Transient acoustic vaporization signatures unique to low boiling point phase change contrast agents enable super-resolution ultrasound imaging without spatiotemporal filtering.

The unique activation signal of phase-change contrast agents (PCCAs or droplets) can be separated from the tissue signal and localized to generate super-resolution (SR) ultrasound (US) images. Lipid-shelled, perfluorocarbon PCCAs can be stochastically vaporized (activated) by a plane wave US transmission thereby enabling them to be used as separable targets for ultrasound localization microscopy. The unique signature of droplet vaporization imaging and the transient inherent nature of this signature increases signal contrast and therefore localization confidence, while the poor resolution of the low-frequency vaporization signal is overcome by the super-resolution result. Furthermore, our proposed PCCA SR technique does not require the use of user-dependent and flow-dependent spatio-temporal filtering via singular-value decomposition. Rather, matched filters selected by Fourier-domain analysis are able to identify and localize PCCA activations. Droplet SR was demonstrated in a crossed-microtube water phantom by localizing the activation signals of octafluoropropane nanodroplets (OFP, C3F8, -37 °C boiling point) to resolve 100 µm diameter fluorinated ethylene propylene tubes, which are ordinarily 35% smaller than the native diffraction-limited resolution of the imaging system utilized.

Variability in circulating gas emboli after a same scuba diving exposure.

PURPOSE: A reduction in ambient pressure or decompression from scuba diving can result in ultrasound-detectable venous gas emboli (VGE). These environmental exposures carry a risk of decompression sickness (DCS) which is mitigated by adherence to decompression schedules; however, bubbles are routinely observed for dives well within these limits and significant inter-personal variability in DCS risk exists. Here, we assess the variability and evolution of VGE for 2 h post-dive using echocardiography, following a standardized pool dive in calm warm conditions. METHODS: 14 divers performed either one or two (with a 24 h interval) standardized scuba dives to 33 mfw (400 kPa) for 20 min of immersion time at NEMO 33 in Brussels, Belgium. Measurements were performed at 21, 56, 91 and 126 min post-dive: bubbles were counted for all 68 echocardiography recordings and the average over ten consecutive cardiac cycles taken as the bubble score. RESULTS: Significant inter-personal variability was demonstrated despite all divers following the same protocol in controlled pool conditions: in the detection or not of VGE, in the peak VGE score, as well as time to VGE peak. In addition, intra-personal differences in 2/3 of the consecutive day dives were seen (lower VGE counts or faster clearance). CONCLUSIONS: Since VGE evolution post-dive varies between people, more work is clearly needed to isolate contributing factors. In this respect, going toward a more continuous evaluation, or developing new means to detect decompression stress markers, may offer the ability to better assess dynamic correlations to other physiological parameters.

Pulse sequences for uniform perfluorocarbon droplet vaporization and ultrasound imaging.

Phase-change contrast agents (PCCAs) consist of liquid perfluorocarbon droplets that can be vaporized into gas-filled microbubbles by pulsed ultrasound waves at diagnostic pressures and frequencies. These activatable contrast agents provide benefits of longer circulating times and smaller sizes relative to conventional microbubble contrast agents. However, optimizing ultrasound-induced activation of these agents requires coordinated pulse sequences not found on current clinical systems, in order to both initiate droplet vaporization and image the resulting microbubble population. Specifically, the activation process must provide a spatially uniform distribution of microbubbles and needs to occur quickly enough to image the vaporized agents before they migrate out of the imaging field of view. The development and evaluation of protocols for PCCA-enhanced ultrasound imaging using a commercial array transducer are described. The developed pulse sequences consist of three states: (1) initial imaging at sub-activation pressures, (2) activating droplets within a selected region of interest, and (3) imaging the resulting microbubbles. Bubble clouds produced by the vaporization of decafluorobutane and octafluoropropane droplets were characterized as a function of focused pulse parameters and acoustic field location. Pulse sequences were designed to manipulate the geometries of discrete microbubble clouds using electronic steering, and cloud spacing was tailored to build a uniform vaporization field. The complete pulse sequence was demonstrated in the water bath and then in vivo in a rodent kidney. The resulting contrast provided a significant increase (>15 dB) in signal intensity.

