Single Microbubbles Research Articles

Controlled permeation of cell membrane by single bubble acoustic cavitation

Sonoporation is the membrane disruption generated by ultrasound and has been exploited as a non-viral strategy for drug and gene delivery. Acoustic cavitation of microbubbles has been recognized to play an important role in sonoporation. However, due to the lack of adequate techniques for precise control of cavitation activities and real-time assessment of the resulting sub-micron process of sonoporation, limited knowledge has been available regarding the detail processes and correlation of cavitation with membrane disruption at the single cell level. In the current study, we developed a combined approach including optical, acoustical, and electrophysiological techniques to enable synchronized manipulation, imaging, and measurement of cavitation of single bubbles and the resulting cell membrane disruption in real-time. Using a self-focused femtosecond laser and high frequency ultrasound (7.44 MHz) pulses, a single microbubble was generated and positioned at a desired distance from the membrane of a Xenopus oocyte. Cavitation of the bubble was achieved by applying a low frequency (1.5 MHz) ultrasound pulse (duration 13.3 or 40 μs) to induce bubble collapse. Disruption of the cell membrane was assessed by the increase in the transmembrane current (TMC) of the cell under voltage clamp. Simultaneous high-speed bright field imaging of cavitation and measurements of the TMC were obtained to correlate the ultrasound-generated bubble activities with the cell membrane poration. The change in membrane permeability was directly associated with the formation of a sub-micrometer pore from a local membrane rupture generated by bubble collapse or bubble compression depending on ultrasound amplitude and duration. The impact of the bubble collapse on membrane permeation decreased rapidly with increasing distance (D) between the bubble (diameter d) and the cell membrane. The effective range of cavitation impact on membrane poration was determined to be D/d = 0.75. The maximum mean radius of the pores was estimated from the measured TMC to be 0.106 ± 0.032 μm (n = 70) for acoustic pressure of 1.5 MPa (duration 13.3 μs), and increased to 0.171 ± 0.030 μm (n = 125) for acoustic pressure of 1.7 MPa and to 0.182 ± 0.052 μm (n = 112) for a pulse duration of 40 μs (1.5 MPa). These results from controlled cell membrane permeation by cavitation of single bubbles revealed insights and key factors affecting sonoporation at the single cell level.

Optical observation of cell sonoporation with low intensity ultrasound

Sonoporation is a promising drug delivery technique with great potential in medicine. However, its applications have been limited mostly by the lack of understanding its underlying biophysical mechanism, partly due to the inadequacy of the existing models for coupling with highly sensitive imaging techniques to directly observe the actual precursor events of cell–microbubble interaction under low intensity ultrasound. Here, we introduce a new in vitro method utilizing capillary-microgripping system and micro-transducer to achieve maximum level of experimental flexibility for capturing real time highly magnified images of cell–microbubble interaction, hitherto unseen in this context. Insonation of isolated single cells and microbubbles parallel with high speed microphotography and fluorescence microscopy allowed us to identify dynamic responses of cell-membrane/microbubble in correlation with sonoporation. Our results showed that bubble motion and linear oscillation in close contact with the cell membrane can cause local deformation and transient porosity in the cell membrane without rupturing it. This method can also be used as an in situ gene/drug delivery system of targeted cells for non-invasive clinical applications.

Measurement of the Resonant Characteristics of a Single Bubble Vibration by Using a Laser Doppler Vibrometer

We constructed the experimental system with a laser Doppler vibrometer (LDV) for measuring the vibration of a single microbubble. It was demonstrated that the system enabled the capture of the vibration with an amplitude of nanometer order. We attempted to experimentally measure the resonant characteristics of a bubble attached to a wall by using the system. As a result, we succeeded in measuring the characteristics and evaluating the Q factor and the resonant radius at a driving frequency of 27.8 kHz, although these values are different from those predicted on the basis of the theory for a single free bubble. The LDV measurement system is expected to an effective tool for evaluating bubble vibrations with very small displacement amplitudes, such as the vibration of a microcapsule.

Air bubble contact with endothelial cells in vitro induces calcium influx and IP3-dependent release of calcium stores.

