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Characterization of Acoustic Droplet Vaporization for Control of Bubble Generation Under Flow Conditions
Ultrasound in Med. & Biol., Vol. 40, No. 3, pp. 551–561, 2014 Copyright Ó 2014 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/$ - see front matter
http://dx.doi.org/10.1016/j.ultrasmedbio.2013.10.020
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Original Contribution CHARACTERIZATION OF ACOUSTIC DROPLET VAPORIZATION FOR CONTROL OF BUBBLE GENERATION UNDER FLOW CONDITIONS SHIH-TSUNG KANG, YI-LUAN HUANG, and CHIH-KUANG YEH Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, Taiwan (Received 3 June 2013; revised 20 October 2013; in final form 21 October 2013)
Abstract—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. (E-mail: [email protected]) Ó 2014 World Federation for Ultrasound in Medicine & Biology. Key Words: Acoustic droplet vaporization, Flow condition, Diffusion-assisted bubble growth, Perfluorocarbon droplet, Size control and manipulation.
INTRODUCTION
gas nucleation and/or cavitation that triggers the transformation of these droplets into gaseous bubbles, with the ADV threshold being higher for smaller droplets (Kripfgans et al. 2004; Shpak et al. 2013; Wong et al. 2011). The droplet-to-bubble transition results in an instant volume expansion, permitting substantial bubble formation from small droplet doses (Kripfgans et al. 2000). The ideal expansion ratio falls within the range of 3.5 to 5.4 times in diameter, which decreases with decreasing droplet size because of increased Laplace pressure (Sheeran et al. 2011b). Several studies have indicated that ADV produces localized ultrasound contrast enhancement (as point sources) that can be used in the correction of transcranial ultrasound phase aberrations (Carneal et al. 2011; Haworth et al. 2008). In possible therapeutic applications, the produced bubbles could serve as gas emboli to reduce or stop the blood supply to tumors or to enhance high-intensity focused ultrasound (HIFU) ablation by increasing the amount of cavitation
Acoustic droplet vaporization (ADV) has received increasing attention in recent years because of its ability to produce microbubbles on demand in a non-invasive and localized manner (Kawabata et al. 2010; Kripfgans et al. 2000; Rapoport et al. 2007; Reznik et al. 2012; Sheeran et al. 2011a). Such droplets are often based on liquid perfluorocarbons (PFCs) with low boiling points, which can be maintained in a superheated state without vaporization after they have been emulsified into micron to submicron droplets. However, exposure to ultrasound pulses with acoustic pressures above a particular level (termed the ADV threshold) can initiate
Address correspondence to: Chih-Kuang Yeh, Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, 101, Section 2, Kuang-Fu Road, Hsinchu, Taiwan 30013, ROC. E-mail: [email protected] 551
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nuclei in the sonicated region (Zhang et al. 2010, 2011). In addition, droplets can carry drugs that could be released at particular locations by the application of ADV (Fabiilli et al. 2010; Rapoport et al. 2009b; Wang et al. 2012). With the aid of targeting ligands such as aptamers, ADV can produce cavitation bubbles immediately adjacent to target cells that will cause membrane damage or permeabilization by mechanical stretching (Kang and Yeh 2011; Wang et al. 2012, 2013). In comparison to bubble-based agents, droplets are able to sustain higher mechanical stress in vivo and carry a larger drug payload and, therefore, are potential ultrasound-responsive agents with versatile diagnostic and therapeutic usefulness (Dayton et al. 2006). The control and manipulation of the bubble population are of great importance for the correct use of ADV in different applications. For example, large bubbles are advantageous for gas embolotherapy, whereas large quantities of small bubbles are far superior for HIFU ablation therapy and ultrasound contrast agent-enhanced imaging. However, the occurrence of ADV is accompanied by a rapid reduction in the surface density of surfactant molecules, which means that the resulting bubbles are highly vulnerable to further changes, such as coalescence into larger bubbles when they contact each other (Kripfgans et al. 2000; Rapoport et al. 2009a). The diffusion of gases such as O2, CO2, Ar and N2 from the host medium may cause post-ADV bubble growth (Kripfgans et al. 2000). The characteristics of the bubble population might also vary under different acoustic and hemodynamic conditions and, thus, might not be predictable simply on the basis of the properties of the initial droplet population. High-speed photography has been used to observe the transient ADV of single static droplets (Kripfgans et al. 2004; Reznik et al. 2012; Wong et al. 2011). The effects of acoustic parameters on the ADV of a group of droplets under static conditions (in gels) and flow conditions (in flow chambers) have also been investigated using ultrasound contrast enhancement, but these studies have not provided information about the bubble population (Fabiilli et al. 2009; Kawabata et al. 2010; Lo et al. 2007). No study has systematically investigated the effects of varying acoustic parameters on ADV and the resultant bubble population in dynamic flows that imitate in vivo conditions. In this study we simultaneously conducted optical microscopy and ultrasound imaging to monitor the ADV of a group of droplets under clinically relevant flow conditions. Temporal evolution of the diameter and number of ADV-generated bubbles was recorded optically after the onset of ADV. The dependence of the bubble population on droplet concentration, flow velocity, fluid viscosity and acoustic parameters such as acoustic
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pressure, pulse duration and pulse repetition frequency (PRF) were investigated. The enhancement of ultrasound contrast by the produced bubbles was assessed for comparison with the results of previous studies. The main aim of the present study was to extend our understanding of how ADV bubbles can be optimally manipulated for different applications. METHODS Droplet preparation and characterization Perfluorocarbon droplets were composed of a core of perfluoropentane (PFP, bulk boiling point of 29 C) with a shell comprising a thin layer of phospholipids. The materials were purchased from ABCR (Karlsruhe, Germany) and Avanti Polar Lipids (Alabaster, AL, USA). To fabricate droplet emulsions, a thin phospholipid film containing 95 mol% distearoylphosphatidylcholine and 5 mol% distearoylphosphatidylethanolamine-polyethylene glycol 2000 was prepared in a 2-mL vial using the following procedures: The vial was filled with 1 mL of degassed phosphate-buffered saline (PBS) and then immersed in a sonication bath at 60 C until the lipid film was completely dissolved. After the vial had cooled to 4 C, an ice-cold gas-tight syringe (Model HA-81000, Hamilton, Tokyo, Japan) was used to inject 100 mL of PFP into the vial; this procedure was performed at a low temperature to minimize the evaporation of PFP. The vial was then sealed and sonicated at 20 C for 5 min to produce a droplet emulsion. To remove excess phospholipids, the droplet emulsion was subjected to four 30-s washing/centrifugation cycles at 2000g with fresh PBS. The purified droplet emulsion was stored at 4 C overnight before experiments to allow the clearance of unstable droplets that could undergo spontaneous vaporization to become bubbles. Typical number and volume distributions of the produced droplets are illustrated in Figure 1. The number and volume concentrations were measured with a Coulter counter (Multisizer 3, Beckman Coulter, Fullerton, CA, USA) to be about (2.54 6 0.51) 3 1010 droplets/mL (mean 6 SD) and 40.7 6 4.09 mL/mL, respectively (range: 0.7–8 mm, N 5 17). The number and volume mean diameters were 1.3 6 0.1 and 2.1 6 0.2 mm, respectively. Less than 0.001% of the droplets were larger than 6 mm. Experimental setup Experiments were performed in an integrated acousto-optical system comprising an HIFU transmission system, a high-frequency ultrasound imaging system and an optical microscope (Kang and Yeh 2011). The system was based on an inverted microscope (Model IX71, Olympus, Tokyo, Japan) attached to a custom-made water tank (Fig. 2). The water tank contained de-ionized
Acoustic droplet vaporization for control of bubble generation d S.-T. KANG et al.
