Quantitative Measurement of Ultrasound Disruption of Polymer-Shelled Microbubbles

Quantitative Measurement of Ultrasound Disruption of Polymer-Shelled Microbubbles

Ultrasound in Med. & Biol., Vol. 33, No. 11, pp. 1777–1786, 2007 Copyright © 2007 World Federation for Ultrasound in Medicine & Biology Printed in the...

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Ultrasound in Med. & Biol., Vol. 33, No. 11, pp. 1777–1786, 2007 Copyright © 2007 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/07/$–see front matter

doi:10.1016/j.ultrasmedbio.2007.05.013

● Original Contribution QUANTITATIVE MEASUREMENT OF ULTRASOUND DISRUPTION OF POLYMER-SHELLED MICROBUBBLES PETER D. BEVAN,* RAFFI KARSHAFIAN,* E. GLENN TICKNER,‡ and PETER N. BURNS*† *Department of Medical Biophyscs, University of Toronto and †Sunnybrook Health Sciences Centre, Toronto, ON, Canada, and ‡Point Biomedical Corporation, San Carlos, CA, USA (Received 24 January 2006; revised 8 May 2007; in final form 17 May 2007)

Abstract—The goal of this study was to assess the threshold of disruption and subsequent time-course of acoustic response of four experimental nitrogen-filled polymer-shelled microbubbles. Using an in vitro measurement system, a sequence of low-amplitude detection pulses measured the change in echo caused by a high-amplitude disruption pulse on a dilute suspension of bubbles. Detection pulses were transmitted 0.5 ms before disruption and between 1 and 200 ms after disruption. Separate transducers, aligned confocally and orthogonally, were used to transmit and receive bubble echoes. After disruption, all agents demonstrated a transient increase in scattered power. Above the disruption threshold, highly echogenic, nonlinear scatterers were observed. Their echoes slowly disappeared after disruption with median decay times from 7.4 to 13.6 ms, calculated by fitting to a monoexponential decay. This is consistent with a process wherein the shell is disrupted, releasing the gas and generating free gas bubbles, which cause high-amplitude nonlinear scattering followed by relatively slow diffusion of the gas into solution. This picture has been observed optically with single bubbles and differs from the concept of “stimulated acoustic emission” from disrupted bubbles. (E-mail: [email protected]) © 2007 World Federation for Ultrasound in Medicine & Biology. Key Words: Ultrasound, Contrast agents, Microbubbles, Bubble disruption, Nonlinear ultrasound imaging.

ing tissue perfusion used high-MI power or colour Doppler (Burns et al. 1994). It was recognized that the disruption of the microbubbles was associated with a high amplitude “flash” in a Doppler imaging frame. UCA disruption results in large phase shifts between successive echoes that appear as a high Doppler power or a mosaic of random colour Doppler velocity estimates. Quantitative studies have shown that the power Doppler enhancement in the “flash” frame is proportional to the UCA concentration (Tiemann et al. 2000). Terms such as “stimulated acoustic emission” (Hauff et al. 1997; Blomley et al. 1998) reflect the view that the bright flash seen on B-mode or colour imaging after disruption has its origin in some sort of energy release, much as a balloon pops when it is burst. The apparent release of power has also been described as “cavitation” energy (Porter 1997). In this study, we show that the origin of these signals is scatter from bubbles of free gas that are released into the medium by shell fracture. The imaging signal derived from this echo can either be detected using a method such as harmonic imaging (Porter and Xie 1995; de Jong et al. 1996) or by decorrelation of echoes from successive pulses in Dopp-

INTRODUCTION The development of microbubble ultrasound contrast agents (UCAs) has made it possible to detect and image blood flowing in small vessels. The nonlinear behaviour of the bubbles in an acoustic field allows them to be detected even in small capillaries and at low velocities. Nonlinear scattering characteristics are related to the peak negative pressure amplitude of the incident ultrasound. At low mechanical index (MI), the bubbles undergo stable harmonic oscillation which results in a nonlinear echo that can be used to provide tissue-to-UCA contrast. At a sufficiently high pressure, the same sound that is used to image these microbubbles can rupture the shell, releasing the gas inside. This bubble disruption is an important phenomenon associated with the response of UCAs at high MI. Many UCA imaging techniques have been developed that either rely on or are made more sensitive by bubble disruption. One of the earliest methods for imagAddress correspondence to: Peter N. Burns, Sunnybrook Health Sciences Centre S660, 2075 Bayview Ave., Toronto, Ontario, Canada, M4N 3M5. E-mail: [email protected] 1777

