Effect of Acoustic Parameters and Microbubble Concentration on the Likelihood of Encapsulated Microbubble Coalescence

Microbubble contrast agents are commonly used for therapeutic and diagnostic imaging applications. Under certain conditions, these contrast agents can coalesce on ultrasound application and form larger bubbles than the initial population. The formation of large microbubbles potentially influences therapeutic outcomes and imaging quality. We studied clinically relevant ultrasound parameters related to low-pressure therapy and contrast-enhanced ultrasound imaging to determine their effect on microbubble coalescence and subsequent changes in microbubble size distributions in vitro. Results indicate that therapeutic ultrasound at low frequencies, moderate pressures and high duty cycles are capable of forming bubbles greater than two times larger than the initial bubble distribution. Furthermore, acoustic parameters related to contrast-enhanced ultrasound imaging that are at higher frequency, low-pressure and low-duty cycle exhibit no statistically significant changes in bubble diameter, suggesting that standard contrast ultrasound imaging does not cause coalescence. Overall, this work suggests that the microbubble coalescence phenomenon can readily occur at acoustic parameters used in therapeutic ultrasound, generating bubbles much larger than those found in commercial contrast agents, although coalescence is unlikely to be significant in diagnostic contrast-enhanced ultrasound imaging. This observation warrants further expansion of parameter ranges and investigation of resulting effects.     

Ultrasound-induced encapsulated microbubble phenomena

When encapsulated microbubbles are subjected to high-amplitude ultrasound, the following phenomena have been reported: oscillation, translation, coalescence, fragmentation, sonic cracking and jetting. In this paper, we explain these phenomena, based on theories that were validated for relatively big, free (not encapsulated) gas bubbles. These theories are compared with high-speed optical observations of insonified contrast agent microbubbles. Furthermore, the potential clinical applications of the bubble-ultrasound interaction are explored. We conclude that most of the results obtained are consistent with free gas bubble theory. Similar to cavitation theory, the number of fragments after bubble fission is in agreement with the dominant spherical harmonic oscillation mode. Remarkable are our observations of jetting through contrast agent microbubbles. The pressure at the tip of a jet is high enough to penetrate any human cell. Hence, liquid jets may act as remote-controlled microsyringes, delivering a drug to a region-of-interest. Encapsulated microbubbles have (potential) clinical applications in both diagnostics and therapeutics.     

Characterization of Acoustic Droplet Vaporization for Control of Bubble Generation Under Flow Conditions

This study investigated the manipulation of bubbles generated by acoustic droplet vaporization (ADV) under clinically relevant flow conditions. Optical microscopy and high-frequency ultrasound imaging were used to observe bubbles generated by 2-MHz ultrasound pulses at different time points after the onset of ADV. The dependence of the bubble population on droplet concentration, flow velocity, fluid viscosity and acoustic parameters, including acoustic pressure, pulse duration and pulse repetition frequency, was investigated. The results indicated that post-ADV bubble growth spontaneously driven by air permeation markedly affected the bubble population after insonation. The bubbles can grow to a stable equilibrium diameter as great as twice the original diameter in 0.5–1 s, as predicted by the theoretical calculation. The growth trend is independent of flow velocity, but dependent on fluid viscosity and droplet concentration, which directly influence the rate of gas uptake by bubbles and the rate of gas exchange across the wall of the semipermeable tube containing the bubbles and, hence, the gas content of the host medium. Varying the acoustic pressure does not markedly change the formation of bubbles as long as the ADV thresholds of most droplets are reached. Varying pulse duration and pulse repetition frequency markedly reduces the number of bubbles. Lengthening pulse duration favors the production of large bubbles, but reduces the total number of bubbles. Increasing the PRF interestingly provides superior performance in bubble disruption. These results also suggest that an ADV bubble population cannot be assessed simply on the basis of initial droplet size or enhancement of imaging contrast by the bubbles. Determining the optimal acoustic parameters requires careful consideration of their impact on the bubble population produced for different application scenarios.     