Fig. 1. Typical number and volume distributions of perfluoropentane droplets.
water that had been exposed to room air for 1 h to equilibrate the gas content, and it was maintained at 37 C to simulate body temperature. The gas content was assessed by measuring oxygen saturation using a dissolvedoxygen probe (Model DO-166 MT-1 SXS, Lazar Research Laboratories, Los Angeles, CA, USA). A 200mm hollow cellulose tube (Spectrum Labs, Rancho Dominguez, CA, USA) that was semipermeable to gases was immersed in the water at the focus of a waterproof objective (Achroplan 63 X, Carl Zeiss, Tokyo, Japan). Diluted droplet emulsions were infused into the tube using a syringe pump (Model KDS100, KD Scientific, Holliston, MA, USA). A stirrer was placed in the syringe and driven by a rotating magnet to mix the emulsions cautiously. The infusion was performed for 15 min before each experimental test to ensure not only a steady flow, but also the clearance of unstable droplets that had not been removed
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after overnight storage. A 2-MHz HIFU transducer (Model SU-101, Sonic Concepts, Bothell, WA, USA) and a homemade 40-MHz ultrasound transducer were positioned confocally with the objective and with their beam axes perpendicular to the tube. The specifications of both transducers are summarized in Table 1. The HIFU transducer was driven by an arbitrary-waveform generator (Model WG 2005, Tektronix, Santa Clara, CA, USA) and a 53-dB radiofrequency (RF) power amplifier (Model A150, E&I, Long Island City, NY, USA) to transmit high-pressure ultrasound pulses to induce ADV. Positioning the HIFU focus at different distances (0, 5, 10, 15, 20 and 25 mm) upstream from the mutual focus permitted simultaneous acoustic and optical observations of flowing bubbles at different times after the onset of ADV with the downstream 40-MHz transducer and objective. Note that 40-MHz ultrasound has been widely used in pre-clinical studies (Foster et al. 2009). The high frequency provides high spatial resolution that can improve the sensitivity of bubble detection in the small tube and also prevent the resonance of large ADV bubbles that can facilitate bubble disruption, coalescence and radiation-force interactions during the observations (Dayton et al. 1999; Kang and Yeh 2011; Yeh and Su 2008). For optical observations, a high-speed camera (FASTCAM SA4, Photron, Tokyo, Japan) was used to acquire serial optical images of the produced bubbles at 15 fps. This capture rate was slow enough to ensure that the same group of bubbles appeared once in the optical field of view. For acoustic observation, the 40-MHz transducer was operated in a pulse/echo mode at a PRF of 2000 Hz to assess the echogenicity of the produced bubbles (Yeh and Su 2008). The transducer was driven by an arbitrary-waveform generator (Model AWG 2040, Tektronix) and a RF power amplifier (Model
Fig. 2. Schematic diagram of an integrated acousto-optical system for monitoring acoustic droplet vaporization under flow conditions using optical microscopy and ultrasound imaging.
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Table 1. Specifications of the ultrasound transducers used in this study No. Center frequency (MHz) Focal length (mm) Active aperture (mm) Frequency bandwidth (MHz) Lateral beam width (mm) Depth of focus (mm) 1 2
2 40
55 12.7
35 4
325 LA, Electronic Navigation, Canandaigua, NY, USA) to transmit one-cycle acoustic pulses with a peak negative pressure of 1.6 MPa at its focus. The ultrasound echoes were received using the same transducer through a diode limiter/transformer diplexer circuit (Model DIP-3, Matec Instruments NDT, Northborough, MA, USA). The acquired RF signals were amplified with a 32-dB preamplifier (Model AU-1114, MITEQ, Hauppauge, NY, USA) and then digitized with an oscilloscope (Model LT354, LeCroy, Chestnut Ridge, NY, USA) at a sampling rate of 1 Gsample/s. The digitized signal was stored in M-mode (i.e., motion mode) format using a computer via a GPIB and then processed off-line using MATLAB software (The MathWorks, Natick, MA, USA). Data acquisition and analysis Optical images were acquired at 15 fps for 1 min (i.e., a total of 900 images) for each data set. Each experimental case comprised 5–10 data sets that could be used for statistical analysis of the mean diameter and total number and volume of the counted bubbles. Bubbles in the acquired images generally appeared as dark circles
1.5–2.6 21–49
1.2 0.12
14.0 2.32
whose diameters and numbers could be estimated using MATLAB software. The procedures are illustrated in Figure 3(a–c). Each image was first subtracted from a background image to minimize errors. The color depth of the subtracted image was then reduced to two colors using an appropriate threshold for delineating the contours of bubbles for counting. The diameters of counted bubbles were estimated from the areas of the extracted contours with a minimum identifiable diameter of 3.5 mm. The 3-D structure of the tube meant that the produced bubbles may have been not located within the same plane of optical focus. To minimize the error in estimating the mean diameter of bubbles within the tube, the condenser diaphragm was reduced to maximize the optical depth of field and contrast in the obtained images. For bubbles observed at different distances downstream from the HIFU focus, the plane of optical focus was accordingly lifted to where the detected number of bubbles was maximized so as to counterbalance the effect of bubble flotation. A trial analysis for static single bubbles with diameters of 8–65 mm yielded standard deviations below 14% of the estimated diameters in different depths within the tube. The presence of smaller bubbles
Fig. 3. Processing of optical and acoustic data. (a) Bright-field microscopy image of vaporized droplets. (b) Binary picture of (a). (c) Bubble counts and diameters. (d) Radiofrequency M-mode image. (e) Filtered and envelope-detected Mmode image. (f) Integrated echo power (IEP) over the time interval 0–1 s in (e).
