James Chomas

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.

Mechanisms of contrast agent destruction

Various applications of contrast-assisted ultrasound, including blood vessel detection, perfusion estimation, and drug delivery, require controlled destruction of contrast agent microbubbles. The lifetime of a bubble depends on properties of the bubble shell, the gas core, and the acoustic waveform impinging on the bubble. Three mechanisms of microbubble destruction are considered: fragmentation, acoustically driven diffusion, and static diffusion. Fragmentation is responsible for rapid destruction of contrast agents on a time scale of microseconds. The primary characteristics of fragmentation are a very large expansion and subsequent contraction, resulting in instability of the bubble. Optical studies using a novel pulsed-laser optical system show the expansion and contraction of ultrasound contrast agent microbubbles with the ratio of maximum diameter to minimum diameter greater than 10. Fragmentation is dependent on the transmission pressure, occurring in over 55% of bubbles insonified with a peak negative transmission pressure of 2.4 MPa and in less than 10% of bubbles insonified with a peak negative transmission pressure of 0.8 MPa. The echo received from a bubble decorrelates significantly within two pulses when the bubble is fragmented, creating an opportunity for rapid detection of bubbles via a decorrelation-based analysis. Preliminary findings with a mouse tumor model verify the occurrence of fragmentation in vivo. A much slower mechanism of bubble destruction is diffusion, which is driven by both a concentration gradient between the concentration of gas in the bubble compared with the concentration of gas in the liquid, as well as convective effects of motion of the gas-liquid interface. The rate of diffusion increases during insonation, because of acoustically driven diffusion, producing changes in diameter on the time scale of the acoustic pulse length, thus, on the order of microseconds. Gas bubbles diffuse while they are not being insonified, termed static diffusion.

Threshold of fragmentation for ultrasonic contrast agents.

Ultrasound contrast agents are small microbubbles that can be readily destroyed with sufficient acoustic pressure, typically, at a frequency in the low megaHertz range. Microvascular flow rate may be estimated by destroying the contrast agent in a vascular bed, and estimating the rate of flow of contrast agents back into the vascular bed. Characterization of contrast agent destruction provides important information for the design of this technique. In this paper, high-speed optical observation of an ultrasound contrast agent during acoustic insonation is performed. The resting diameter is shown to be a significant parameter in the prediction of microbubble destruction, with smaller diameters typically correlated with destruction. Pressure, center frequency, and transmission phase are each shown to have a significant effect on the fragmentation threshold. A linear prediction for the fragmentation threshold as a function of pressure, when normalized by the resting diameter, has a rate of change of 300 kPa/microm for the range of pressures from 310 to 1200 kPa, and a two-cycle excitation pulse with a center frequency of 2.25 MHz. A linear prediction for the fragmentation threshold as a function of frequency, when normalized by the resting diameter, has a rate of change of -1.2 MHz/microm for a transmission pressure of 800 kPa, and a two-cycle excitation pulse with a range of frequencies from 1 to 5 MHz.

Nondestructive subharmonic imaging

Ultrasound contrast agent microbubbles are intravascular agents that can be used to estimate blood perfusion. Blood perfusion may be estimated by destroying the bubbles in a vascular bed and observing the refresh of contrast agents back into the vascular bed. Contrast agents can be readily destroyed by traditional imaging techniques. The design of a nondestructive imaging technique is necessary for the accurate quantification of contrast agent refresh. In this work, subharmonic imaging is investigated as a method for nondestructive imaging with the contrast agent microbubble MP1950 (Mallinckrodt, Inc., St. Louis, MO). Optical observation during insonation, in conjunction with a modified Rayleigh-Plesset (R-P) analysis, provides insight into the mechanisms of and parameters required for subharmonic frequency generation. Subharmonic imaging with a transmission frequency that is the same as the resonant frequency of the bubble is shown to require a minimum pressure of insonation that is greater than the experimentally-observed bubble destruction threshold. Subharmonic imaging with a transmission frequency that is twice the resonant frequency of the bubble produces a subharmonic frequency response while minimizing bubble instability. Optimization is performed using optical experimental analysis and R-P analysis.

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.

Optical observation of contrast agent destruction

Fragmentation of an ultrasound contrast agent on the time scale of microseconds provides opportunities for the advancement of microvascular detection, blood flow velocity estimation, and targeted drug delivery. Images captured by high-speed imaging systems show destruction of a microbubble during compression. Peak wall velocity of −700 m/s and peak acceleration of 1.2×1012 m/s2 is observed for insonation with a peak pressure of −1.1 MPa and a center frequency of 2.4 MHz. Theoretical calculations of wall velocity and acceleration using a modified Rayleigh–Plesset model predict a peak negative wall velocity of −680 m/s and peak acceleration of 2×1012 m/s2.