Automated production and analysis of echo contrast agents

To develop and standardize contrast agents for use in contrast echocardiographic imaging, microbubble size, concentration, decay, and ultrasound backscatter must be known. These parameters were assessed with a scanning laser particle counter, a commercial ultrasound unit, and various sonicated intravenous solutions. The scanning laser particle counter proved to be a fast and effective means of evaluating microbubble size, concentration, and stability. In addition, sonication was found to be a reliable and reproducible technique for preparing standardized echo contrast agent solutions containing uniformly small microbubbles. The bubbles generated ranged in size from 1 to 15 micron in diameter. All solutions had mean bubble diameters less than 6 micron. The half life of solutions ranged from 44 +/- 12 seconds for Hypaque 50%, to 253 +/- 73 seconds for Iopamidol. Addition of the surfactant to dextrose 70% prolonged bubble half life from 58 +/- 12 seconds to 1018 +/- 276 seconds. Phased array two-dimensional echocardiography of sonicated microbubble solutions, and subsequent videodensitometric analysis, revealed that bubble concentration was directly proportional to echo-reflective properties, and that the solutions have significant ultrasound reflective properties in vitro at concentrations of less than 1500 bubbles/ml.     

Evaluation of Transfection Efficiency in Skeletal Muscle Using Nano/Microbubbles and Ultrasound

Recent studies have revealed that ultrasound contrast agents with low-intensity ultrasound, namely, sonoporation, can noninvasively deliver therapeutic molecules into target sites. However, the efficiency of molecular delivery is relatively low and the methodology requires optimization. Here, we investigated three types of nano/microbubbles (NMBs)—human albumin shell bubbles, lipid bubbles and acoustic liposomes—to evaluate the efficiency of gene expression in skeletal muscle as a function of their physicochemical properties and the number of bubbles in solution. We found that acoustic liposomes showed the highest transfection and gene expression efficiency among the three types of NMBs under ultrasound-optimized conditions. Liposome transfection efficiency increased with bubble volume concentration; however, neither bubble volume concentration nor their physicochemical properties were related to the tissue damage detected in the skeletal muscle, which was primarily caused by needle injection. (E-mail: kodama@bme.tohoku.ac.jp)     

Bubble cycling and standing waves in ultrasonic cell lysis.

Two quite different but potentially complementary hypothetical mechanisms have been proposed to explain the dependence of ultrasonic cell lysis in vitro on exposure vessel rotation. One mechanism proposes that resonant-size bubbles are trapped in planar arrays by standing waves in the sample and that exposure vessel rotation sweeps cells through the bubble arrays. The other mechanism, for which there is now considerable support, proposes that exposure vessel rotation simply allows bubbles to recycle after having been driven through the medium by the ultrasound. We report here the results of efforts to further discriminate between, and quantify, the relative contributions of these two mechanisms. Murine P388 cells were exposed to 1.07 MHz continuous-wave (CW) ultrasound for 5 min at 5 W/cm2 (ISP) using various exposure vessel compositions and exposure configurations. Experimental treatments were designed either to minimize or maximize standing waves in the sample tube, or to minimize the potential for bubble recycling while maximizing standing wave formation. The results of these experiments indicate that bubble cycling is responsible for the majority of cell lysis occurring in the rotating exposure vessels, but that a significant contribution to total lysis is provided by trapped bubbles under some conditions.     

Rapid Shrinkage of Lipid-Coated Bubbles in Pulsed Ultrasound

Many researchers have observed rapid shrinkage of lipid-coated microbubbles subjected to brief, MHz ultrasound pulses. The shrinkage is sometimes, but not always, accompanied by the shedding of visible fragments of the coat. It has been suggested that the shedding of the lipid coat alone is sufficient to explain the rapid shrinkage, as that loss increases bubble surface tension and, thus, internal pressure, increasing gas loss even between pulses. We have determined, however, that the shedding of the coat lipid must also entrain some of the gas content of the bubble, to account for the observed shrinkage rates. The evidence for this is that insonated bubbles typically shrink much faster than the Epstein-Plesset (diffusion) limit for gas dissolution and diffusion, whereas uncoated quiescent bubbles shrink more slowly. We have also modeled the diffusion of gas in the moving liquid surrounding the bubble and find no advective enhancement of diffusive loss of gas from the bubble. Thus, bubble gas loss through diffusion alone is insufficient to account for rapid shrinkage.     