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produced a higher standard deviation in the diameter estimation. Each M-mode image comprised 2000 RF signals. Ten M-mode images were acquired for each experimental test to allow statistical analysis of the echogenicity of ADV-generated bubbles. The two sets of prominent horizontal lines in Figure 3(d) represent the stationary signals arising from the primary and reverberation echoes from the tube walls. A high-pass Butterworth filter with a cutoff frequency of 100 Hz was applied over a slow time index to minimize these stationary signals. Further processing with baseband demodulation yielded the envelope-detected images representing the bubble contrast, an example of which is provided in Figure 3(e). The echogenicity of bubbles was assessed by integrating the power of the envelope-detected signals. The integration was first performed over the time interval of a single M-mode image (i.e., 1 s), yielding a curve of the integrated echo power versus depth, as illustrated in Figure 3(f). The integration was then performed over a suitable region of interest at a depth clear of the reverberation echoes from the tube walls. The integrated echo power obtained was positively correlated with the total bubble volume within the tube. Experimental parameters Initial experimental conditions were as follows: The droplet dilution ratio and flow velocity were set to be 1:6000 and 21.4 mm/s, respectively. The acoustic parameters—acoustic pressure (peak negative pressure), pulse duration and PRF—were set to be 10 MPa, 3 cycles and 18 Hz, respectively. This fairly high acoustic pressure was used to ensure that ADV was induced in most droplets in the diluted emulsions. These conditions were separately varied in each experimental case to investigate their independent effects on the population of ADVgenerated bubbles. When flow velocity was varied to imitate different hemodynamic conditions, the PRF was accordingly regulated to ensure that droplets were insonated once within the focal volume of the HIFU sonication. The droplet concentrations used in all experimental tests were at least 10 times lower than the critical doses reported to produce adverse bio-effects such as pulmonary hyperinflation (Kripfgans et al. 2005). Such low concentrations also helped to reduce the probability of bubbles overlapping in the acquired optical images. To evaluate the effect of fluid viscosity on the population of ADV-generated bubbles, droplets were diluted in either PBS or porcine blood plasma (with viscosities of 0.7 and 1.4–2.0 mPa$s, respectively) in some experimental cases (Windberger et al. 2003). The porcine blood plasma was prepared according to the Muskiet et al. (1981). Oxygen content of the PBS and blood plasma after flowing through the semipermeable tube were
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measured to be 6.89 6 0.21 and 5.72 6 0.32 ppm (N 5 3), respectively. RESULTS Flow velocity was varied to assess the effects of ADV under different conditions of arteriole blood flow (Wang et al. 2007). Figure 4(a) illustrates the temporal evolution of mean bubble diameter at different flow velocities after the onset of ADV for droplets diluted in PBS. Owing to the short length of the cellulose tube, the results for higher flow velocities are presented over shorter periods with the same amount of data points. The mean bubble diameter at t 5 0 s was approximately 9 mm for all flow velocities. Because the mean diameter of the vaporized droplets was measured to be about 1.2 mm, the expansion ratios of droplets to bubbles at t 5 0 s are slightly higher than those (i.e., an increase in diameter of at most 5- to 5.5-fold) reported for single droplets vaporized under static conditions (Kripfgans et al. 2000; Sheeran et al. 2011b; Wong et al. 2011). After ADV, mean bubble diameter slowly increased before reaching plateaus around 16–18 mm at t 5 0.7 s. The independence of growth rate from flow velocity suggests that the post-ADV bubble expansion is a consequence of diffusion of dissolved gas from the host medium (Kripfgans et al. 2000; Reznik et al. 2012; Shpak et al. 2013). Moreover, the similarity in plateau levels suggests the presence of an equilibrium size after this growth phase, which corresponds to about twice the mean diameter relative to that observed at t 5 0 s. On the other hand, varying the droplet concentration (within the range tested in this study) did not markedly affect temporal evolution of the mean bubble diameter, as illustrated in Figure 4(b). Mean bubble diameter at t 5 0 s for a dilution ratio of 1:2000 was slightly larger than those of the other cases (with a p-value of 0.02 between 1:2000 and 1:10000). The slight difference might be due to the shorter distance between adjacent droplets, which increases the probability of bubble coalescence at the onset of ADV, and/or the shielding of ultrasound energy, which hinders the ADV of small droplets. In contrast, the mean diameter after t 5 0.7 s for a dilution ratio of 1:2000 was smaller than for the other cases, which might be attributable to a reduction of dissolved gas within the tube as a result of its rapid uptake by the produced bubbles. In Figure 4(a and b), the droplets diluted in blood plasma exhibit a slower growth rate and a lower plateau relative to those diluted in PBS. Because the diffusion constant is inversely proportional to fluid viscosity according to the Stokes-Einstein equation, the gas diffusion rate is two to three times slower in blood plasma than in PBS (Cussler 1984; Windberger et al. 2003). The initially lower gas content
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Fig. 4. Temporal evolution of mean bubble diameter after the onset of acoustic droplet vaporization (ADV) for perfluoropentane droplets in phosphate-buffered saline solution (a) under different flow velocities and (b) with different dilution ratios (N 5 10). The case marked with an asterisk was diluted in blood plasma. The acoustic pressure, pulse duration and pulse repetition frequency were set to 10 MPa, 3 cycles and 18 Hz, respectively.
and the suppressed rate of gas exchange across the wall of the semipermeable tube might be responsible for the reduced equilibrium bubble size in blood plasma. The results presented below are derived from detailed investigations of the effects of three acoustic parameters: acoustic pressure, pulse duration and PRF. Varying these parameters may exert various effects, such as size-selective ADV caused by the threshold effect, bubble disruption caused by unstable radial oscillation, bubble dissolution caused by rectified gas diffusion and bubble coalescence assisted by acoustic radiation forces, all of which can greatly influence the characteristics of the initial population of ADV-generated bubbles (Chomas
et al. 2001; Eller 1965; Fabiilli et al. 2009; Rapoport et al. 2009a). The effect of each acoustic parameter on temporal evolution of mean bubble diameter was first evaluated. Acoustic and detailed optical observations were then performed for analysis of integrated echo power, diameter distribution (in percentage terms) and total number and volume of the counted bubbles at t 5 0.93 s. This time point was chosen to ensure that the resulting bubble populations, in most cases, had reached their equilibrium sizes (corresponding to the plateau regions of the growth curves). Because of the polydispersive nature of the droplet population and the size-selective ADV, the
Fig. 5. (a) Temporal evolution of mean bubble diameter after acoustic droplet vaporization (ADV) for perfluoropentane droplets insonated at different acoustic pressures (N 5 10). (b–e) Bubble diameter distribution (in percentage terms), total bubble number (TBN), total bubble volume (TBV) and integrated echo power (IEP), respectively, as functions of the acoustic pressure at t 5 0.93 s after ADV. Each diameter distribution in (b) consists of only one typical data set, and the mean value is denoted by an open circle.
