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.     

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. An air bubble with initial diameter of 2 microns in water at 37 degrees C is predicted to fully dissolve within 25 ms. Clinical ultrasound contrast agents are often designed with a high molecular weight core in an attempt to decrease the diffusion rate. C3F8 and C4F10 gas bubbles of the same size are predicted to fully dissolve within 400 ms and 4000 ms, respectively. Optical experiments involving gas diffusion of a contrast agent support the theoretical predictions; however, shelled agents diffuse at a much slower rate without insonation, on the order of minutes to hours. Shell properties play a significant role in the rate of static diffusion by blocking the gas-liquid interface and decreasing the transport of gas into the surrounding liquid. Static diffusion decreases the diameter of albumin-shelled agents to a greater extent than lipid-shelled agents after insonation.     

Simulations and measurements of optical images of insonified ultrasound contrast microbubbles

Ultrasound contrast agents (UCAs) are used in a clinical setting to enhance the backscattered signal from the blood pool to estimate perfusion and blood flow. The UCAs consist of encapsulated microbubbles, measuring 1-10 /spl mu/m in diameter. Acoustic characterization of UCAs is generally carried out from an ensemble of bubbles. The measured signal is a complicated summation of all signals from the individual microbubbles. Hence, characterization of a single bubble from acoustic measurements is complex. In this study, 583 optical observations of freely flowing, oscillating, individual microbubbles from an experimental UCA were analyzed. The excursions during ultrasound exposure were observed through a microscope. Images were recorded with a high frame rate camera operating at 3 MHz. Microbubbles on these images were measured offline, and maximal excursions were determined. A technique is described to determine the diameters of the bubbles observed. We compared the maximal excursions of microbubbles of the same initial size in an ultrasound field with a 500 kHz center frequency at acoustic amplitudes ranging from 0.06 MPa to 0.85 MPa. It was concluded that maximal excursions of identical bubbles can differ by 150% at low acoustic pressures (mechanical index or MI<0.2). At a high acoustic pressure (MI=1.2) an image sequence was recorded on which a bubble collapsed, but an apparently identical bubble survived.     

Stable and transient subharmonic emissions from isolated contrast agent microbubbles

Ultrasound contrast agents (UCAs) have been widely studied in recent years in order to improve and develop new, sophisticated imaging techniques for clinical applications. In order to improve the understanding of microbubble-ultrasound interactions, an acoustic dynamic characterization of UCA microbubble behavior was performed in this work using a high frame-rate acquiring and processing system. This equipment is connected to a commercial scanner that provides RF beam-formed data with a frame-rate of 30 Hz. Acquired RF sequences allows us to follow the dynamics of cavitation mechanisms in its temporal evolution during different insonifying conditions. The experimental setup allowed us to keep the bubbles free in a spatial region of the supporting medium, thus avoiding boundary effects that can alter the ultrasound field and the scattered echo from bubbles. The work focuses on the study of subharmonic emission from an isolated bubble of contrast agent. In particular, the acoustic pressure threshold for a subharmonic stable emission was evaluated for a subset of 50 microbubbles at 3.3 MHz and at 5 MHz of insonation frequencies. An unexpected second pressure threshold, which caused the stand still of the subharmonic emission, was detected at 3.3 MHz and 5 MHz excitation frequencies. A transient subharmonic emission, which is hypothesized as being related to the formation of new free gas bubbles, was detected during the ultrasound-induced destruction of microbubbles. An experimental procedure was devised in order to investigate these behaviors and several sequences of RF echo signals and the related spectra, acquired from an isolated bubble in different insonation conditions, are presented and discussed in this paper.     

Trapped Bubble Dynamics in Cryogenic Fluids

Initial studies of the properties of helium bubbles acoustically trapped in liquid nitrogen at 66 K have been carried out. Laser scattering is used to monitor the dynamics of the bubble radius as a function of the acoustic driving field. In this apparatus, no sonoluminescence has yet been observed from helium bubbles in liquid nitrogen, although sonoluminescence was previously observed from xenon bubbles in ethanol cooled to 165 K. It is found that bubbles can be stably trapped at quite low acoustic drive levels, enabling the observation of new dynamical bubble behavior.     