A comparative evaluation of ultrasound molecular imaging, perfusion imaging, and volume measurements in evaluating response to therapy in patient-derived xenografts.

Most pre-clinical therapy studies use the change in tumor volume as a measure for disease response. However, tumor size measurements alone may not reflect early changes in tumor physiology that occur as a response to treatment. Ultrasonic molecular imaging (USMI) and Dynamic Contrast Enhanced-Perfusion Imaging (DCE-PI) with ultrasound are two attractive alternatives to tumor volume measurements. Since these techniques can provide information prior to the appearance of gross phenotypic changes, it has been proposed that USMI and DCE-PI could be used to characterize response to treatment earlier than traditional methods. This study evaluated the ability of tumor volume measurements, DCE-PI, and USMI to characterize response to therapy in two different types of patient-derived xenografts (known responders and known non-responders). For both responders and non-responders, 7 animals received a dose of 30 mg/kg of MLN8237, an investigational aurora-A kinase inhibitor, for 14 days or a vehicle control. Volumetric USMI (target integrin:α av ß3) and DCE-PI were performed on day 0, day 2, day 7, and day 14 in the same animals. For USMI, day 2 was the earliest point at which there was a statistical difference between the untreated and treated populations in the responder cohort (Untreated: 1.20 ±â€…0.53 vs. Treated: 0.49 ±â€…0.40; p < 0.05). In contrast, statistically significant differences between the untreated and treated populations as detected using DCE-PI were not observed until day 14 (Untreated: 0.94 ±â€…0.23 vs. Treated: 1.31 ±â€…0.22; p < 0.05). Volume measurements alone suggested no statistical differences between treated and untreated populations at any readpoint. Monitoring volumetric changes is the "gold standard" for evaluating treatment in pre-clinical studies, however, our data suggests that volumetric USMI and DCE-PI may be used to earlier classify and robustly characterize tumor response.

WE-C-218-01: Ultrasound Contrast Agents.

Medical ultrasound has long been used in clinical applications both as a primary modality and as a supplement to other diagnostic procedures. The basis for ultrasound imaging is the transmission of high frequency (megaHertz) sound waves that propagate through tissue. These sound waves backscatter from the interfaces between tissue components with different acoustic properties and are detected by the imaging system, allowing the creation of images based on tissue characteristics and spatial location. Thus, traditional ultrasound has focused primarily on the imaging of anatomical structures and analysis of blood flow in large vessels. Unfortunately, blood is a weak scatterer, which can make vascular diagnostic applications (example: echocardiography) challenging especially with larger patients. Contrast agents help to improve on this shortcoming by enhancing the visualization of blood flow, thus improving the quality of diagnostics. The use of contrast agents for ultrasound was first reported in 1968 when Gramiak and Shah discovered that there was an increased backscatter of ultrasound caused by injected microbubbles. This is because the mismatch in acoustic impedance (a function of an object's density and compressibility) between the microbubble gas core and blood (or tissue) is several orders of magnitude, which results in substantially higher scattering from a bubble than an equivalent volume of tissue or blood. Additionally, microbubbles oscillate in response to an ultrasound field, and respond non-linearly to acoustic pulses even at low energies, unlike tissue. The non-linear property of microbubbles in an ultrasound field allows for the use of various pulsing and signal processing strategies to detect the backscattered signal from contrast agents and segment it from tissue, thus providing a high contrast-to- noise ratio. Due to these unique acoustic properties, a clinical ultrasound system can detect even single microbubble contrast agents, providing exquisite sensitivity and the ability to perform advanced diagnostic procedures. Over the last several decades, ultrasound contrast agents have been improved for enhanced stability and increased persistence times. Although preclinical studies as well as clinical use in Europe and Asia strongly suggest that the use of contrast ultrasound can substantially improve diagnostic capabilities in both cardiology and radiology applications, contrast use in the US is still very limited. Obstacles to the widespread use of microbubbles include safety concerns, the need for optimization of approaches for contrast use, and general understanding of their potential by physicians. This course covers the basic principles of contrast agents used in ultrasound imaging including their stability, shell properties and their behavior within an acoustic field. In addition, we will cover many new techniques that are being evaluated in preclinical studies including: p er fus ion-based techniques, molecular imaging, gene therapy, drug delivery, and acoustic angiography. Finally, basic safety concerns and biological effects will be reviewed. LEARNING OBJECTIVES: 1. Understand the basic principles of ultrasound contrast agents a. What are microbubble contrast agents? b. Properties of microbubbles c. Safety concerns and biological effects 2. Understand basic contrast imaging techniques a. Harmonic and suharmonic imaging techniques b. Pulse inversion techniques 3. Understand the use of contrast agents in various vascular applications a. Traditional methods (Cardiovascular, Abdominal) b. Advanced perfusion imaging techniques 4. Understand the role of contrast agents in preclinical applications a. Ultrasound molecular imaging b. Gene therapy c. Drug delivery d. Acoustic angiography.