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Fig. 6. (a) Temporal evolution of mean bubble diameter after acoustic droplet vaporization (ADV) for perfluoropentane droplets insonated under different pulse durations (N 5 10). (b–e) Bubble diameter distribution (in percentage terms), total bubble number (TBN), total bubble volume (TBV) and integrated echo power (IEP), respectively, as functions of the pulse duration at t 5 0.93 s after ADV. Each diameter distribution in (b) consists of only one typical data set, and the mean value is denoted by an open circle.
mean diameter of ADV-generated bubbles decreased with increasing acoustic pressure at t 5 0 s, as illustrated in Figure 5(a). Insonation at 7 MPa was only able to vaporize large droplets, meaning that the resulting bubble population contained a relatively large proportion of larger bubbles at all periods. However, the small size differences caused by size-selective ADV were smoothed during bubble growth when the pressures were higher than 8 MPa, which should have exceeded the ADV thresholds of most of the droplet population. The simultaneous acoustic and optical observations performed at t 5 0.93 s indicated strong correlations (R2 . 0.99) between the integrated echo power, total bubble number and total bubble volume, but not mean bubble diameter, as depicted in Figure 5(b–e). The values of integrated echo power, total bubble number and total bubble volume increased as acoustic pressure increased up to 8 MPa; at higher acoustic pressures, the characteristics of the bubble population and integrated echo power remained approximately constant. These results suggest that insonation with a three-cycle pulse at pressures far above ADV threshold levels does not affect the formation of intact bubbles. For droplets insonated under varying pulse durations, the mean diameter of bubbles formed at t 5 0 s after ADV decreased slightly with increasing pulse duration, presumably because of the decreased ADV thresholds and the presence of slight bubble disruption (Fig. 6a). The prolonged insonation might also have enhanced rectified gas diffusion to reduce the bubbles toward a resonance size, which is suggested to be about 1.5–2.5 mm in diameter for the given acoustic frequency
(Chatterjee and Sarkar 2003; Eller 1965). However, this correlation became negative after t 5 0.93 s, when the mean bubble diameter for longer pulse durations was much greater than the stable equilibrium size observed in the previous experiments. At t 5 0.93 s, the diameters of bubbles for pulse durations shorter than five cycles exhibited an intact unimodal distribution, whereas those for longer pulse durations exhibited a bimodal distribution, as illustrated in Figure 6(b). Most of the bubbles were larger than twice (40– 50 mm) the stable equilibrium diameter. Integrated echo power, total bubble number and total bubble volume, but not mean bubble diameter, were found to be negatively correlated with pulse duration, as illustrated in Figure 6(b–e). The absence of a stable diameter and the considerable decrease in total bubble number and total bubble volume for different pulse durations suggest that prolonged insonation has an adverse effect on the stability of ADV bubbles, making them more susceptible to gas transfer (both inward and outward), which causes bubble growth and dissolution. Increasing the pulse duration favors the production of fewer but larger bubbles. Varying the PRF had a much greater impact than varying the pulse duration on the population of ADV bubbles. The mean diameter of bubbles formed at t 5 0 s after ADV was independent of PRF, as illustrated in Figure 7(a). A positive correlation between mean bubble diameter and PRF was observed only when PRF was in the range 18–36 Hz. At PRFs , 18 Hz, droplets were insonated once only within the beam width of the HIFU, producing bubbles with a unimodal diameter distribution at t 5 0.93 s, as illustrated in Figure 7(b). In contrast, at
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Fig. 7. (a) Temporal evolution of mean bubble diameter after acoustic droplet vaporization (ADV) for perfluoropentane droplets insonated at different pulse repetition frequencies (N 5 10). (b–e) Bubble diameter distribution (in percentage terms), total bubble number (TBN), total bubble volume (TBV) and integrated echo power (IEP), respectively, as functions of the pulse repetition frequency (PRF) at t 5 0.93 s after ADV. Each diameter distribution in (b) consists of only one typical data set, and the mean value is denoted by an open circle.
PRFs . 36 Hz, droplets were insonated twice and more within the beam width of the HIFU, in which case the produced bubbles presented a multimodal diameter distribution at t 5 0.93 s with large fluctuations in their mean diameters for different PRFs. As illustrated in Figure 7(c–e), integrated echo power, total bubble number and total bubble volume decreased rapidly with increasing PRF at a rate that was significantly faster than when increasing the pulse duration for a similar total pulse energy. The small number of bubbles accounts for the large standard deviations of the growth curves in Figure 7(a). These results suggest that increasing the PRF provides superior performance in disrupting ADV-generated bubbles. DISCUSSION This study investigated the effects of varying droplet concentration, flow velocity and various acoustic parameters on the ADVof polydisperse droplets under clinically relevant flow conditions. Acoustic and optical observations were conducted to evaluate this dependence and the temporal evolution of bubble populations after the onset of ADV. Because the HIFU and optical microscopy systems were not synchronized, observation delays of up to 16 ms might have resulted in calculated expansion ratios at t 5 0 s slightly higher than the reported upper limits of 5.2 to 5.4 caused by gas diffusion (Sheeran et al. 2011b). Nevertheless, during t 5 0–0.7 s, the bubbles absorbed ambient dissolved gases and grew to an equilibrium diameter that was as much as twice their initial diameter. The expansion ratio is consistent with previous studies (Reznik et al. 2012; Sheeran et al.
2011b), and the duration is of the same order as those (2 s) predicted for uncoated PFC bubbles (Kabalnov et al. 1998a). Post-ADV bubble growth is inevitable because ADV-generated bubbles initially contain pure PFP vapor that is maintained against multiple inward-acting pressures, including atmospheric pressure, blood pressure and Laplace pressure (Kabalnov et al. 1998a; Schutt et al. 2003). The high purity of PFP vapor establishes a high concentration gradient for both PFP and air at the water–bubble interface. The rapid decrease in surface density of lipid molecules during ADV increases the permeability of the protective shell to gas (Reznik et al. 2012). For instance, assume that a PFP bubble with a diameter of 10 mm stays in a 100% air-saturated medium. The low Ostwald coefficient (6.6 3 1025) of PFP contributes to an extremely low diffusional flux (1.3 3 10218 mol/s) that maintains the content of PFP inside the bubble (Kabalnov et al. 1998a, 1998b). However, the high Ostwald coefficients of air components (1.6 3 1022 for nitrogen and 3.1 3 1022 for oxygen) contribute to high diffusional fluxes (1.5 3 10213 mol/s for nitrogen and 8.0 3 10214 mol/s for oxygen) inward to the bubble. The intake of dissolved air from the host medium gradually increases the bubble size until the diffusional flux of air decreases to zero when the air content reaches equilibrium between the host medium and the bubble. The temporary cessation of air influx and the weak protection provided by dilute lipids in the shell result in an osmotically stabilized bubble population. A simple theoretical calculation can be performed to estimate the equilibrium bubble size. The
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pressure equilibrium before and after air permeation into a pure PFP bubble produced by ADV can be written as Pair 1PPFP 1Pvapor 2PL 5 Patm 1Pblood 5 P0air 1P0PFP 1P0vapor 2P0L ;
(1)