Gas embolism is a serious complication of decompression events and clinical procedures, but the mechanism of resulting injury remains unclear. Previous work has demonstrated that contact between air microbubbles and endothelial cells causes a rapid intracellular calcium transient and can lead to cell death. Here we examined the mechanism responsible for the calcium rise. Single air microbubbles (50-150 μm), trapped at the tip of a micropipette, were micromanipulated into contact with individual human umbilical vein endothelial cells (HUVECs) loaded with Fluo-4 (a fluorescent calcium indicator). Changes in intracellular calcium were then recorded via epifluorescence microscopy. First, we confirmed that HUVECs rapidly respond to air bubble contact with a calcium transient. Next, we examined the involvement of extracellular calcium influx by conducting experiments in low calcium buffer, which markedly attenuated the response, or by pretreating cells with stretch-activated channel blockers (gadolinium chloride or ruthenium red), which abolished the response. Finally, we tested the role of intracellular calcium release by pretreating cells with an inositol 1,4,5-trisphosphate (IP3) receptor blocker (xestospongin C) or phospholipase C inhibitor (neomycin sulfate), which eliminated the response in 64% and 67% of cases, respectively. Collectively, our results lead us to conclude that air bubble contact with endothelial cells causes an influx of calcium through a stretch-activated channel, such as a transient receptor potential vanilloid family member, triggering the release of calcium from intracellular stores via the IP3 pathway.

Modeling subharmonic response from contrast microbubbles as a function of ambient static pressure.

Variation of subharmonic response from contrast microbubbles with ambient pressure is numerically investigated for non-invasive monitoring of organ-level blood pressure. Previously, several contrast microbubbles both in vitro and in vivo registered approximately linear (5-15 dB) subharmonic response reduction with 188 mm Hg change in ambient pressure. In contrast, simulated subharmonic response from a single microbubble is seen here to either increase or decrease with ambient pressure. This is shown using the code BUBBLESIM for encapsulated microbubbles, and then the underlying dynamics is investigated using a free bubble model. The ratio of the excitation frequency to the natural frequency of the bubble is the determining parameter--increasing ambient pressure increases natural frequency thereby changing this ratio. For frequency ratio below a lower critical value, increasing ambient pressure monotonically decreases subharmonic response. Above an upper critical value of the same ratio, increasing ambient pressure increases subharmonic response; in between, the subharmonic variation is non-monotonic. The precise values of frequency ratio for these three different trends depend on bubble radius and excitation amplitude. The modeled increase or decrease of subharmonic with ambient pressure, when one happens, is approximately linear only for certain range of excitation levels. Possible reasons for discrepancies between model and previous experiments are discussed.

“Compression-only” behavior: A second-order nonlinear response of ultrasound contrast agent microbubbles

Oscillating phospholipid-coated ultrasound contrast agent microbubbles display a so-called "compression-only" behavior, where it is observed that the bubbles compress efficiently while their expansion is suppressed. Here, a theoretical understanding of the source of this nonlinear behavior is provided through a weakly nonlinear analysis of the shell buckling model proposed by Marmottant et al. [J. Acoust. Soc. Am. 118, 3499-3505 (2005)]. It is shown that the radial dynamics of the bubble can be considered as a superposition of a linear response at the fundamental driving frequency and a second-order nonlinear low-frequency response that describes the negative offset of the mean bubble radius. The analytical solution deduced from the weakly nonlinear analysis shows that the compression-only behavior results from a rapid change of the shell elasticity with bubble radius. In addition, the radial dynamics of single phospholipid-coated microbubbles was recorded as a function of both the amplitude and the frequency of the driving pressure pulse. The comparison between the experimental data and the theory shows that the magnitude of compression-only behavior is mainly determined by the initial phospholipids concentration on the bubble surface, which slightly varies from bubble to bubble.

기포군 영상분석을 통한 초음파 캐비테이션 현상의 변화 관찰

In this study, in order to evaluate the yield of bubble by ultrasonic cavitation in HIFU sonication, the bubble image analysis was performed. The changing phenomenon of cavitation effect according to the sonication condition was discussed by analyzing the bubble image. Especially the appearance of bubble cloud, the size of micro-bubble, and the yield of bubble were considered. The 500 KHz and 1.1 MHz concave type ultrasonic transducers were used for HIFU sonication. Computer controlled digital camera was used to obtain the bubble image, and the binary image processing(binarization coefficient : 0.15) was performed to analyze them. In results of 500 KHz and 1.1 MHz transducer, the area of bubble cloud was increased in proportion to the rise in sonication intensity(<TEX>$R^2$</TEX> : 0.7031 and 0.811). The mean size of single microbubble was measured as 98.18 um in 500 KHz sonication, and 63.38 um in 1.1 MHz sonication. In addition, the amount of produced bubble was increased in proportion to sonication intensity. Through the result of this study and further study for variable image processing method, the quantitative evaluation of ultrasonic cavitation effects in HIFU operation could be possible with the linearity associated with the sonication conditions.