Characterisation of polymer shelled microbubbles in wall less flow phantom using high frequency ultrasound and video microscopy.

A high frequency ultrasound pulse echo system and a video microscope were combined to investigate the relationship between backscatter from polymer shelled ultrasound contrast agents (UCAs) and their diameter. Individual UCAs (manufactured by Point Biomedical or Philips Research) were imaged while being sonicated with 40 MHz tone bursts. The backscatter magnitude produced by the Philips UCAs was proportional to UCA size, which is consistent with theoretically predicted behaviour of encapsulated microbubbles driven at frequencies above resonance. Despite being smaller, the Point UCAs produced a backscatter magnitude twice that of Philips UCAs, indicating that Point UCAs might behave quasi-resonantly when excited at 40 MHz.

Effects of body positioning on swallowing and esophageal transit in healthy dogs.

BACKGROUND: Contrast videofluoroscopy is the imaging technique of choice for evaluating dysphagic dogs. In people, body position alters the outcome of videofluoroscopic assessment of swallowing. HYPOTHESIS/OBJECTIVE: That esophageal transit in dogs, as measured by a barium esophagram, is not affected by body position. ANIMALS: Healthy dogs (n=15). METHODS: Interventional, experimental study. A restraint device was built to facilitate imaging of dogs in sternal recumbency. Each dog underwent videofluoroscopy during swallowing of liquid barium and barium-soaked kibble in sternal and lateral recumbency. Timing of swallowing, pharyngeal constriction ratio, esophageal transit time, and number of esophageal peristaltic waves were compared among body positions. RESULTS: Transit time in the cervical esophagus (cm/s) was significantly delayed when dogs were in lateral recumbency for both liquid (2.58+/-1.98 versus 7.23+/-3.11; P=.001) and kibble (4.44+/-2.02 versus 8.92+/-4.80; P=.002). In lateral recumbency, 52+/-22% of liquid and 73+/-23% of kibble swallows stimulated primary esophageal peristalsis. In sternal recumbency, 77+/-24% of liquid (P=.01 versus lateral) and 89+/-16% of kibble (P=.01 versus lateral) swallows stimulated primary esophageal peristalsis. Other variables were not significantly different. CONCLUSIONS AND CLINICAL IMPORTANCE: Lateral body positioning significantly increases cervical esophageal transit time and affects the type of peristaltic wave generated by a swallow.

Selective imaging of adherent targeted ultrasound contrast agents.