where Patm is atmospheric pressure; Pblood is blood pressure; Pair, PPFP and Pvapor are the partial pressures of air, PFP and vapor, respectively, in the bubble; and PL is the Laplace pressure of the bubble. The final pressures after bubble growth are denoted with primes. When the bubble expands by a factor of E in diameter, it is reasonable to assume that the variation in surface tension of the dilute lipid molecules (near 50 mN/m) is negligible (Kabalnov et al. 1998a; Sheeran et al. 2011b). The decrease in Laplace pressure and partial pressure of PFP during bubble expansion can be calculated as PPFP’ 5 PPFP/E3 and PL’ 5 PL/E. Rearranging the left and right parts of eqn (1) gives 1 1 PPFP 12 3 5 P0air 1P0vapor 1PL 12 : (2) E E Pair and Pvapor approach zero as the bubble initially contains only PFP, so that PPFP equals the sum of Patm, Pblood and PL. Rearranging eqn (2) and replacing PPFP such that only environmental and droplet conditions are involved yield Pblood 1Patm 2P0air 2P0vapor E3 (3) 1PL E2 2ðPblood 1Patm 1PL Þ 5 0 Solving the real and positive root of this cubic equation yields the expansion ratio. According to Henry’s law, 0 Pair is proportional to the concentration of dissolved 0 air. This means that Pair finally reaches 1 atm (101.325 kPa) for a 100% air-saturated medium. For bubbles with initial diameters of 5–20 mm subject to 0 Patm, Pblood and Pvapor of 101.325, 12.36 and 6.15 kPa, respectively, the expansion ratios fall within the range 1.74–2.25 and are very close to the experimental results. This equation suggests that bubble expansion caused by air permeation depends on initial bubble size, ambient pressures and the concentration of dissolved air. A decrease in the concentration of dissolved air by 10% can reduce the post-ADV expansion ratio of a 10-mm bubble by about 12%. The volume growth trend is expected to be independent of flow velocity as long as the bubbles remain in the same medium for the same period, because the whole process is associated with classic gas diffusion (Kabalnov et al. 1998a). However, the volume growth trend is dependent on droplet concentration and fluid
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viscosity, which directly influence the rate of gas uptake by bubbles and the rate of gas exchange across the wall of the tube. Insufficient replenishment of dissolved gas in the tube, which causes local gas depletion, may also reduce the rate of gas uptake by bubbles and hence retard the volume growth of bubbles. Note that the presence of osmotic equilibrium does not guarantee a permanently maintained bubble population, as the lack of sufficient protection means that the bubbles can experience further air permeation enhanced by flow disturbance to grow more than expected because of a tendency to decrease Laplace pressure. Large bubbles are barely subject to rapid dissolution presumably because of the decrease in Laplace pressure after diffusion-assisted volume growth and the low solubility (0.77 mg/L at 25 C) of PFP in water (Kabalnov et al. 1998a). Moreover, it has been found that the ADV of albumin-stabilized droplets results in fragmented shells that are no longer able to stabilize the produced bubbles (Reznik et al. 2012). These bubbles are more susceptible to external disturbances than bubbles having dilute but uniform shells, resulting in the absence of a temporarily stable size during post-ADV bubble growth. The role of surfactants in post-ADV bubble growth behavior may need to be considered in clinical applications. Other results from this study, as described below, provide information that is valuable for manipulating the population of ADV-generated bubbles by varying acoustic parameters. Applying an acoustic pressure that exceeds the threshold level will maintain the bubble population, but varying both pulse duration and PRF has a much greater impact on the bubble population. Lengthening the pulse duration favors the production of large bubbles, presumably driven by enhanced gas exchange rather than bubble coalescence. The evidence is that the bubbles exhibited a unimodal diameter distribution irrespective of the pulse duration at t 5 0 s after ADV, but subsequently exhibited a bimodal diameter distribution at t 5 0.93 s for pulse durations longer than 10 cycles (Fig. 8). The concurrent growth and dissolution of bubbles are indicative of an enhanced two-way gas diffusion. We speculated that the prolonged insonation might partly result in the exclusion of surfactant molecules from the bubble surfaces (Yount 1982). On the other hand, varying the PRF might affect the stability of produced bubbles in a similar manner, but is much more effective for bubble disruption than varying the pulse duration for a similar transmitted acoustic energy. A sufficiently high PRF will result in insonation of bubbles more than once within the focal volume of the HIFU. Insonation at low acoustic pressures will disrupt bubbles and also hinder their ability to aid the ADV of remaining droplets that have not yet been vaporized (Lo et al. 2007). This speculation
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long pulse duration and high PRF leads to a low integrated echo power, which may reduce the accuracy and sensitivity of the obtained results. The results also suggest that integrated echo power does not adequately reflect the characteristics of the bubble population. The reported strategies for reducing the ADV threshold by increasing pulse duration and PRF need to be carefully considered in terms of their impact on the bubble population produced in a particular application scenario. CONCLUSIONS
Fig. 8. Bubble diameter distributions (in percentage terms) at (a, c) t 5 0 s and (b, d) t 5 0.93 s after acoustic droplet vaporization (ADV) for perfluoropentane droplets insonated under pulse durations of (a, b) 3 cycles and (c, d) 20 cycles. The transition from unimodal distribution to bimodal distribution is an indication of enhanced gas exchange.
implies that applying a subsequent acoustic pulse at pressures lower than ADV thresholds may be advantageous to bubble clearance. Moreover, the intervals between successive acoustic pulses allow bubbles to grow larger than their initial sizes. Any change in the effects of cavitation, disruption and coalescence of bubbles in response to the size difference should also be considered to account for the enhanced bubble disruption. Elucidation of a detailed mechanism requires further investigation. Accordingly, determining the optimal acoustic parameters is of great importance for different application scenarios. For example, ultrasound contrast agentenhanced imaging requires high concentrations of small bubbles, which may be achieved by using high acoustic pressures, low pulse durations and matching the PRF with the velocity of the blood flow. Lengthening the pulse duration at a sufficient acoustic pressure and a matched PRF may produce the larger bubbles that are beneficial for gas embolotherapy. Moreover, several studies have monitored the extent of ADV and measured the threshold pressure based on an increase in imaging contrast caused by the presence of bubbles (Fabiilli et al. 2009; Kripfgans et al. 2000; Lo et al. 2007; Reznik et al. 2011). However, our results suggest that insufficient bubble generation at a
The main aim of this study was to extend our understanding of how ADV-generated bubbles can be optimally manipulated under flow conditions. For droplets with concentrations below 1 3 107 droplets/mL insonated under short pulse durations (,5 cycles) above their threshold pressures (.8 MPa), the principal aspect that affects the characteristics of the bubble population is the post-ADV bubble growth driven by air permeation, and not size-selective ADVor coalescence and disruption of bubbles. The results obtained suggest that there is a stable equilibrium size during the growth phase for vaporized droplets with phospholipid shells. The growth trend may be influenced by parameters that affect the gas content of the host medium, including droplet concentration (affecting the rate of gas uptake) and fluid viscosity (affecting the rate of gas exchange across the wall of the semipermeable tube). Varying pulse duration and PRF, but not acoustic pressure, markedly reduces the number of bubbles. Lengthening pulse duration favors the production of a smaller number of larger bubbles, whereas increasing PRF interestingly provides superior bubble disruption. These results suggest that the ADV bubble population may not be assessable simply on the basis of initial droplet sizes or their enhancement of imaging contrast. Determining the optimal acoustic parameters requires careful consideration of their impact on the bubble population produced for different application scenarios. Future studies should be conducted to provide in vivo validation of the effects observed in the present work. Acknowledgments—The authors acknowledge the support of the National Science Council of Taiwan under Grants 101-2628-E-007-001 and 101-2221-E-007-035-MY3 and National Tsing Hua University under Grant 102 N2046 E1.