High-frequency dynamics of ultrasound contrast agents

Ultrasound contrast agents enhance echoes from the microvasculature and enable the visualization of flow in smaller vessels. Here, we optically and acoustically investigate microbubble oscillation and echoes following insonation with a 10 MHz center frequency pulse. A high-speed camera system with a temporal resolution of 10 ns, which provides two-dimensional (2-D) frame images and streak images, is used in optical experiments. Two confocally aligned transducers, transmitting at 10 MHz and receiving at 5 MHz, are used in acoustical experiments in order to detect subharmonic components. Results of a numerical evaluation of the modified Rayleigh-Plesset equation are used to predict the dynamics of a microbubble and are compared to results of in vitro experiments. From the optical observations of a single microbubble, nonlinear oscillation, destruction, and radiation force are observed. The maximum bubble expansion, resulting from insonation with a 20-cycle, 10-MHz linear chirp with a peak negative pressure of 3.5 MPa, has been evaluated. For an initial diameter ranging from 1.5 to 5 microm, a maximum diameter less than 8 microm is produced during insonation. Optical and acoustical experiments provide insight into the mechanisms of destruction, including fragmentation and active diffusion. High-frequency pulse transmission may provide the opportunity to detect contrast echoes resulting from a single pulse, may be robust in the presence of tissue motion, and may provide the opportunity to incorporate high-frequency ultrasound into destruction-replenishment techniques.     

Synergistic cytotoxicity of low-energy ultrasound and innovative mesoporous silica-based sensitive nanoagents

Low-energy ultrasound (LEUS) shows distinct potential as a safe therapeutic strategy for cancer treatment. Herein, mesoporous silica nanoparticles with closed-end cavities as sensitive nanoagents are prepared for effective cancer cell killing, when synergistically combined with mild LEUS (1 MHz, ≤1.0 W cm^?2). The closed-end cavities can entrap gas bubbles, and provide a large number of cavitation nucleation sites, which could lead to drastically amplify ultrasonic cavitation effect by responding to the mild LEUS (1 MHz, ≤1.0 W cm^?2). Significant killing effect against cancer cells is observed, when cells are treated by synergetic combination of mild LEUS and the nanoagents with closed-end cavities, showing distinct dose dependency on the nanoagents and irradiation intensity. Nevertheless, the killing effect is disappeared when the closed-end cavities are destructed. Moreover, no obvious cytotoxicity is observed when either the nanoagents or the LEUS is applied alone. The research may open up application opportunities of mild low-energy ultrasound for cancer therapy.     

Ultrasonic contrast agent shell rupture detected by inertial cavitation and rebound signals

Determining the rupture pressure threshold of ultrasound contrast agent microbubbles has significant applications for contrast imaging, development of therapeutic agents, and evaluation of potential bioeffects. Using a passive cavitation detector, this work evaluates rupture based on acoustic emissions from single, encapsulated, gas-filled microbubbles. Sinusoidal ultrasound pulses were transmitted into weak solutions of Optison at different center frequencies (0.9, 2.8, and 4.6 MHz), pulse durations (three, five, and seven cycles of the center frequencies), and peak rarefactional pressures (0.07 to 5.39 MPa). Pulse repetition frequency was 10 Hz. Signals detected with a 13-MHz, center-frequency transducer revealed postexcitation acoustic emissions (between 1 and 5 micros after excitation) with broadband spectral content. The observed acoustic emissions were consistent with the acoustic signature that would be anticipated from inertial collapse followed by "rebounds" when a microbubble ruptures and thus generates daughter/free bubbles that grow and collapse. The peak rarefactional pressure threshold for detection of these emissions increased with frequency (e.g., 0.53, 0.87, and 0.99 MPa for 0.9, 2.8, and 4.6 MHz, respectively; five-cycle pulse duration) and decreased with pulse duration. The emissions identified in this work were separated from the excitation in time and spectral content, and provide a novel determination of microbubble shell rupture.     