Transfection effect of microbubbles on cells in superposed ultrasound waves and behavior of cavitation bubble

The combination of ultrasound and ultrasound contrast agents (UCAs) is able to induce transient membrane permeability leading to direct delivery of exogenous molecules into cells. Cavitation bubbles are believed to be involved in the membrane permeability; however, the detailed mechanism is still unknown. In the present study, the effects of ultrasound and the UCAs, Optison on transfection in vitro for different medium heights and the related dynamic behaviors of cavitation bubbles were investigated. Cultured CHO-E cells mixed with reporter genes (luciferase or beta-gal plasmid DNA) and UCAs were exposed to 1 MHz ultrasound in 24-well plates. Ultrasound was applied from the bottom of the well and reflected at the free surface of the medium, resulting in the superposition of ultrasound waves within the well. Cells cultured on the bottom of 24-well plates were located near the first node (displacement node) of the incident ultrasound downstream. Transfection activity was a function determined with the height of the medium (wave traveling distance), as well as the concentration of UCAs and the exposure time was also determined with the concentration of UCAs and the exposure duration. Survival fraction was determined by MTT assay, also changes with these values in the reverse pattern compared with luciferase activity. With shallow medium height, high transfection efficacy and high survival fraction were obtained at a low concentration of UCAs. In addition, capillary waves and subsequent atomized particles became significant as the medium height decreased. These phenomena suggested cavitation bubbles were being generated in the medium. To determine the effect of UCAs on bubble generation, we repeated the experiments using crushed heat-treated Optison solution instead of the standard microbubble preparation. The transfection ratio and survival fraction showed no additional benefit when ultrasound was used. These results suggested that cavitation bubbles created by the collapse of UCAs were a key factor for transfection, and their intensities were enhanced by the interaction of the superpose ultrasound with the decreasing the height of the medium. Hypothesizing that free cavitation bubbles were generated from cavitation nuclei created by fragmented UCA shells, we carried out numerical analysis of a free spherical bubble motion in the field of ultrasound. Analyzing the interaction of the shock wave generated by a cavitation bubble and a cell membrane, we estimated the shock wave propagation distance that would induce cell membrane damage from the center of the cavitation bubble.     

Effect of macroscopic air bubbles on cell lysis by shock wave lithotripsy in vitro.

In studies of cells or stones in vitro, the material to be exposed to shock waves (SWs) is commonly contained in plastic vials. It is difficult to remove all air bubbles from such vials. Because SWs reflect at an air-fluid interface, and because existing gas bubbles can serve as nuclei for cavitation events, we sought to determine in our system whether the inclusion of small, visible bubbles in the specimen vial has an effect on SW-induced cell lysis. We found that even small bubbles led to increased lysis of red blood cells (1- to 3-mm diameter bubbles, 9.8+/-0.5% lysis, n = 7; no bubbles, 4.4+/-0.8%, n = 4), and that the degree of lysis increased with bubble size. Damage could not be reduced by centrifuging the cells to the opposite end of the vial, away from the bubble. B-scan ultrasound imaging of blood in polypropylene pipette bulbs showed that, with each SW, bubbles were recruited from the air interface, mixing throughout the fluid volume, and these appeared to serve as nuclei for increased echogenicity during impact by subsequent SWs; thus, bubble effects in vials could involve the proliferation of cavitation nuclei from existing bubbles. Whereas injury to red blood cells was greatly increased by the presence of bubbles in vials, lytic injury to cultured epithelial cells (LLC-PK1, which have a more complex cytoarchitecture than red blood cells) was not increased by the presence of small air bubbles. This suggests different susceptibility to SW damage for different types of cells. Thus, the presence of even a small air bubble can increase SW-induced cell damage, perhaps by increasing the number of cavitation nuclei throughout the vial, but this effect is variable with cell type.     

The influence of fragmentation on the acoustic response from shrinking bubbles

Bubble disruption is associated with the response of ultrasound contrast agents (UCAs) exposed to high acoustic pressures. This behavior is important for bubble detection techniques as well as flow quantitation and some proposed therapeutic applications. Previous work has measured acoustically the disruption threshold and postdisruption echo from populations of microbubbles. This suggests a model for UCA disruption whereby ultrasound breaks their shell, leaving free gas bubbles. Diffusion of gas causes the bubbles to shrink and, consequently, reduces the measured backscatter echo over time. In this work, similar bubbles containing three different gases were measured and their echo behavior with time compared with a simple simulation based on diffusion of gas out of the bubble. It was found that, in general, the simulations and experiments compared well at low disruption pressures. Incorporating bubble fragmentation in the simulation model brought its results closer to experiment.     