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Acoustic droplet vaporization for control of bubble generation d S.-T. KANG et al. Chomas JE, Dayton P, Allen J, Morgan K, Ferrara KW. Mechanisms of contrast agent destruction. IEEE Trans Ultrason Ferroelectr Freq Control 2001;48:232–248. Cussler EL. Diffusion, mass transfer in fluid systems. Cambridge/New York: Cambridge University Press; 1984. Dayton PA, Morgan KE, Klibanov AL, Brandenburger GH, Ferrara KW. Optical and acoustical observations of the effects of ultrasound on contrast agents. IEEE Trans Ultrason Ferroelectr Freq Control 1999;46:220–232. Dayton PA, Zhao S, Bloch SH, Schumann P, Penrose K, Matsunaga TO, Zutshi R, Doinikov A, Ferrara KW. Application of ultrasound to selectively localize nanodroplets for targeted imaging and therapy. Mol Imaging 2006;5:160–174. Eller A. Rectified diffusion during nonlinear pulsations of cavitation bubbles. J Acoust Soc Am 1965;37:493. Fabiilli ML, Haworth KJ, Fakhri NH, Kripfgans OD, Carson PL, Fowlkes JB. The role of inertial cavitation in acoustic droplet vaporization. IEEE Trans Ultrason Ferroelectr Freq Control 2009;56: 1006–1017. Fabiilli ML, Haworth KJ, Sebastian IE, Kripfgans OD, Carson PL, Fowlkes JB. Delivery of chlorambucil using an acousticallytriggered perfluoropentane emulsion. Ultrasound Med Biol 2010; 36:1364–1375. Foster FS, Mehi J, Lukacs M, Hirson D, White C, Chaggares C, Needles A. A new 15–50 MHz array-based micro-ultrasound scanner for preclinical imaging. Ultrasound Med Biol 2009;35:1700–1708. Haworth KJ, Fowlkes JB, Carson PL, Kripfgans OD. Towards aberration correction of transcranial ultrasound using acoustic droplet vaporization. Ultrasound Med Biol 2008;34:435–445. Kabalnov A, Klein D, Pelura T, Schutt E, Weers J. Dissolution of multicomponent microbubbles in the bloodstream: 1. Theory. Ultrasound Med Biol 1998a;24:739–749. Kabalnov A, Bradley J, Flaim S, Klein D, Petura B, Otto S, Reynolds J, Schutt E, Weers J. Dissolution of multicomponent microbubbles in the bloodstream: 2. Experiment. Ultrasound Med Biol 1998b;24: 751–760. Kang ST, Yeh CK. Intracellular acoustic droplet vaporization in a single peritoneal macrophage for drug delivery applications. Langmuir 2011;27:13183–13188. Kawabata K, Asami R, Azuma T, Umemura S. Acoustic response of microbubbles derived from phase-change nanodroplet. Jpn J Appl Phys 2010;49:07HF18. Kripfgans OD, Fabiilli ML, Carson PL, Fowlkes JB. On the acoustic vaporization of micrometer-sized droplets. J Acoust Soc Am 2004; 116:272–281. Kripfgans OD, Fowlkes JB, Miller DL, Eldevik OP, Carson PL. Acoustic droplet vaporization for therapeutic and diagnostic applications. Ultrasound Med Biol 2000;26:1177–1189. Kripfgans OD, Orifici CM, Carson PL, Ives KA, Eldevik OP, Fowlkes JB. Acoustic droplet vaporization for temporal and spatial control of tissue occlusion: A kidney study. IEEE Trans Ultrason Ferroelectr Freq Control 2005;52:1101–1110. Lo AH, Kripfgans OD, Carson PL, Rothman ED, Fowlkes JB. Acoustic droplet vaporization threshold: Effects of pulse duration and contrast agent. IEEE Trans Ultrason Ferroelectr Freq Control 2007;54: 933–946. Muskiet FA, Stratingh MC, Stob GJ, Wolthers BG. Simultaneous determination of the four major catecholamine metabolites and estimation of a serotonin metabolite in urine by capillary gas chromatography of their tert-butyldimethylsilyl derivatives. Clin Chem 1981;27:223–227.
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Rapoport N, Gao Z, Kennedy A. Multifunctional nanoparticles for combining ultrasonic tumor imaging and targeted chemotherapy. J Natl Cancer Inst 2007;99:1095–1106. Rapoport NY, Efros AL, Christensen DA, Kennedy AM, Nam KH. Microbubble generation in phase-shift nanoemulsions used as anticancer drug carriers. Bubble Sci Eng Technol 2009a;1: 31–39. Rapoport NY, Kennedy AM, Shea JE, Scaife CL, Nam KH. Controlled and targeted tumor chemotherapy by ultrasound-activated nanoemulsions/microbubbles. J Control Release 2009b;138:268–276. Reznik N, Seo M, Williams R, Bolewska-Pedyczak E, Lee M, Matsuura N, Gariepy J, Foster FS, Burns PN. Optical studies of vaporization and stability of fluorescently labelled perfluorocarbon droplets. Phys Med Biol 2012;57:7205–7217. Reznik N, Williams R, Burns PN. Investigation of vaporized submicron perfluorocarbon droplets as an ultrasound contrast agent. Ultrasound Med Biol 2011;37:1271–1279. Schutt EG, Klein DH, Mattrey RM, Riess JG. Injectable microbubbles as contrast agents for diagnostic ultrasound imaging: The key role of perfluorochemicals. Angew Chem Int Ed 2003;42: 3218–3235. Sheeran PS, Luois S, Dayton PA, Matsunaga TO. Formulation and acoustic studies of a new phase-shift agent for diagnostic and therapeutic ultrasound. Langmuir 2011a;27:10412–10420. Sheeran PS, Wong VP, Luois S, McFarland RJ, Ross WD, Feingold S, Matsunaga TO, Dayton PA. Decafluorobutane as a phase-change contrast agent for low-energy extravascular ultrasonic imaging. Ultrasound Med Biol 2011b;37:1518–1530. Shpak O, Stricker L, Versluis M, Lohse D. The role of gas in ultrasonically driven vapor bubble growth. Phys Med Biol 2013;58: 2523–2535. Wang CH, Kang ST, Lee YH, Luo YL, Huang YF, Yeh CK. Aptamerconjugated and drug-loaded acoustic droplets for ultrasound theranosis. Biomaterials 2012;33:1939–1947. Wang CH, Kang ST, Yeh CK. Superparamagnetic iron oxide and drug complex-embedded acoustic droplets for ultrasound targeted theranosis. Biomaterials 2013;34:1852–1861. Wang Y, Bower BA, Izatt JA, Tan O, Huang D. In vivo total retinal blood flow measurement by Fourier domain Doppler optical coherence tomography. J Biomed Opt 2007;12:041215. Windberger U, Bartholovitsch A, Plasenzotti R, Korak KJ, Heinze G. Whole blood viscosity, plasma viscosity and erythrocyte aggregation in nine mammalian species: Reference values and comparison of data. Exp Physiol 2003;88:431–440. Wong ZZ, Kripfgans OD, Qamar A, Fowlkes JB, Bull JL. Bubble evolution in acoustic droplet vaporization at physiological temperature via ultra-high speed imaging. Soft Matter 2011;7:4009–4016. Yeh CK, Su SY. Effects of acoustic insonation parameters on ultrasound contrast agent destruction. Ultrasound Med Biol 2008;34: 1281–1291. Yount DE. On the evolution, generation, and regeneration of gas cavitation nuclei. J Acoust Soc Am 1982;71:1473–1481. Zhang M, Fabiilli ML, Haworth KJ, Fowlkes JB, Kripfgans OD, Roberts WW, Ives KA, Carson PL. Initial investigation of acoustic droplet vaporization for occlusion in canine kidney. Ultrasound Med Biol 2010;36:1691–1703. Zhang M, Fabiilli ML, Haworth KJ, Padilla F, Swanson SD, Kripfgans OD, Carson PL, Fowlkes JB. Acoustic droplet vaporization for enhancement of thermal ablation by high intensity focused ultrasound. Acad Radiol 2011;18:1123–1132.