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ler modes such as harmonic power Doppler (Burns et al. 1992) or pulse inversion imaging (Hope Simpson et al. 1999). Bubble disruption also plays an important role in the quantitative measurement of perfusion in, for example, the myocardium. In a “triggered” or “intermittent” imaging mode, bubbles that replenish the myocardium after different delays are detected by disrupting them (Porter and Xie 1995). By measuring the replenishment rate, estimates of relative blood flow rate and vascular volume can be deduced (Wei et al. 1998). Newer, low MI methods (Tiemann et al. 1999) allow this “negative bolus” method to be implemented in real-time. Therapeutic applications have also been proposed that rely on the ability to disrupt bubbles. Encapsulated genes or drugs might be delivered to a specific target site (Lanza et al. 1996; Unger et al. 1996; Lindner et al. 2000). Targeting ligands would attach to clinically relevant receptors; any encapsulated therapeutic agent could be released locally by disrupting the targeted bubbles (Postema et al. 2004; Shortencarier et al. 2004; Rychak et al. 2005). Finally, there is evidence to suggest that the behaviour of bubbles near cells and endothelial linings, oscillating and collapsing under the influence of ultrasound, can potentiate the uptake of macromolecules and therapeutic agents (Kudo et al. 2002; Wolfrum et al. 2002; Van Wamel et al. 2004; Karshafian et al. 2005). In order for such applications to be realised in practice, the induction of bubble disruption and the subsequent behaviour of the targets need to be understood. Theoretical models and optical observations of single bubbles undergoing resonant oscillation and disruption provide information that complements the acoustical study of a population of bubbles: the latter is presented in this work. Here, no new model is proposed. Instead, we study the experimental behaviour of a population of bubbles comprising several formulations of air-polymer formulation, one of which (PB127) has also been examined using high-speed optical video-microscopy (Bouakaz et al. 2005; Postema et al. 2005). In this study, acoustic measurements were made over a range of pressures and the postdisruption acoustic response for each bubble formulation was measured.

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interface) (Ottoboni et al. 2001). The agents have the same mean bubble diameter (3.9 ⫾ 1.0 ␮m; measurement using 30 ␮m aperture on Multisizer 3 from Beckman Coulter, Fullterton, CA, USA, shown in Fig. 1), contain the same gas (N2) and have shell thicknesses that vary over a factor of five as follows: M1204 ⬍ PB127 ⬍ M1211 ⬍ M1212. The agents were all handled the same way. First, by suspending in 10 mL of deionised water and swirling by hand, then by dilution by adding 0.2 mL to 1000 mL deionised water at room temperature, in gas equilibrium with room air. Experimental setup The acoustic setup for this experiment is shown in Fig. 2a. A 100 M sample/S arbitrary waveform generator (Sony Tektronix AWG520, Richardson, TX, USA) produced two types of pulses: (1) low amplitude detection pulses, to interrogate the bubble population and (2) high amplitude disruption pulses, that could disrupt the bubbles. The detection pulses had 2.0 MHz centre-frequency and Gaussian-envelope, full-width-half-maximum (FWHM) of 0.33 MHz. The disruption pulses were 2.0 MHz centre-frequency eight-cycle tone bursts. The pulses were amplified (ENI, model 240L, Rochester, NY, USA, or Amplifier Research, model 150A1008, Souderton, PA, USA) to a peak negative pressure of 30 kPa for the detection pulses and up to 2.8 MPa for the disruption pulses. The UCAs were exposed to peak negative pressures of 0, 480, 1130 and 1950 kPa. The thickest-shelled agents (M1211 and M1212) were also exposed to a peak negative pressure of 2.8 MPa. The pressures were measured in a water tank using a calibrated membrane hydrophone (Sonora Medical Systems, Longmont, CO, USA) placed at the acoustic focus of the transducer. Separate transmit (2.25 MHz, 1.0 inch diameter,

METHODS Contrast agents Four different agents were studied, each with a polymer shell encapsulating nitrogen gas. They were developed by Point Biomedical (San Carlos, CA, USA) and have an inner shell of biodegradable polymer (to provide physical structure and control the acoustic response) and an outer shell (to function as a biological

Fig. 1. Bubble size distribution (M1212 bubbles shown) measured using multisizer 3 (Beckman Coulter Inc., Fullerton, CA, USA).