Method for microbubble characterization using primary radiation force

Medical ultrasound contrast agents (UCAs) have evolved from straight image enhancers to pathophysiological markers and drug delivery vehicles. However, the exact dynamic behavior of the encapsulated bubbles composing UCAs is still not entirely known. In this article, we propose to characterize full populations of UCAs, by looking at the translational effects of ultrasound radiation force on each bubble in a diluted population. The setup involves a sensitive, fully programmable transmitter/receiver and two unconventional, real-time display modes. Such display modes are used to measure the displacements produced by irradiation at frequencies in the range 2-8 MHz and pressures between 150 kPa and 1.5 MPa. The behavior of individual bubbles freely moving in a water tank is clearly observed, and it is shown that it depends on the bubble physical dimensions as well as on the viscoelastic properties of the encapsulation. A new method also is distilled that estimates the viscoelastic properties of bubble encapsulation by fitting the experimental bubble velocities to values simulated by a numerical model based on the modified Herring equation and the Bjerknes force. The fit results are a shear modulus of 18 MPa and a viscosity of 0.23 Pas for a thermoplastic PVC-AN shell. Phospholipid shell elasticity and friction parameter of the experimental contrast agent are estimated as 0.8 N/m and 1 10(-7) kg/s, respectively (shear modulus of 32 MPa and viscosity of 0.19 Pas, assuming 4-nm shell thickness).     

Reduction of cavitation using pseudorandom signals [therapeutic US]

It is known that the scattering of ultrasound by cavitation bubbles reduces the efficiency of treatment by high-intensity focused ultrasound. In the authors' experiments striving to reduce grating lobe levels of annular arrays they observed less microbubble formation at the focus of the transducer when pseudorandom phase-modulated CW signals were used rather than single-frequency CW signals. To confirm this unexpected result, the authors performed an experiment in a solution of luminol which is known to be a cavitation detector. A 5-cm diameter spherical transducer (1.1 MHz center frequency and 0.6 MHz bandwidth), focused at 197 mm was used. The ratio of the sonoluminescence intensity produced by a single-frequency signal to that produced by a pseudorandom phase-modulated signal increased with the intensity of the applied held and attained a value of nearly 50 for an intensity of 4.6 W cm^-2      

Enhanced heat deposition using ultrasound contrast agent--modeling and experimental observations

Ultrasound contrast agents (UCA), created originally for visualization and diagnostic purposes, recently have been suggested as efficient enhancers of ultrasonic power deposition in tissue. The ultrasonic energy absorption by the contrast agents, considered as problematic in diagnostic imaging, might have beneficial impact in therapeutic applications such as targeted hyperthermia-based or ablation treatments. Introduction of gas microbubbles into the tissue to be treated can improve the effectiveness of current treatments by limiting the temperature rise to the treated site and minimizing the damage to the surrounding healthy tissues. To this end, proper assessment of the governing parameters of energy absorption by ultrasonically induced stabilized bubbles is important for both diagnostic and therapeutic ultrasound applications. The current study was designed to predict theoretically and measure experimentally the dissipation and heating effects of encapsulated UCA in a well-controlled and calibrated environment. The ultrasonic effects of the microbubble concentration, transmitted intensity, and frequency on power dissipation and stability of the UCA have been studied. The maximal temperature elevation obtained during 300 s experiments was 21 degrees C, in a 10 ml volume target containing UCA, insonifled by unfocused 3.2 MHz continuous wave (CW) at spatial average intensity of 1.1 W/cm2 (182 kPa). The results also suggest that higher frequencies are more efficiently absorbed by commonly used UCA. In particular, for spatial average intensity of 1.1 W/cm2 and concentration of 5 x 10(6) microspheres/cm3, no significant reduction of UCA absorption was noticed during the first 150 s for insonation at 3.2 MHz and the first 100 s for insonation at 1 MHz. In addition, when lower average intensity of 0.5 W/cm2 (160 kPa) at 3.2 MHz was used, the UCA absorptivity sustained for almost 200 s. Thus, when properly activated, UCA may be suitable for localized hyperthermic therapies.     