Role of Primary and Secondary Capture for Leukocyte Accumulation In Vivo

Leukocyte accumulation during inflammation depends on the concerted action of selectin and integrin adhesion molecules, which promote capture, rolling, and arrest of these cells on activated endothelium. In addition to interacting with endothelial cells, leukocytes can also adhere to already adherent leukocytes through an L-selectin-dependent mechanism. Initiation of adhesion through this mechanism has been called nucleation and leads to characteristic geometric patterns (ie, clusters and strings) of adherent leukocytes in flow chambers. We have used intravital microscopy of tumor necrosis factor-alpha (TNF-alpha)-treated mouse cremaster muscles to quantitatively investigate the potential role of leukocyte-leukocyte adhesion in initiating and maintaining the leukocyte clusters that are commonly observed in inflamed venules. Our data show that in TNF-alpha-treated venules with diameters between 23 and 108 microm, leukocyte adhesion occurs in clusters that are 19 to 50 microm long and 8 to 44 microm wide. They are almost entirely made up of slow-rolling leukocytes. Of all leukocytes recruited into a cluster (100%), the majority enter the cluster rolling along the endothelium and sharply reduce their velocity in the absence (59%) or presence (15%) of other leukocytes in proximity (one cell diameter). Some of the rolling leukocytes (17%) pass through the cluster without reducing their velocity. Recruitment of leukocytes from the free flow regime into a cluster is a rare event and accounts for only 7 (1.2%) of 476 leukocytes arriving in the cluster. However, of the leukocytes captured from the free flow, 6 initiated contact with a slow-rolling leukocyte rather than making direct contact with the endothelium. Our data show that leukocyte-leukocyte interactions can occur in vivo but are not important for cluster formation. This is confirmed by the observation of normal cluster formation in L-selectin-deficient mice, in which leukocyte-leukocyte interactions under flow are abolished. We conclude that leukocyte-mediated nucleation contributes little to leukocyte recruitment during inflammation in vivo. Cluster formation appears to be dominated by areas of endothelium with a higher expression of E-selectin, because cluster formation is greatly reduced in E-selectin-deficient mice.

Subharmonic phase-inversion for tumor perfusion estimation

A perfusion estimation scheme based on the destruction and wash-in of ultrasound contrast agents enables the estimation of microvascular flow velocities below those estimated by traditional Doppler and colorflow methods. The observation of low-flow microvascular beds is useful in the monitoring of therapy in a research setting. Perfusion estimation may be described in four steps: bubble destruction, non-destructive imaging, detection, and estimation. The destructive and non-destructive imaging modes are implemented in real-time on a modified clinical ultrasound scanner. In-vivo results in a chronic tumor study show that the system is capable of detecting microvascular flow that is otherwise undetectable with Doppler or colorflow imaging methods. The resulting maps of increased vascular density correlate with regions of active tumor cells on histology. Maps of increased blood flow correlate with computed tomography. Microvascular heterogeneities on the scale of 800 /spl mu/m are observed in tumors, with a ring of perfusion developing as the tumor grows. Vascular regions in the periphery of the tumor require less than 4 seconds to achieve 80% perfusion, while other regions in the tumor require up to 30 seconds.

Ultrasound contrast agents phagocytosed by neutrophils demonstrate acoustic activity

Ultrasound contrast agents are microbubbles composed of a thin lipid or albumin shell filled with air or a high molecular weight gas. These microbubbles are used for contrast-enhanced ultrasound (CEU) assessment of organ perfusion. In regions of inflammation, microbubbles are phagocytosed intact by activated neutrophils adherent to the venular wall. The authors hypothesized that microbubbles remain acoustically active following phagocytosis. Accordingly, they assessed the physical responses of both phagocytosed and free microbubbles by direct microscopic observation during delivery of repetitive single pulses of ultrasound at various acoustic pressures. Insonation results in oscillation in the bubbles volume. Microbubbles were optically recorded during insonation with a high-speed imaging system and diameter-time curves were analyzed to determine the effect of phagocytosis. Phagocytosed microbubbles retained their acoustic activity, although the intracellular environment increased viscoelastic damping experienced by microbubbles. With a pulse of high acoustic intensity (>1 MPa), phagocytosed microbubbles expanded up to 500% of their initial radii, which occasionally resulted in neutrophil rupture. Primary radiation force displaced phagocytosed microbubbles a distance of 100 microns with an acoustic pressure of -240 kPa and a pulse repetition frequency of 10 kHz, thus providing further evidence of acoustic activity. The authors conclude that phagocytosed microbubbles exhibit viscoelastic damping and yet are susceptible to acoustic destruction. They can generate non-linear echoes on the same order of magnitude as free microbubbles. These results indicate that CEU may be used to identify and assess regions of inflammation by detecting acoustic signals from microbubbles that are phagocytosed by activated neutrophils. In addition, the rapid expansion of a microbubble at high acoustic pressure may present a means to rupture a neutrophil or drug capsule at a specific site, resulting in delivery of a drug.

Correlation analysis of received echoes from contrast agents in-vitro and in-vivo

Ultrasound contrast agents can be used to receive signals from vessels significantly smaller than the effective resolution of an ultrasound system. The signal from these microbubbles is coherent across pulses as long as the microbubble is intact, looking like a moving reflector that is more echogenic than blood cells and shifted in spectral mean. When the microbubble is destroyed, the received signal decorrelates between pulses. In-vitro optical and acoustical experiments observing single bubble echoes were conducted. Three mechanisms of destruction were observed optically: static diffusion, pressure-driven diffusion, and fragmentation. Fragmentation was often observed at pressures above 1.4 MPa, while static diffusion and acoustical diffusion were most frequently responsible for bubble destruction at low transmitted pressure. Correlation analysis between echoes received from the same bubble provided analytical evidence of bubble destruction; moreover, a detection scheme based on decorrelation was designed and tested.