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Fig. 2. (a) Schematic of experimental setup. (b) Pulse sequence for the experiment: Eight low-amplitude detection pulses and one disruption pulse.

2.0-inch spherical focus; Matec Inc., Northborough, MA, USA) and receive (3.5 MHz, 0.75-inch diameter, 1.5inch spherical focus; Matec) transducers were placed in a water tank and aligned confocally at a 90° angle. The receive transducer was connected to an analogue-todigital (A/D) acquisition system (eight-bit A/D oscilloscope (LC574AL LeCroy Inc., Chestnut Ridge, NY, USA) or 12-bit A/D high-speed PCI bus acquisition system [DC440, Acqiris, Monroe, NY, USA]). The exposure and acquisition sequences were controlled using specifically designed software (Labview, National Instruments, Austin TX, USA). A small flow cell with mylar windows was placed at the intersection of the focal zones of the two transducers. The acoustic active volume, or region of interest (ROI), situated near the centre of the flow cell, was approximately 3 mm3. Contrast agent flowed from a reservoir above the flow cell and discharged below it. EXPERIMENTAL PROCEDURES The diluted suspension of contrast agent was allowed to flow initially for two min, to replenish the flow cell completely. Between measurements, diluted agent flowed for 30 s to refresh the bubbles within the ROI; flow was then stopped for 60 s. The sequence of pulses transmitted for each measurement is shown in Fig. 2b, comprising a predisruption

detection pulse at t ⫽ ⫺0.5 ms, a disruption pulse at t ⫽ 0 ms and a series of detection pulses at t ⫽ 1, 10, 20, 30, 50, 100 and 200 ms. The peak negative pressure (Pneg) of the detection pulses was constant, 30 kPa (MI ⫽ 0.02). This was weak enough to not disrupt the bubbles, yet strong enough to maintain a reasonable signal-to-noise ratio. A test was carried out to determine if the detection pulses influenced the disappearance time of the bubbles: the echo and decay was measured when only three of the eight pulses were transmitted. Fourteen measurements were made for each of: (1) the full eight-pulse detection sequence and (2) a truncated three-pulse sequence with detection pulses at ⫺0.5, 1 and 50 ms. The data were compared using a matched two-way analysis of variance (ANOVA). For each sequence of measurements, the disruption pulse pressure was adjusted to one of five values: Pneg ⫽ 0, 0.480, 1.13, 1.95 or 2.8 MPa (MI ⫽ 0, 0.34, 0.8, 1.4 and 2.0, respectively). The measurement was repeated at least 14 (and up to 68) times for each disruption pulse amplitude, depending on the availability and in vitro stability of each agent. Data analysis The acquired echoes were analysed in MATLAB (Mathorks Inc., Natick, MA, USA) by integrating the noise-subtracted power spectrum from 1.5 to 4.5 MHz.

Noise was measured by repeating the experiments with no bubbles in the ROI. The resulting mean noise power spectrum was subtracted from the bubble power spectrum. The bubble amplitude spectrum was corrected to compensate for the receive transducer response, which was measured from the two-way broadband response with a quartz flat at the acoustic focus of the transducer, using a pulser-receiver (model 5052PR, Panametrics Inc, Waltham, MA, USA). Following this procedure, a relative scattered power, ␴rel, was calculated for each measurement time point:

␴rel ⫽



f⫽4.5MHz

f⫽1.5MHz

Pt( f ) ⫺ Pnoise( f ) df, Ptrans( f )

(1)

where Pt共 f 兲 is the scattered echo power spectrum at detection pulse time t, Pnoise共 f 兲 is the noise power spectrum and Ptrans共 f 兲 is the transducer response power spectrum. For each acquisition, the ␴rel versus time curves were fitted, from the peak echo power to the end of the time series, to a mono-exponential decay, as suggested by Tickner et al. (2001) and Sboros et al. (2001): g(t) ⫽ Ae⫺(t⫺Dt) ⁄ ␶ ⫹ C.

