound and suppression of bladder contractions was 3 min 23 s (±51 s), the average time of contraction suppression was 13 min and 50 s (±2 min 25 s) and the average time from the end of ultrasound to return of contractions was 9 min 37 s (±2 min and 30 s). CONCLUSION In this work, we demonstrate the ability of targeted transcutaneous ultrasound to inhibit rhythmic bladder contractions in anesthetized rats. Due to its non-invasive nature and ease of application, we believe ultrasound mediated suppression of the micturition reflex is potentially an ideal outpatient treatment of overactive bladder and dysfunctional elimination.

Background MR-guided focused ultrasound or high-intensity focused ultrasound (MRgFUS/MRgHIFU) is a non-invasive therapeutic modality with many potential applications in areas such as cancer therapy, drug delivery, and blood-brain barrier opening. However, the large financial costs involved in developing preclinical MRgFUS systems represent a barrier to research groups interested in developing new techniques and applications. We aim to mitigate these challenges by detailing a validated, open-source preclinical MRgFUS system capable of delivering thermal and mechanical FUS in a quantifiable and repeatable manner under real-time MRI guidance. Methods A hardware and software package was developed that includes closed-loop feedback controlled thermometry code and CAD drawings for a therapy table designed for a preclinical MRI scanner. For thermal treatments, the modular software uses a proportional integral derivative controller to maintain a precise focal temperature rise in the target given input from MR phase images obtained concurrently. The software computes the required voltage output and transmits it to a FUS transducer that is embedded in the delivery table within the magnet bore. The delivery table holds the FUS transducer, a small animal and its monitoring equipment, and a transmit/receive RF coil. The transducer is coupled to the animal via a water bath and is translatable in two dimensions from outside the magnet. The transducer is driven by a waveform generator and amplifier controlled by real-time software in Matlab. MR acoustic radiation force imaging is also implemented to confirm the position of the focus for mechanical and thermal treatments. Results The system was validated in tissue-mimicking phantoms and in vivo during murine tumor hyperthermia treatments. Sonications were successfully controlled over a range of temperatures and thermal doses for up to 20 min with minimal temperature overshoot. MR thermometry was validated with an optical temperature probe, and focus visualization was achieved with acoustic radiation force imaging. Conclusions We developed an MRgFUS platform for small-animal treatments that robustly delivers accurate, precise, and controllable sonications over extended time periods. This system is an open source and could increase the availability of low-cost small-animal systems to interdisciplinary researchers seeking to develop new MRgFUS applications and technology.

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

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.

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.

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.

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

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.

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.