The goal of ultrasonic molecular imaging is the detection of targeted contrast agents bound to receptors on endothelial cells. We propose imaging methods that can distinguish adherent microbubbles from tissue and from freely circulating microbubbles, each of which would otherwise obscure signal from molecularly targeted adherent agents. The methods are based on a harmonic signal model of the returned echoes over a train of pulses. The first method utilizes an 'image-push-image' pulse sequence where adhesion of contrast agents is rapidly promoted by acoustic radiation force and the presence of adherent agents is detected by the signal change due to targeted microbubble adhesion. The second method rejects tissue echoes using a spectral high-pass filter. Free agent signal is suppressed by a pulse-to-pulse low-pass filter in both methods. An overlay of the adherent and/or flowing contrast agents on B-mode images can be readily created for anatomical reference. Contrast-to-tissue ratios from adherent microbubbles exceeding 30 dB and 20 dB were achieved for the two methods proposed, respectively. The performance of these algorithms is compared, emphasizing the significance and potential applications in ultrasonic molecular imaging.

Optical and acoustical dynamics of microbubble contrast agents inside neutrophils.

Acoustically active microbubbles are used for contrast-enhanced ultrasound assessment of organ perfusion. In regions of inflammation, contrast agents are captured and phagocytosed by activated neutrophils adherent to the venular wall. Using direct optical observation with a high-speed camera and acoustical interrogation of individual bubbles and cells, we assessed the physical and acoustical responses of both phagocytosed and free microbubbles. Optical analysis of bubble radial oscillations during insonation demonstrated that phagocytosed microbubbles experience viscous damping within the cytoplasm and yet remain acoustically active and capable of large volumetric oscillations during an acoustic pulse. Fitting a modified version of the Rayleigh-Plesset equation that describes mechanical properties of thin shells to optical radius-time data of oscillating bubbles provided estimates of the apparent viscosity of the intracellular medium. Phagocytosed microbubbles experienced a viscous damping approximately sevenfold greater than free microbubbles. Acoustical comparison between free and phagocytosed microbubbles indicated that phagocytosed microbubbles produce an echo with a higher mean frequency than free microbubbles in response to a rarefaction-first single-cycle pulse. Moreover, this frequency increase is predicted using the modified Rayleigh-Plesset equation. We conclude that contrast-enhanced ultrasound can detect distinct acoustic signals from microbubbles inside of neutrophils and may provide a unique tool to identify activated neutrophils at sites of inflammation.

Noninvasive imaging of inflammation by ultrasound detection of phagocytosed microbubbles.

BACKGROUND: We have previously shown that microbubbles adhere to leukocytes in regions of inflammation. We hypothesized that these microbubbles are phagocytosed by neutrophils and monocytes and remain acoustically active, permitting their detection in inflamed tissue. METHODS AND RESULTS: In vitro studies were performed in which activated leukocytes were incubated with albumin or lipid microbubbles and observed under microscopy. Microbubbles attached to the surface of activated neutrophils and monocytes, were phagocytosed, and remained intact for up to 30 minutes. The rate of destruction of the phagocytosed microbubbles on exposure to ultrasound was less (P

Experimental and theoretical evaluation of microbubble behavior: effect of transmitted phase and bubble size.

Ultrasound contrast agents provide new opportunities to image vascular volume and flow rate directly. To accomplish this goal, new pulse sequences can be developed to detect specifically the presence of a microbubble or group of microbubbles. We consider a new scheme to detect the presence of contrast agents in the body by examining the effect of transmitted phase on the received echoes from single bubbles. In this study, three tools are uniquely combined to aid in the understanding of the effects of transmission parameters and bubble radius on the received echo. These tools allow for optical measurement of radial oscillations of single bubbles during insonation, acoustical study of echoes from single contrast agent bubbles, and the comparison of these experimental observations with theoretical predictions. A modified Herring equation with shell terms is solved for the time-dependent bubble radius and wall velocity, and these outputs are used to formulate the predicted echo from a single encapsulated bubble. The model is validated by direct comparison of the predicted radial oscillations with those measured optically. The transient bubble response is evaluated with a transducer excitation consisting of one-cycle pulses with a center frequency of 2.4-MHz. The experimental and theoretical results are in good agreement and predict that the transmission of two pulses with opposite polarity will yield similar time domain echoes with the first significant portion of the echo generated when the rarefactional half-cycle reaches the bubble.