(2)

In eqn 2, the most important parameters are ␶, the decay time constant, and Dt, the time to peak enhancement postdisruption. For each measurement, the time to peak was measured by hand. Although generally Dt was chosen to be the maximum postdisruption measurement, if a prominent secondary peak was obvious in the data then that was used instead. If there was no obvious peak, Dt was chosen to be at t ⫽ 1 ms, the first measurement after the disruption pulse. ANOVA statistical tests (and posttests) were used to compare between agents. Values below p ⫽ .05 were considered significant. Predisruption echo powers, postdisruption enhancement, drop in echo power after disruption, and decay time constants (␶ from monoexponential fits) were analysed. Theoretical considerations As bubbles shrink slowly, there is a mass transfer from inside the bubble to the surrounding liquid. Epstein and Plesset (1950) describe a differential equation to describe the change in radius of a free bubble as the gas diffuses into the surrounding liquid over time:

再 冑 冎

d␧ (1 ⫺ f ) ⫹ ␦ ⁄ ␧ x ⫽⫺ ⫹2 dx 1 ⫹ 2␦ ⁄ 3␧ ␧

cs , ␳2 ␲

a , x⫽ a0

time t, a0 is the initial bubble radius, ␬ is the gas diffusivity (20 ⫻ 10⫺6 cm2/s), ␳ is the gas density (0.0012 g/cm3), M is the molecular weight of the gas (28 g/mol), ␴ is the surface tension (72 dyne/cm), B is the universal gas constant, T is the temperature (293°K), and ci and cs are the initial and saturation gas concentrations in the liquid respectively. Here, it is assumed that the ratio between these concentrations, f, is one for a saturated solution of deionised water at gas equilibrium with air. The numerical solution of eqn 3 for a 4 ␮m diameter N2-filled bubble is shown in Fig. 3a. It is expected, then, that the echo from this bubble will change as the bubble shrinks. The theory by Keller and Kolodner (1980) can be used to estimate this low-amplitude scattered echo:

冉 冊 冉 冊 冉 冊

aa¨ 1 ⫺

˙ a 3 2 1 a˙ ⫹ ˙ a 1⫺ c 2 3c ⫺ 1⫹

(3)

冑冉 冊

2␬ cs 2M ␴ ci t, ␦ ⫽ , and f ⫽ . BTa0␳(⬁) cs ␳a02

In these equations, a is the bubble radius as a function of

˙ a pL ⫺ p0共t ⫹ a ⁄ c兲 a ⫺ ˙ p ⫽ 0, c ␳ ␳c L

(4)

where c is the speed of sound, pL is the pressure in the liquid at the bubble wall, p0 is the ambient pressure and pi is the incident acoustic pressure. In Fig. 3b, the pre-

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Fig. 3. (a) The numerical solution to the Epstein-Plesset equation for a shrinking air bubble in water with an initial diameter of 4 ␮m demonstrates that the bubble has completely dissolved by 140 ms after the onset of dissolution. (b) The scattered echo power (predicted by the Keller-Miksis model of bubble behaviour) over time for a shrinking air bubble in water that has an initial diameter of 4 ␮m shows an increase shortly after the onset of dissolution and the effective disappearance of the echo by approximately 100 ms.

Disruption of polymer microbubbles ● P. D. BEVAN et al.