Controlling the Size Distribution of Lipid-Coated Bubbles via Fluidity Regulation

Lipid-coated bubbles exhibit oscillation responses capable of enhancing the sensitivity of ultrasound imaging by improving contrast. Further improvements in performance enhancement require control of the size distribution of bubbles to promote correspondence between their resonance frequency and the frequency of the ultrasound. Here we describe a size-controlling technique that can shift the size distribution using a currently available agitation method. This technique is based on regulating the membrane dynamic fluidity of lipid mixtures and provides a general size-controlling variable that could also be applied in other fabrication methods. Three materials (1,2-dihexadecanoyl-sn-glycero-3-phosphocholine, 1,2-dioctadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) and polyethylene glycol 40 stearate) with distinct initial fluidities and phase behaviors were used to demonstrate the use of fluidity regulation to control bubble sizes. Bubble size distributions of different formulations were determined by electrical impedance sensing, and bubble volumes and surface areas were calculated. To confirm the relationship between regulated fluidity and mean bubble size, the membrane fluidity of each composition was determined by fluorescence anisotropy, with the results indicating linear relations in the compositions with similar main transition temperatures. Compositions with a higher molar proportion of polyethylene glycol 40 stearate showed higher fluidities and larger bubbles. B-mode ultrasound imaging was performed to investigate the echogenicity and lifetime of the fabricated bubbles, with the results indicating that co-mixing a high-transition-temperature charged lipid (i.e., 1,2-dioctadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol)) extends the tailoring range of this fluidity regulation technique, allowing refined and continuous changes in mean bubble size (from 0.93 to 2.86 μm in steps of ∼0.5 μm), and also prolongs bubble lifetime. The polydispersity of each composition was also determined to evaluate practicality in particular applications. Our study demonstrates a feasible approach to naturally controling bubble size distribution and provides a practical reference for other fabrication systems and ultrasound imaging applications.     

The ring-down artifact

"Ring-down" is an ultrasound artifact that appears as a solid streak or a series of parallel bands radiating away from abdominal gas collections. Using an in vitro system of bubbles in water or gelatin, it was found that the ring-down artifact originated from the center of a cluster of four bubbles (bubble tetrahedron), three on top and one nestled beneath. Entrapped between the bubbles is a horn- or bugle-shaped fluid collection that we theorize emits a continuous sound wave back to the transducer when struck by an ultrasound pulse. Electronic processing by the scanner converts this continuous sound wave into the series of bands seen in the ring-down artifact.     

Use of overpressure to assess the role of bubbles in focused ultrasound lesion shape in vitro

Overpressure--elevated hydrostatic pressure--was used to assess the role of gas or vapor bubbles in distorting the shape and position of a high-intensity focused ultrasound (HIFU) lesion in tissue. The shift from a cigar-shaped lesion to a tadpole-shaped lesion can mean that the wrong area is treated. Overpressure minimizes bubbles and bubble activity by dissolving gas bubbles, restricting bubble oscillation and raising the boiling temperature. Therefore, comparison with and without overpressure is a tool to assess the role of bubbles. Dissolution rates, bubble dynamics and boiling temperatures were determined as functions of pressure. Experiments were made first in a low-overpressure chamber (0.7 MPa maximum) that permitted imaging by B-mode ultrasound (US). Pieces of excised beef liver (8 cm thick) were treated in the chamber with 3.5 MHz for 1 to 7 s (50% duty cycle). In situ intensities (I(SP)) were 600 to 3000 W/cm(2). B-mode US imaging detected a hyperechoic region at the HIFU treatment site. The dissipation of this hyperechoic region following HIFU cessation corresponded well with calculated bubble dissolution rates; thus, suggesting that bubbles were present. Lesion shape was then tested in a high-pressure chamber. Intensities were 1300 and 1750 W/cm(2) ( +/- 20%) at 1 MHz for 30 s. Hydrostatic pressures were 0.1 or 5.6 MPa. At 1300 W/cm(2), lesions were cigar-shaped, and no difference was observed between lesions formed with or without overpressure. At 1750 W/cm(2), lesions formed with no overpressure were tadpole-shaped, but lesions formed with high overpressure (5.6 MPa) remained cigar-shaped. Data support the hypothesis that bubbles contribute to the lesion distortion.     