http://dx.doi.org/10.1016/j.ultrasmedbio.2013.10.020
d
Original Contribution CHARACTERIZATION OF ACOUSTIC DROPLET VAPORIZATION FOR CONTROL OF BUBBLE GENERATION UNDER FLOW CONDITIONS SHIH-TSUNG KANG, YI-LUAN HUANG, and CHIH-KUANG YEH Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, Taiwan (Received 3 June 2013; revised 20 October 2013; in final form 21 October 2013)
Abstract—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. (E-mail: [email protected]) Ó 2014 World Federation for Ultrasound in Medicine & Biology. Key Words: Acoustic droplet vaporization, Flow condition, Diffusion-assisted bubble growth, Perfluorocarbon droplet, Size control and manipulation.
INTRODUCTION
gas nucleation and/or cavitation that triggers the transformation of these droplets into gaseous bubbles, with the ADV threshold being higher for smaller droplets (Kripfgans et al. 2004; Shpak et al. 2013; Wong et al. 2011). The droplet-to-bubble transition results in an instant volume expansion, permitting substantial bubble formation from small droplet doses (Kripfgans et al. 2000). The ideal expansion ratio falls within the range of 3.5 to 5.4 times in diameter, which decreases with decreasing droplet size because of increased Laplace pressure (Sheeran et al. 2011b). Several studies have indicated that ADV produces localized ultrasound contrast enhancement (as point sources) that can be used in the correction of transcranial ultrasound phase aberrations (Carneal et al. 2011; Haworth et al. 2008). In possible therapeutic applications, the produced bubbles could serve as gas emboli to reduce or stop the blood supply to tumors or to enhance high-intensity focused ultrasound (HIFU) ablation by increasing the amount of cavitation
Acoustic droplet vaporization (ADV) has received increasing attention in recent years because of its ability to produce microbubbles on demand in a non-invasive and localized manner (Kawabata et al. 2010; Kripfgans et al. 2000; Rapoport et al. 2007; Reznik et al. 2012; Sheeran et al. 2011a). Such droplets are often based on liquid perfluorocarbons (PFCs) with low boiling points, which can be maintained in a superheated state without vaporization after they have been emulsified into micron to submicron droplets. However, exposure to ultrasound pulses with acoustic pressures above a particular level (termed the ADV threshold) can initiate
Address correspondence to: Chih-Kuang Yeh, Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, 101, Section 2, Kuang-Fu Road, Hsinchu, Taiwan 30013, ROC. E-mail: [email protected] 551
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nuclei in the sonicated region (Zhang et al. 2010, 2011). In addition, droplets can carry drugs that could be released at particular locations by the application of ADV (Fabiilli et al. 2010; Rapoport et al. 2009b; Wang et al. 2012). With the aid of targeting ligands such as aptamers, ADV can produce cavitation bubbles immediately adjacent to target cells that will cause membrane damage or permeabilization by mechanical stretching (Kang and Yeh 2011; Wang et al. 2012, 2013). In comparison to bubble-based agents, droplets are able to sustain higher mechanical stress in vivo and carry a larger drug payload and, therefore, are potential ultrasound-responsive agents with versatile diagnostic and therapeutic usefulness (Dayton et al. 2006). The control and manipulation of the bubble population are of great importance for the correct use of ADV in different applications. For example, large bubbles are advantageous for gas embolotherapy, whereas large quantities of small bubbles are far superior for HIFU ablation therapy and ultrasound contrast agent-enhanced imaging. However, the occurrence of ADV is accompanied by a rapid reduction in the surface density of surfactant molecules, which means that the resulting bubbles are highly vulnerable to further changes, such as coalescence into larger bubbles when they contact each other (Kripfgans et al. 2000; Rapoport et al. 2009a). The diffusion of gases such as O2, CO2, Ar and N2 from the host medium may cause post-ADV bubble growth (Kripfgans et al. 2000). The characteristics of the bubble population might also vary under different acoustic and hemodynamic conditions and, thus, might not be predictable simply on the basis of the properties of the initial droplet population. High-speed photography has been used to observe the transient ADV of single static droplets (Kripfgans et al. 2004; Reznik et al. 2012; Wong et al. 2011). The effects of acoustic parameters on the ADV of a group of droplets under static conditions (in gels) and flow conditions (in flow chambers) have also been investigated using ultrasound contrast enhancement, but these studies have not provided information about the bubble population (Fabiilli et al. 2009; Kawabata et al. 2010; Lo et al. 2007). No study has systematically investigated the effects of varying acoustic parameters on ADV and the resultant bubble population in dynamic flows that imitate in vivo conditions. In this study we simultaneously conducted optical microscopy and ultrasound imaging to monitor the ADV of a group of droplets under clinically relevant flow conditions. Temporal evolution of the diameter and number of ADV-generated bubbles was recorded optically after the onset of ADV. The dependence of the bubble population on droplet concentration, flow velocity, fluid viscosity and acoustic parameters such as acoustic
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pressure, pulse duration and pulse repetition frequency (PRF) were investigated. The enhancement of ultrasound contrast by the produced bubbles was assessed for comparison with the results of previous studies. The main aim of the present study was to extend our understanding of how ADV bubbles can be optimally manipulated for different applications. METHODS Droplet preparation and characterization Perfluorocarbon droplets were composed of a core of perfluoropentane (PFP, bulk boiling point of 29 C) with a shell comprising a thin layer of phospholipids. The materials were purchased from ABCR (Karlsruhe, Germany) and Avanti Polar Lipids (Alabaster, AL, USA). To fabricate droplet emulsions, a thin phospholipid film containing 95 mol% distearoylphosphatidylcholine and 5 mol% distearoylphosphatidylethanolamine-polyethylene glycol 2000 was prepared in a 2-mL vial using the following procedures: The vial was filled with 1 mL of degassed phosphate-buffered saline (PBS) and then immersed in a sonication bath at 60 C until the lipid film was completely dissolved. After the vial had cooled to 4 C, an ice-cold gas-tight syringe (Model HA-81000, Hamilton, Tokyo, Japan) was used to inject 100 mL of PFP into the vial; this procedure was performed at a low temperature to minimize the evaporation of PFP. The vial was then sealed and sonicated at 20 C for 5 min to produce a droplet emulsion. To remove excess phospholipids, the droplet emulsion was subjected to four 30-s washing/centrifugation cycles at 2000g with fresh PBS. The purified droplet emulsion was stored at 4 C overnight before experiments to allow the clearance of unstable droplets that could undergo spontaneous vaporization to become bubbles. Typical number and volume distributions of the produced droplets are illustrated in Figure 1. The number and volume concentrations were measured with a Coulter counter (Multisizer 3, Beckman Coulter, Fullerton, CA, USA) to be about (2.54 6 0.51) 3 1010 droplets/mL (mean 6 SD) and 40.7 6 4.09 mL/mL, respectively (range: 0.7–8 mm, N 5 17). The number and volume mean diameters were 1.3 6 0.1 and 2.1 6 0.2 mm, respectively. Less than 0.001% of the droplets were larger than 6 mm. Experimental setup Experiments were performed in an integrated acousto-optical system comprising an HIFU transmission system, a high-frequency ultrasound imaging system and an optical microscope (Kang and Yeh 2011). The system was based on an inverted microscope (Model IX71, Olympus, Tokyo, Japan) attached to a custom-made water tank (Fig. 2). The water tank contained de-ionized
Acoustic droplet vaporization for control of bubble generation d S.-T. KANG et al.