210402 マイクロバブルの非線形振動におけるバブル間相互作用の影響(OS14 非線形振動,オーガナイズド・セッション)

Recently, microbubbles attract attention as ultrasound contrast agents in the medical field because of their excellent ultrasound scattering including superharmonic response. Such superharmonic signals obtained by detecting ultrasound waves can be used for distinguish responses of contrast agents from those of surrounding tissues. Therefore, nonlinear oscillation of insonified microbubbles has been studied actively. However, most of such studies focus on dynamics of single microbubble. This research investigates how interaction between a pair of microbubbles affects their oscillation. Our numerical simulation shows that I to 2 natural frequency ratio of the two microbubbles can result in their superharmonic resonance. Its mechanism was discussed on the basis of their equations of motion coupled through nonlinear terms.

Producing single microbubbles with controlled size using microfiber

Microbubble-mediated pore formation in sonoporation includes a combination of complex processes such as collision or coalescence of translating or collapsing microbubbles with vascular cells. Although understanding the pore formation mechanisms may improve drug delivery efficiency, their details are still poorly understood. In the present study, we describe an experimental model that produces single air bubbles with controllable size. A carbon microfiber in liquids is illuminated by an infrared laser to produce individual bubbles having size comparable to that of the microfiber. The microbubbles can be physically isolated from the fiber for placing at arbitrary positions in the liquids. The lifetime of the bubbles is several tens of minutes depending on the intensity of the laser used. The preparation of the controllable air bubbles may be useful in future investigations of ultrasound-mediated microbubble–cell interactions.

Zeta potential of single and polymer-coated microbubbles using an adapted microelectrophoresis technique

The zeta potential of microbubbles, generated by injecting pressurised air into water and then releasing the pressure, was determined in the absence and presence of different polyacrylamides. Air was dissolved in either water or solutions of cationic, anionic, amphoteric or nonionic polymers at a constant pressure of four atmospheres. The charge of the bubbles at the shear-zeta plane was measured using a modified microelectrophoresis glass cell, held at a stationary level, at varying pH (2.0–12.0). Known practical problems with this technique were solved, and these solutions are described in detail. The anionic, amphoteric and nonionic polymers increased the negative charge of the bubbles, but the isoelectric point (iep) remained constant at about pH 2.0. Conversely, in the presence of the cationic polymer, the bubbles exhibited positive surface charges between pH 2.0 and 8.0 and an iep of pH 8.0. The results revealed the existence of an important interaction mechanism between air bubbles and polymeric macromolecules; neither this mechanism nor its practical implications have been reported in the literature to date, especially in the treatment of wastewater by flocculation followed by dissolved air flotation (DAF).

A Statistical Model for the Quantification of Microbubbles in Destructive Imaging

Quantification of targeted ultrasound contrast agents allows for the monitoring of endothelial marker expressions on a molecular level. In this study, a statistical correction is provided, which allows for improved precision in estimating the concentration of microbubbles (MBs) from Doppler images. Doppler imaging can be used to display the destruction of single MBs. However, concentrations will generally be too high to distinguish the individual events resulting in an inaccurate microbubble (MB) quantification. Therefore, a mathematical description of destruction events in Doppler images is developed which yields a correction formula for the concentration estimate from the color pixel density. The mathematical model is experimentally verified in gelatin phantoms using a high resolution imaging system (Vevo 770) and experimental cyanoacrylate MBs. Sensitive Particle Acoustic Quantification (SPAQ) is used to quantify MB in a defined volume. The SPAQ step size is varied from 32 to 127 μm to demonstrate the validity of the model for high color pixel densities. The corrected acoustic quantification shows the expected linear dependence on the step size and thus the amount of MBs in the images (R = 0.95). At SPAQ step sizes up to 127 μm, a MB concentration of 2.7 × 10 MBs/mL can be quantified. The results demonstrate the validity of the proposed correction. Quantification results of the SPAQ technique were considerably improved. The resulting formula is readily applied to SPAQ measurements at no additional expense.