A search for ultrasonic cavitation within the canine cardiovascular system

Resonant bubble detectors (RBD) were used to search for both pre-existing bubbles and bubbles created by cavitation within the cardiovascular system of 22 dogs. No pre-existing bubbles of 3.8 micron diam or larger were found, nor were any created by exposing the left ventricle to 0.51-1.61 MHz ultrasound of up to 16 W/cm2 spatial-peak intensity. Bubbles introduced into the arterial system by high speed injection were readily detected and could be held in the heart by 1 MHz ultrasound at 1-2 W/cm2 or above. A surprising observation was that gas bubbles of resonant size injected into the left ventricle and held by ultrasound did not multiply continuously as happens in saline or water in vitro. This in vivo system was designed to assess the potential for cavitation bioeffects and the essentially negative results obtained may limit the expected or potential risk of this mechanism in regard to medical applications of ultrasound.     

Dynamics of bubble oscillation in constrained media and mechanisms of vessel rupture in SWL

Rupture of small blood vessels is a primary feature of the vascular injury associated with shock-wave lithotripsy (SWL) and cavitation has been implicated as a potential mechanism. To understand more precisely the underlying mechanical cause of the injury, the dynamics of SWL-induced bubble dynamics in constrained media were investigated. Silicone tubing and regenerated cellulose hollow fibers of various inner diameters (0.2 to 1.5 mm) were used to fabricate vessel phantoms, which were placed in a test chamber filled with castor oil so that cavitation outside the phantom could be suppressed. Degassed water seeded with 0.2% Albunex contrast agent was circulated inside the vessel phantom, and intraluminal bubble dynamics during SWL were examined by high-speed shadowgraph imaging and passive cavitation detection via a 20-MHz focused transducer. It was observed that, in contrast to the typical large and prolonged expansion and violent inertial collapse of SWL-induced bubbles in a free field, the expansion of the bubbles inside the vessel phantom was significantly constrained, leading to asymmetric elongation of the bubbles along the vessel axis and, presumably, much weakened collapse. The severity of the constraint is vessel-size dependent, and increases dramatically when the inner diameter of the vessel becomes smaller than 300 microm. Conversely, the rapid, large intraluminal expansion of the bubbles causes a significant dilation of the vessel wall, leading to consistent rupture of the hollow fibers (i.d. = 200 microm) after less than 20 pulses of shock wave exposure in a XL-1 lithotripter. The rupture is dose-dependent, and varies with the spatial location of the vessel phantom in the lithotripter field. Further, when the large intraluminal bubble expansion was suppressed by inversion of the lithotripter pressure waveform, rupture of the hollow fiber could be avoided even after 100 shocks. Theoretical calculation of SWL-induced bubble dynamics in blood confirms that the propensity of vascular injury due to intraluminal bubble expansion increases with the tensile pressure of the lithotripter shock wave, and with the reduction of the inner diameter of the vessel. It is suggested that selective truncation of the tensile pressure of the shock wave may reduce tissue injury without compromising the fragmentation capability of the lithotripter pulse.     

Ultrasonically induced gas bubble production in agar based gels: Part I. Experimental investigation

Macroscopically visible gas bubbles can be produced in an agar based gel by irradiation with either continuous or pulsed ultrasound at frequencies from 0.75 to 3.0 MHz. The variation in the number of bubbles formed with frequency, acoustic pressure, pulse length, duty cycle, and temperature closely resembles that seen in vivo. Furthermore, the acoustic pressure required to initiate bubble formation is also close to that required in vivo. It has been observed that alterations in the concentration and pH of the gels can have a profound effect on the nature and quantity of bubbles. This suggests that not only is this gel model suitable for the representation of the macroscopic features of bubble formation in vivo, but can be used to gain information about the preexisting bubble nuclei. Based on the experimental results obtained it can be suggested that for peak negative acoustic pressures of up 1 MPa (equivalent, for a plane travelling sinusoidal wave, to a time averaged intensity of 30 W/cm2) bubble formation can be avoided by the use of high frequencies, short pulse lengths and long duty cycles.     