Influence of detection pulses on bubbles The low amplitude pulse did not significantly influence the disappearance time of the bubbles: the measured echo and decay was not significantly different when only three of the seven pulses were transmitted. This is shown graphically in Fig. 4 for M1204 and Pneg ⫽ 1130 kPa. No significant statistical difference was found between the two pulsing sequences. Data from detection pulses An example of the eight detection pulses recorded for one bubble measurement is shown in Fig. 5a. In the measurements where bubble disruption was evident (as here), an increase in echo power was observed a short time after disruption. After this peak, there was a decay of the echo to lower than the initial (predisruption) level. The integrated echo power versus time (and corresponding mono-exponential fit) for these data are shown in Fig. 5b as a plot of ␴rel, normalised to the predisruption level (at t ⫽ ⫺0.5 ms), against detection pulse time. Integrated power of predisruption detection echoes In general, the predisruption (t ⫽ ⫺0.5 ms) detection pulse integrated power was higher for thinnerwalled agents. A comparison of the predisruption echo powers is shown in Fig. 6. The scattered echo power decreased as shell thickness increased with M1204 ⬎

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dicted integrated echo power over time (calculated for the same low-amplitude broadband detection pulse used in the experiments described in this work) is shown for the shrinking bubble of Fig. 3a.

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Fig. 5. (a) An example of the detected signal from eight detection pulses. This is one acquisition for the Point Biomedical M1204 bubbles, with a 1.13 MPa peak negative pressure pulse between the first two detection pulses, at t ⫽ 0. The eight detection pulses have been temporally windowed with a Hanning window. (b) Integrated power representation of the detected signal in (a), normalized to the predisruption value. The integration range for this analysis was between 1.5 and 4.5 MHz, after correcting the receive power spectrum for electronic noise and receive transducer response. Also shown is the monoexponential fit where Dt ⫽ 1 ms.

PB127 ⬎ M1211. A significant difference was seen (p ⬍ 0.001) between all of the predisruption scattered echo powers except PB127 and M1212.

Fig. 4. Comparing full eight-pulse detection sequence with a shorter three-pulse sequence (M1204, Pneg ⫽ 1130 kPa; median echo power and IQR shown). The full pulse sequence does not appear to cause the bubbles to disappear faster. (For clarity, the error bars for the “three-pulse” data are not shown in the graph.)

Integrated power of detection echoes following disruption All bubble populations showed a similar pattern of behaviour during the 200 ms after disruption. As an example, the echo power versus time curves for M1204 are shown in Fig. 7. This shows that at all disruption pressures above zero, there is a transient increase in the echo (postdisruption enhancement) followed by decay between 1 ms and 200 ms after disruption. With no disruption pulse, there was no significant change in the echo over time. Qualitatively, the other three agents showed the same behaviour. Immediately after disruption, the targets became highly echogenic and caused nonlinear echoes. In Fig. 8a, a bubble mean amplitude spectrum (at t ⫽ 1 ms for M1204 and Pneg ⫽ 1950 kPa) is compared with the amplitude spectrum for a linear scattering target (a nee-

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Fig. 6. Predisruption echo power as a function of agent (ordered left to right from thinnest to thickest walled bubble). M1204 predisruption echo power is significantly greater than PB127 (p ⬍ 0.001) and also PB127 ⬎ M1211 (p ⬍ 0.001).

dle placed in the detection zone). The spectra have been corrected for the transducer response and normalized to their respective fundamental (2 MHz) peaks to demonstrate the enhanced second harmonic detected from the bubbles. In Fig. 8b, the mean amplitude spectra for select time points after disruption are plotted, normalized to the fundamental peak of the t ⫽ 1 ms spectrum. They are the

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average for all measurements on M1204 at Pneg ⫽ 1950 kPa. This series of spectra demonstrate how, over the full detection bandwidth, the echo increases immediately after disruption and then decreases as a function of time. The postdisruption enhancement at t ⫽ 1 ms for each of the agents and disruption pulse pressures is shown in Fig. 9. Postdisruption enhancement was compared first within each agent and then across all agents and disruption pressures. For each agent, a significant increase was seen for Pneg ⱖ 1130 kPa compared with the Pneg ⫽ 480 kPa enhancement. For Pneg ⱖ 1130 kPa, however, no significant difference was seen as a function of disruption pressure. Comparing M1204 and PB127 at 1130 and 1950 kPa disruption, no significant difference was seen in the postdisruption enhancement. The M1211 and M1212 enhancement at these pressures, however, was significantly less than for the thinner-walled agents. Even at the highest pressure (2800 kPa), the enhancement for M1211 or M1212 was significantly less than the 1130 or 1950 kPa enhancement for M1204 or PB127. Bubble disruption and disappearance At the end of the period of enhancement following disruption, an irreversible drop in echo power was seen. In the echo power versus time curves for M204 (Fig. 7), this drop can be seen for all Pneg ⱖ 480 kPa. The