Acoustic Sizing of an Ultrasound Contrast Agent

Because the properties of ultrasound contrast agent populations after administration to patients are largely unknown, methods able to study them noninvasively are required. In this study, we acoustically performed a size distribution measurement of the ultrasound contrast agent Definity ®. Single lipid-shelled microbubbles were insonified at 25 MHz, which is considerably higher than their resonance frequency, so that their acoustic responses depended on their geometrical cross sections only. We calculated the size of each microbubble from their measured backscattered pressures. The acoustic size measurements were compared with optical reference size measurements to test their accuracy. Our acoustic sizing method was applied to 88 individual Definity ® bubbles to derive a size distribution of this agent. The size distribution obtained acoustically showed a mean diameter (2.5 μm) and a standard deviation (0.9 μm) in agreement within 8% with the optical reference measurement. At 25 MHz, this method can be applied to bubble sizes larger than 1.2 μm in diameter. It was observed that similar sized bubbles can give different responses (up to a factor 1.5), probably because of shell differences. These limitations should be taken into account when implementing the method in vivo. This acoustic sizing method has potential for estimating the size distribution of an ultrasound contrast agent noninvasively. (E-mail: d.maresca@erasmusmc.nl)

The Acoustic Scatter from Single biSphere Microbubbles

Single microbubble acoustic acquisitions provide information on the behaviour of microbubble populations by enabling the generation of large amounts of data. Acoustic signals from single polylactide-shelled and albumin coated biSphere™ microbubbles have been acquired. The responses observed from a range of incident frequencies and acoustic pressures varied in duration. Partial echoes shorter than the incident pulse duration have been observed for low frequency pulses of sufficient amplitude, suggesting release of gas from bubbles. The results presented suggest that the mechanism of scatter from hard shelled agents may be shell disruption and gas release, or partly from gas leaking from defected shell sites, which has previously not been observed optically. These results can provide the basis for improved imaging through optimization of incident pulse parameters, with potential benefits to both diagnostic and therapeutic techniques. (E-mail: d.h.thomas@ed.ac.uk)

Determination of postexcitation thresholds for single ultrasound contrast agent microbubbles using double passive cavitation detection

This work presents experimental responses of single ultrasound contrast agents to short, large amplitude pulses, characterized using double passive cavitation detection. In this technique, two matched, focused receive transducers were aligned orthogonally to capture the acoustic response of a microbubble from within the overlapping confocal region. The microbubbles were categorized according to a classification scheme based on the presence or absence of postexcitation signals, which are secondary broadband spikes following the principle oscillatory response of the ultrasound contrast agent and are indicative of the transient collapse of the microbubble. Experiments were conducted varying insonifying frequencies (0.9, 2.8, 4.6, and 7.1 MHz) and peak rarefactional pressures (200 kPa to 6.2 MPa) for two types of contrast agents (Definity and Optison). Results were fit using logistic regression analysis to define pressure thresholds where at least 5% and 50% of the microbubble populations collapsed for each frequency. These thresholds were found to occur at lower pressures for Definity than for Optison over the range of frequencies studied; additionally, the thresholds occurred at lower pressures with lower frequencies for both microbubble types in most cases, though this trend did not follow a mechanical index scaling.

Probing microbubble targeting with atomic force microscopy

Microbubble science is expanding beyond ultrasound imaging applications to biological targeting and drug/gene delivery. The characteristics of molecular targeting should be tested by a measurement system that can assess targeting efficacy and strength. Atomic force microscopy (AFM) is capable of piconewton force resolution, and is reported to measure the strength of single hydrogen bonds. An in-house targeted microbubble modified using the biotin–avidin chemistry and the CD31 antibody was used to probe cultures of Sk-Hep1 hepatic endothelial cells. We report that the targeted microbubbles provide a single distribution of adhesion forces with a median of 93 pN. This interaction is assigned to the CD31 antibody–antigen unbinding event. Information on the distances between the interaction forces was obtained and could be important for future microbubble fabrication. In conclusion, the capability of single microbubbles to target cell lines was shown to be feasible with AFM.

Nonlinear Emission from Individual Bound Microbubbles at High Frequencies

Targeted microbubbles detected with high-frequency ultrasound can establish the molecular expression of blood vessels with submillimeter resolution. To improve microbubble-specific imaging at high frequencies, the subharmonic and second harmonic signal from individual microbubbles were measured as a function of size and pressure. Single phospholipid-shell microbubbles (1.1 to 5.0 μm in diameter) bound to gelatin, co-aligned with an optical microscope and transducer, were insonated with 30 MHz Gaussian-enveloped pulses at pressures from 20 kPa to 1 MPa with –6 dB one-way bandwidths of 11%, 20% and 45%. A subharmonic signal (15 MHz) was detected above a pressure threshold of 110 kPa—independent of bandwidth. The signal peaked for microbubbles 1.60 μm in diameter subject to 20% and 11% bandwidth pulses, and 1.80 μm for 45% bandwidth pulses, for pressures up to 400 kPa, agreeing with the notion that microbubbles insonated at twice their resonant frequency preferentially emit a subharmonic component. For pressures between 400 kPa and 1 MPa, a broader range of microbubbles emitted a subharmonic signal, and microbubbles below 1.70 μm in diameter were disrupted. The second harmonic signal measured, within the limited experimental conditions, was consistent with nonlinear propagation. Further, the results shed light on the effect of the shell on the phase of the subharmonic signal with respect to the fundamental signal. (E-mail: michael.sprague@sri.utoronto.ca)