Towards aberration correction of transcranial ultrasound using acoustic droplet vaporization

We report on the first experiments demonstrating the transcranial acoustic formation of stable gas bubbles that can be used for transcranial ultrasound aberration correction. It is demonstrated that the gas bubbles can be formed transcranially by phase-transitioning single, superheated, micron-size, liquid dodecafluoropentane droplets with ultrasound, a process known as acoustic droplet vaporization (ADV). ADV was performed at 550 kHz, where the skull is less attenuating and aberrating, allowing for higher-amplitudes to be reached at the focus. Additionally, it is demonstrated that time-reversal focusing at 1 MHz can be used to correct for transcranial aberrations with a single gas bubble acting as a point beacon. Aberration correction was performed using a synthetic aperture approach and verified by the realignment of the scattered waveforms. Under the conditions described below, time-reversal aberration correction using gas bubbles resulted in a gain of 1.9 +/- 0.3 in an introduced focusing factor. This is a small fraction of the gain anticipated from complete transmit-receive of a fully-populated two-dimensional array with sub-wavelength elements. (E-mail: khaworth@umich.edu).     

Attenuation correction in ultrasound contrast agent imaging: elementary theory and preliminary experimental evaluation

Progress in imaging and quantification of tissue perfusion using ultrasound (US) and microbubble contrast agents has been undermined by the lack of an effective automatic attenuation correction technique. In this article, an elementary model of the US attenuation processes for microbubble contrast enhanced imaging is developed. In the model, factors such as nonlinear bubble scattering, nonlinear attenuation, attenuation to both fundamental and harmonic and the US beam profile are considered. Methods are proposed for fast formation of images with automatic attenuation correction based on the model. In the proposed method, linear tissue echoes are extracted and filtered and then used to compensate for the attenuation in nonlinear bubble echoes at the same location to produce quantities that are a truer representation of bubble concentration. The technique does not require additional measurements and can be implemented in real time. Preliminary experiments on laboratory phantoms consisting of bubbles and tissue-mimicking materials are presented and the effectiveness of the proposed method is supported by improvements in image quality compared with unprocessed data. This development is an important step towards real-time quantitative contrast US imaging.     

Numerical and Experimental Study of Mechanisms Involved in Boiling Histotripsy

The aim of boiling histotripsy is to mechanically fractionate tissue as an alternative to thermal ablation for therapeutic applications. In general, the shape of a lesion produced by boiling histotripsy is tadpole like, consisting of a head and a tail. Although many studies have demonstrated the efficacy of boiling histotripsy for fractionating solid tumors, the exact mechanisms underpinning this phenomenon are not yet well understood, particularly the interaction of a boiling vapor bubble with incoming incident shockwaves. To investigate the mechanisms involved in boiling histotripsy, a high-speed camera with a passive cavitation detection system was used to observe the dynamics of bubbles produced in optically transparent tissue-mimicking gel phantoms exposed to the field of a 2.0-MHz high-intensity focused ultrasound (HIFU) transducer. We observed that boiling bubbles were generated in a localized heated region and cavitation clouds were subsequently induced ahead of the expanding bubble. This process was repeated with HIFU pulses and eventually resulted in a tadpole-shaped lesion. A simplified numerical model describing the scattering of the incident ultrasound wave by a vapor bubble was developed to help interpret the experimental observations. Together with the numerical results, these observations suggest that the overall size of a lesion induced by boiling histotripsy is dependent on the sizes of (i) the heated region at the HIFU focus and (ii) the backscattered acoustic field by the original vapor bubble.     

Theoretical study on shear stress generated by microstreaming surrounding contrast agents attached to living cells

Numerical calculations have shown that shear stress associated with microstreaming surrounding encapsulated stable bubbles of contrast agents, near living cells driven by 0.12-MPa acoustic pressure amplitude ultrasound (US) at 1 MHz or 2 MHz, may be large enough to generate reparable sonoporation of the cells. Some encapsulated bubbles that have mechanically weak shells may break into free bubbles under the above-mentioned sound field. When that happens, the shear stress caused by microstreaming surrounding the free bubble increases dramatically and may play an important role in lethal sonoporation and fragmentation of cells during the early stage of US exposure.