Fig. 7. Experimental results for M1204 at all disruption pulse pressures, shown as integrated power (median and inter-quartile range) over the frequency range 1.5 to 4.5 MHz. At all disruption pressures above Pneg ⫽ 0, a post disruption enhancement and echo decay is evident.

Disruption of polymer microbubbles ● P. D. BEVAN et al.

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Fig. 8. (a) Comparing the raw amplitude spectra of the bubble postdisruption echo (M1204, Pneg ⫽ 1950 kPa) with scatter off a needle placed in the ROI, normalized to their respective fundamental (2 MHz) peaks. (b) Mean raw amplitude spectra over time for M1204 and Pneg ⫽ 1950 kPa, normalized to the fundamental (2 MHz) peak.

postdisruption drop in echo power was assessed for each agent and for the measurements after the disruption pulse. For all agents and Pneg ⱖ 480 kPa, a significant drop was measured between 1 and 200 ms postdisruption. Furthermore, for M1204 (where a strong predisruption echo was measured), there was also a statistically significant drop between the 200 ms time point and the predisruption echo power. These observations are consistent with the irreversible loss of these agents by 200 ms after disruption. For individual measurements when bubble disruption was evident, the median delay for the fit (Dt) was 1 ms, the first measurement after the disruption pulse. For each agent, no significant difference in decay times was seen as a function of pressure. To compare between agents, the decay times were pooled over all Pneg for a given bubble type. The decay constants for each agent are plotted in Fig. 10. The decay time for PB127 was significantly higher than M1211 (p ⬍ 0.01) and M1212 (p ⬍ 0.001) and the decay time for M1204 was significantly higher than M1212 (p ⬍ 0.001). DISCUSSION Detection pulse integrated echo power The integrated echo power from the predisruption detection pulses reflects the response of intact bubbles to nondisruption ultrasound and is probably dominated by shell stiffness, a major determinant of radial amplitude for a bubble of resonant size (de Jong et al. 2002). At constant dilution, the low-MI acoustic response of the agents was different (see Fig. 6), with the thinner-shelled agents giving a stronger echo. With comparable number density, this suggests that the low-MI scattering crosssection is dependent on shell thickness, with the thinner shells providing the highest cross-section.

Echo behaviour following disruption For agents such as these, intended to be imaged exclusively by disruption, the significance of the shell is in its influence on disruption threshold. Below the disruption threshold, all of the polymer agents were much less responsive than free bubbles. Especially at the highest pressures, there was a significant increase for all agents relative to their respective predisruption scattered echo. The transient increase in scattered echo power observed above the disruption thresholds for these agents is consistent with the release of free gas bubbles after disruption of the shell. This picture is confirmed by the optical observations of Bouakaz et al. (2005) for PB127 (Burns et al. 2004). A clear disruption threshold seems to be evident for these polymer agents. For the narrowband pulse used in these experiments, this appears to be between 480 and 1130 kPa (see Fig. 9) as the postdisruption enhancement plateaus for Pneg ⱖ 1130 kPa. Further experiments, with disruption pulse pressures inside this range, would be required to define the threshold more carefully and determine if there is shell-thickness dependence to the disruption threshold. The observed threshold and plateau in postdisruption echo may be a result of a relatively narrow size distribution and/or well-controlled shell thickness for these bubbles. More careful measurements in the 480 to 1130 kPa range might reveal differences in the disruption threshold as a function of shell thickness. Differences in echo remaining after the disruption echo has decayed, (e.g., for M1204 in Fig. 7), could be due to a dependence of disruption threshold on bubble size, which has been reported by (e.g., Chomas et al. 2001; Bouakaz et al. 2005). Above the disruption threshold, the bubbles showed a significant echo 1 ms postdisruption. The postdisruption enhancement (Fig. 9) suggests a differential amount