Coupled FEM and BEM code for simulating acoustically excited bubbles near deformable structures

An understanding of biotissue–bubble interactions and the stresses induced in the tissue is needed to identify potential mechanisms of tissue damage, such as vessel rupture, by acoustically excited bubbles. Interactions between acoustically excited bubbles and nearby rigid structures have been studied effectively using the boundary element method. However, if the nearby structure is a biotissue, structure deformations will affect the bubble response. In this paper a coupled finite element and boundary element code, developed to investigate the interactions between an acoustically excited bubble and a deformable structure, is presented. In particular, this model was developed to investigate the response of bubbles within deformable tubes. This code is validated by comparison to other simulation and experimental results and employed to obtain the response of an acoustically excited bubble centered within a tube. General characteristics of bubble–tube interactions and stresses induced in the tube wall are described by considering typical simulation results.     

Optical thermocapillary bubble trap

The phenomenon of thermocapillary trapping of gas bubbles by a laser beam in a layer of absorbing liquid confined between two plates is demonstrated. In this system, the laser beam is capable of trapping gas bubbles occurring at a distance of up to ten beam diameters. Acceleration of a trapped bubble was observed, and the bubble velocity was determined as a function of the distance to the beam projection on the layer plane. The force developed by a thermocapillary trap exceeds 10 nN.     

Hydrophone spatial averaging corrections from 1 to 40 MHz

The purpose of this study was to develop and experimentally verify a practical spatial averaging model for frequencies up to 40 MHz. The model is applicable to focused sources of circular geometry, accounts for the effects of hydrophone probe finite aperture, and allows calibration by substitution to be performed when the active elements of reference and tested hydrophone probes differ significantly. Several broadband sources with focal numbers between 3 and 20 were used to produce ultrasound fields with frequencies up to 40 MHz. The effective diameters of the ultrasonic hydrophone probes calibrated in the focal plane of the sources ranged from 150 to 500 μm. Prior to application of the spatial averaging corrections, the hydrophones with diameters smaller than that of the reference hydrophone exhibited experimentally determined absolute sensitivities higher than the true ones. This discrepancy increased with decreasing focal numbers and increasing frequency. It was determined that the error was governed by the cross-section of the beam in the focal plane and the ratio of the effective diameters of the reference and tested hydrophone probes. In addition, the error was found to be reliant on the frequency-dependent effective hydrophone radius. After applying the spatial averaging correction, the overall uncertainty in the hydrophone calibration was on the order of ±1 dB. The model developed is being extended to be applicable to frequencies beyond 40 MHz, which are becoming increasingly important in diagnostic ultrasound imaging applications     

Laboratory batch experiments of the combined effects of ultrasound and air stripping in removing CCl4 and 1,1,1-TCA from water

Ultrasonic and air-stripping techniques for removal of carbon tetrachloride (CCl₄) and 1,1,1-trichloroethane (1,1,1-TCA) from water were studied in batch experiments. Ultrasound (US) is effective for destroying organic compounds in aqueous solutions whereas air stripping (AS) efficiently transfers volatile compounds from the liquid to the gas phase. In simultaneous US and AS experiments, synergistic effects were observed and attributed to the effect of US on the mass transfer process. Using a photographic method, ultrasonic break up of gas bubbles and changes in gas holdup ratios were examined. In the two different gas-sparging systems studied, ultrasonic waves did not break up gas bubbles. In contrast, bubbles from the smaller porous size diffuser were coalesced due to sonication. In addition, both photographic and gas holdup experiments demonstrated that ultrasonic irradiation increased the gas holdup ratio. The enhancement observed in the removal of the compounds appeared to be due to this greater ultrasonic gas holdup ratio.     