Radial modulation of single microbubbles

Radial modulation imaging is a new promising technique to improve contrast-enhanced ultrasound images. The method is based on dual-frequency insonation of contrast agent microbubbles. A low-frequency (LF) pulse is used to modulate the responses of the microbubbles to a high-frequency (HF) imaging pulse. Inverting the LF pulse induces amplitude and phase differences in the HF response of contrast agent microbubbles, which can be detected using Doppler techniques. Although the technique has been successfully implemented, no consensus persists on parameter choice and resulting effects. In a separate study, "compression-only" behavior of coated microbubbles was observed. Compression-only behavior could be beneficial for radial modulation imaging. This was investigated using high-speed camera recordings and simulations. We recorded the vibrations of 78 single microbubbles in a dual-frequency ultrasound field. The results showed that the LF pulse induced significant compression-only behavior, which for microbubble sizes below and at HF resonance resulted in high radial amplitude modulation. It, however, also appeared that, for radial modulation imaging, microbubble size is more important than resonance and compression-only effects.

Wave scattering from encapsulated microbubbles subject to high-frequency ultrasound: Contribution of higher-order scattering modes

The theoretical understanding of encapsulated microbubble response to high-frequency ultrasound (HFUS) excitation is still limited although some novel experimental HFUS contrast imaging techniques have been well developed. In this paper, the higher-order modal (HOM) contributions to the scattered field are studied for such microbubbles driven by 1-100 MHz ultrasound. An exact solution of all small-amplitude vibrational modes of a single encapsulated microbubble in water is given by the wave scattering theory (WST) method and compared to results obtained from Church's Rayleigh-Plesset-like model for the small-amplitude radial oscillation of a microbubble in an incompressible fluid. From numerical results, we show that the HOM field contribution is significant for scattering properties from individual Nycomed microbubbles with normalized frequency > or = 0.2. It is also shown that the multiple scattering is strengthened for monodispersed Definity microbubbles of 3 microm radius at frequencies >40 MHz. However, comparisons between the authors' analyses and known experimental data for polydispersed Definity microbubbles indicate that the HOM contributions are insignificant in attenuation estimation at frequencies <50 MHz. In conclusion, the WST model analysis suggests that HOM scattering is an important consideration for single bubbles but may be less critical in the modeling of polydispersed Definity bubbles at high frequencies.

Acoustic characterization of single ultrasound contrast agent microbubbles

Individual ultrasound contrast agent microbubbles (BR14) were characterized acoustically. The bubbles were excited at a frequency of 2 MHz and at peak-negative pressure amplitudes of 60 and 100 kPa. By measuring the transmit and receive transfer functions of both the transmit and receive transducers, echoes of individual bubbles were recorded quantitatively and compared to simulated data. At 100 kPa driving pressure, a second harmonic response was observed for bubbles with a size close to their resonance size. Power spectra were derived from the echo waveforms of bubbles of different sizes. These spectra were in good agreement with those calculated from a Rayleigh-Plesset-type model, incorporating the viscoelastic properties of the phospholipid shell. Small bubbles excited below their resonance frequency have a response dominated by the characteristics of their phospholipid shell, whereas larger bubbles, excited above resonance, have a response identical to those of uncoated bubbles of similar size.

Phospholipid-stabilized microbubbles: Influence of shell chemistry on cavitation threshold and binding to giant uni-lamellar vesicles

We demonstrate the feasibility of covalently linking a single microbubble to a single, giant uni-lamellar vesicle (GUV). Such a combination of GUV plus microbubble might prove useful as a new drug delivery vehicle involving microbubble cavitation-induced sonoporation of the vesicle bilayer as a release mechanism. We therefore applied the well known methodology of passive cavitation detection to measure the influence of lipid shell chemistry on inertial cavitation thresholds for externally added microbubbles. We find that cavitation threshold changes significantly with changes in either molecular weight or mole fraction of poly(ethylene glycol), historically used to impede gas dissolution and microbubble coalescence. We attribute changes in cavitation threshold to changes in microbubble resonance frequency resulting from changes in microbubble shell bending elasticity. To further demonstrate the influence of shell chemistry on microbubble behavior, we describe how several common bubble phenomena – and some new – respond to changes in lipid chain length.