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Fig. 9. Postdisruption enhancement for the four agents. Significant difference is shown within each agent, comparing to the 480 kPa disruption: ** ⫽ p ⬍ 0.01, *** ⫽ p ⬍ 0.001. No significant difference is demonstrated (within each bubble type) for Pneg ⱖ 1130 kPa. Predisruption echo powers are also shown for comparison.

of gas released from the bubbles. If the number density of the agents was comparable, these results suggest that there may have only been partial disruption of the thickest-walled agents (M1211 and M1212), even at 1.13 MPa. The higher disruption pressure (2.8 MPa, MI ⫽ 2) was used to test this hypothesis. The 1 ms postdisruption echo, however, did not increase, even at this higher pressure. If this was not due to incomplete disruption of the agent, it may have been because some of the gas remained inside the shell. This would be consistent with optical observations by Bouakaz (2005) and Postema (2005) for polymer-shelled bubbles. As a result, the smaller free bubbles, even if at the same number concentration would have a weaker overall echo. Bubble disruption and disappearance After disruption, the decay of the scattered echo is probably due to the bubbles shrinking as the gas within them diffuses freely into water. This disappearance was also seen optically (Bouakaz et al. 2005). The shorter

decay times for M1211 and M1212 are consistent with the hypothesis that smaller free bubbles are released after disruption of this agent. The rapid decrease in acoustic response as bubbles shrink would lead to shorter ␶ measurements for a dilute suspension composed of smaller bubbles. Models which simulate this process in a population of bubbles may help interpret further acoustic measurements. An understanding of the physical parameters related to bubble disruption and subsequent gas diffusion is important for imaging as it determines the optimum pulse repetition frequency (PRF) for decorrelation detection: too high will reduce the perfusion signal, too low will introduce motion artefact. The results of this study suggest that a value can be determined by direct measurement of bubble behaviour. The median ␶ between 7.4 and 13.6 ms for the agents suggests that a typical sampling time for harmonic power Doppler (eight pulses at 5 kHz, i.e., 1.6 ms) might be too short for optimal decorrelation detection of these bubbles. For the optimum

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ruption of the bubbles and their disappearance over time has significance for the optimum design of imaging methods for bubble detection and perfusion measurement. Further work seeks to provide a quantitative model based on the physical properties of the gases found within the bubbles. Acknowledgments—The work was supported by the National Cancer Institute of Canada, the Canadian Institutes of Health Research and a grant from Point Biomedical Inc. The authors are grateful to Nico deJong and Ayache Boukaz for discussions on their optical findings.

Fig. 10. Decay times (from monoexponential fits) for each agent with results for all disruption pressures combined (median and inter-quartile range). Significant differences were measured between PB127 and M1211, PB127 and M1212 and M1204 and M1212.

power Doppler sampling time the almost complete decorrelation of the received signal as the bubbles disappear produces a large Doppler signal. This is the explanation of the “flash” seen in high MI harmonic Doppler imaging of bubbles and emphasizes that there is no emission in “stimulated acoustic emission”. Free bubbles shrinking through resonance Comparing the theoretical prediction of echo versus time for a single shrinking bubble to the measurements on real bubbles shown in this study, there is a striking difference in the shape of these curves. Especially, theory predicts the echo will increase before the bubble shrinks through resonant size (at approximately 10 ms after the onset of bubble dissolution). One important feature that has yet to be considered, however, is the contribution of echoes from many bubbles (with different initial sizes) in the detection zone, slowly shrinking over time. A quantitative numerical simulation of the experimental system must therefore consider a subset of bubbles taken from a known population distribution. Such a model could help explain further how the optical and acoustic measurements are related and, hopefully, reconcile some of the differences already noted in the literature (e.g., Postema et al. 2005). CONCLUSION A study of the disruption behaviour of a group of four polymer-shell, nitrogen gas microbubbles used for clinical contrast imaging shows that the mechanism of echo enhancement is gas release and nonlinear oscillation of the resulting free bubbles. The shell influences the threshold of disruption; the decay time of the resulting bubble is determined by the diffusion of free gas in the surrounding fluid. Such measurements related to the dis-

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