A method for radiation-force localized drug delivery using gas-filled lipospheres

We have developed a method using ultrasound and acoustically active lipospheres (AALs) that might be used to deliver bioactive substances to the vascular endothelium. The AALs consist of a small gas bubble surrounded by a thick oil shell and enclosed by an outermost lipid layer. The AALs are similar to ultrasound contrast agents: they can be nondestructively deflected using ultrasound radiation force, and fragmented with high-intensity ultrasound pulses. The lipid-oil complex might be used to carry bioactive substances at high concentrations. An optimized sequence of ultrasound pulses can deflect the AALs toward a vessel wall then disrupt them, painting their contents across the vascular endothelium. This paper presents results from a series of in vitro and ex vivo experiments demonstrating localization of a fluorescent model drug. In experiments using a human melanoma cell (A2085) monolayer, a specific radiation force-fragmentation ultrasound pulse sequence increased cell fluorescence more than 10-fold over no ultrasound or fragmentation pulses alone, and by 50% over radiation force pulses alone. We observe that dye transfer is limited to cells that are in the region of ultrasonic focus, indicating that the application of radiation force pulses to bring the delivery vehicle into proximity with the cell is required for successful adhesion of the vehicle fragments to the cell membrane. We also demonstrate dye transfer from flowing AALs, both in a mimetic vessel and in excised rat cecum. We believe that this method could be successfully used for drug delivery in vivo.     

Thermocapillary Flow and Coalescences of Heterogeneous Bubble Size Diameter in a Rotating Cylinder: 3D Study

Two dimensional axisymmetric and three-dimensional VOF simulations of gas/liquid transient flow were performed using a multiphase flow algorithm based on the finite-volume method. The results for motion of a multiple bubbles of a heterogeneous sizes aligned horizontally and perpendicular to a hot surface incorporating thermocapillary forces in a rotating liquid in a zero-gravity environment have been presented for the first time. No bubbles broke in any of the cases observed. The results also show that collision and agglomeration of bubbles of unequal sizes diameter are different from those of similar size diameters presented from earlier research work of Alhendal et al. Acta Astronaut. 117, 484–496 (2015). Different flow patterns such as thermocapillary bubble migration, collision, and stream function were observed and presented for the 2-D and 3-D models.     

The solubility of gas bubbles

The solubility of a gas is usually defined in terms of its concentration in a semi-infinite material in equilibrium with the gas phase at a pressure of one atmosphere at a specified temperature. Since bubbles have a finite radius of curvature, it is important to extend this definition to describe their solubility in a way which is analogous to the equation relating the increase in solubility and decrease in radius of solid precipitates.A thermodynamic analysis is presented which evaluates the gas solubility in equilibrium with a bubble and shows it to be independent of bubble radius except in so far as this radius is related to the pressure. The analysis is also applicable to bubbles having crystallographic faces and to bubbles whose shape is modified by intersection with grain-boundaries.     

Determination of the thickness of the surface microlayer from which aerosols are formed by burst of gas bubbles at the liquid–gas interface

The surface diameters of gas bubbles at the liquid–gas interface whose burst leads to the formation of aerosol from a thin surface microlayer of thickness 1 μm and less have been determined experimentally. Precisely the anomalous concentrations of such a microlayer are responsible for the fractionation of substances in the process of ocean–atmosphere exchange.     

Potential of microbubbles for use as point targets in phase aberration correction

Bubbles can be produced by vaporization of perfluorocarbon droplets of a few microm diameter. These bubbles can reach over 100 microm in diameter and their backscatter is calculated to be well more than 10 dB above that of several organ tissues. At appropriate sizes, small acoustic amplitudes, and diagnostic frequencies (2-15 MHz), bubbles can be approximated by the nonrigid sphere-scattering solution employed here. This paper concerns the bubble size and its implications on the backscatter amplitude and the phase error introduced in diagnostic ultrasound when assuming that the bubble acts as a point target for phase aberration correction.