Optical and acoustical observations of the effects of ultrasound on contrast agents.

Optimal use of encapsulated microbubbles for ultrasound contrast agents and drug delivery requires an understanding of the complex set of phenomena that affect the contrast agent echo and persistence. With the use of a video microscopy system coupled to either an ultrasound flow phantom or a chamber for insonifying stationary bubbles, we show that ultrasound has significant effects on encapsulated microbubbles. In vitro studies show that a train of ultrasound pulses can alter the structure of an albumin-shelled bubble, initiate various mechanisms of bubble destruction or produce aggregation that changes the echo spectrum. In this analysis, changes observed optically are compared with those observed acoustically for both albumin and lipid-shelled agents. We show that, when insonified with a narrowband pulse at an acoustic pressure of several hundred kPa, a phospholipid-shelled bubble can undergo net radius fluctuations of at least 15%; and an albumin-shelled bubble initially demonstrates constrained expansion and contraction. If the albumin shell contains air, the shell may not initially experience surface tension; therefore, the echo changes more significantly with repeated pulsing. A set of observations of contrast agent destruction is presented, which includes the slow diffusion of gas through the shell and formation of a shell defect followed by rapid diffusion of gas into the surrounding liquid. These observations demonstrate that the low-solubility gas used in these agents can persist for several hundred milliseconds in solution. With the transmission of a high-pulse repetition rate and a low pressure, the echoes from, contrast agents can be affected by secondary radiation force. Secondary radiation force is an attractive force for these experimental conditions, creating aggregates with distinct echo characteristics and extended persistence. The scattered echo from an aggregate is several times stronger and more narrowband than echoes from individual bubbles.

Direct video-microscopic observation of the dynamic effects of medical ultrasound on ultrasound contrast microspheres.

RATIONALE AND OBJECTIVES: Ultrasound can cause destruction of microbubble contrast agents used to enhance medical ultrasound imaging. This study sought to characterize the dynamics of this interaction by direct visual observation of microbubbles during insonification in vitro by a medical ultrasound imaging system. METHODS: Video microscopy was used to observe air-filled sonicated albumin microspheres adsorbed to a solid support during insonation. RESULTS: Deflation was not observed at lowest transmit power settings. At higher intensities, gas left the microparticle gradually, apparently dissolving into the surrounding medium. Deflation was slower for higher microsphere surface densities. Intermittent ultrasound imaging (0.5 Hz refresh rate) caused slower deflation than continuous imaging (33 Hz). CONCLUSIONS: Higher concentrations of microbubbles, lower ultrasound transmit power settings, and intermittent imaging each can reduce the rate of destruction of microspheres resulting from medical ultrasound insonation.

Changes in the echoes from ultrasonic contrast agents with imaging parameters.

Current harmonic imaging scanners transmit a narrowband signal that limits spatial resolution in order to differentiate the echoes from tissue from the echoes from microbubbles. Because spatial resolution is particularly important in applications, including mapping vessel density in tumors, we explore the use of wideband signals in contrast imaging. It is first demonstrated that microspheres can be destroyed using one or two pulses of ultrasound. Thus, temporal signal processing strategies that use the change in the echo over time can be used to differentiate echoes from bubbles and echoes from tissue. Echo parameters, including intensity and spectral shape for narrowband and wideband transmission, are then evaluated. Through these experiments, the echo intensity received from bubbles after wideband transmission is shown to be at least as large as that for narrowband transmission, and can be larger. In each case, the echo intensity increases in a nonlinear fashion in comparison with the transmitted signal intensity. Although the echo intensity at harmonic multiples of the transmitted wave center frequency can be larger for narrowband insonation, echoes received after wideband insonation demonstrate a broadband spectrum with significant amplitude over a very wide range of frequencies.