Fig. 1. Typical number and volume distributions of perfluoropentane droplets.
water that had been exposed to room air for 1 h to equilibrate the gas content, and it was maintained at 37 C to simulate body temperature. The gas content was assessed by measuring oxygen saturation using a dissolvedoxygen probe (Model DO-166 MT-1 SXS, Lazar Research Laboratories, Los Angeles, CA, USA). A 200mm hollow cellulose tube (Spectrum Labs, Rancho Dominguez, CA, USA) that was semipermeable to gases was immersed in the water at the focus of a waterproof objective (Achroplan 63 X, Carl Zeiss, Tokyo, Japan). Diluted droplet emulsions were infused into the tube using a syringe pump (Model KDS100, KD Scientific, Holliston, MA, USA). A stirrer was placed in the syringe and driven by a rotating magnet to mix the emulsions cautiously. The infusion was performed for 15 min before each experimental test to ensure not only a steady flow, but also the clearance of unstable droplets that had not been removed
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after overnight storage. A 2-MHz HIFU transducer (Model SU-101, Sonic Concepts, Bothell, WA, USA) and a homemade 40-MHz ultrasound transducer were positioned confocally with the objective and with their beam axes perpendicular to the tube. The specifications of both transducers are summarized in Table 1. The HIFU transducer was driven by an arbitrary-waveform generator (Model WG 2005, Tektronix, Santa Clara, CA, USA) and a 53-dB radiofrequency (RF) power amplifier (Model A150, E&I, Long Island City, NY, USA) to transmit high-pressure ultrasound pulses to induce ADV. Positioning the HIFU focus at different distances (0, 5, 10, 15, 20 and 25 mm) upstream from the mutual focus permitted simultaneous acoustic and optical observations of flowing bubbles at different times after the onset of ADV with the downstream 40-MHz transducer and objective. Note that 40-MHz ultrasound has been widely used in pre-clinical studies (Foster et al. 2009). The high frequency provides high spatial resolution that can improve the sensitivity of bubble detection in the small tube and also prevent the resonance of large ADV bubbles that can facilitate bubble disruption, coalescence and radiation-force interactions during the observations (Dayton et al. 1999; Kang and Yeh 2011; Yeh and Su 2008). For optical observations, a high-speed camera (FASTCAM SA4, Photron, Tokyo, Japan) was used to acquire serial optical images of the produced bubbles at 15 fps. This capture rate was slow enough to ensure that the same group of bubbles appeared once in the optical field of view. For acoustic observation, the 40-MHz transducer was operated in a pulse/echo mode at a PRF of 2000 Hz to assess the echogenicity of the produced bubbles (Yeh and Su 2008). The transducer was driven by an arbitrary-waveform generator (Model AWG 2040, Tektronix) and a RF power amplifier (Model
Fig. 2. Schematic diagram of an integrated acousto-optical system for monitoring acoustic droplet vaporization under flow conditions using optical microscopy and ultrasound imaging.
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Table 1. Specifications of the ultrasound transducers used in this study No. Center frequency (MHz) Focal length (mm) Active aperture (mm) Frequency bandwidth (MHz) Lateral beam width (mm) Depth of focus (mm) 1 2
2 40
55 12.7
35 4
325 LA, Electronic Navigation, Canandaigua, NY, USA) to transmit one-cycle acoustic pulses with a peak negative pressure of 1.6 MPa at its focus. The ultrasound echoes were received using the same transducer through a diode limiter/transformer diplexer circuit (Model DIP-3, Matec Instruments NDT, Northborough, MA, USA). The acquired RF signals were amplified with a 32-dB preamplifier (Model AU-1114, MITEQ, Hauppauge, NY, USA) and then digitized with an oscilloscope (Model LT354, LeCroy, Chestnut Ridge, NY, USA) at a sampling rate of 1 Gsample/s. The digitized signal was stored in M-mode (i.e., motion mode) format using a computer via a GPIB and then processed off-line using MATLAB software (The MathWorks, Natick, MA, USA). Data acquisition and analysis Optical images were acquired at 15 fps for 1 min (i.e., a total of 900 images) for each data set. Each experimental case comprised 5–10 data sets that could be used for statistical analysis of the mean diameter and total number and volume of the counted bubbles. Bubbles in the acquired images generally appeared as dark circles
1.5–2.6 21–49
1.2 0.12
14.0 2.32
whose diameters and numbers could be estimated using MATLAB software. The procedures are illustrated in Figure 3(a–c). Each image was first subtracted from a background image to minimize errors. The color depth of the subtracted image was then reduced to two colors using an appropriate threshold for delineating the contours of bubbles for counting. The diameters of counted bubbles were estimated from the areas of the extracted contours with a minimum identifiable diameter of 3.5 mm. The 3-D structure of the tube meant that the produced bubbles may have been not located within the same plane of optical focus. To minimize the error in estimating the mean diameter of bubbles within the tube, the condenser diaphragm was reduced to maximize the optical depth of field and contrast in the obtained images. For bubbles observed at different distances downstream from the HIFU focus, the plane of optical focus was accordingly lifted to where the detected number of bubbles was maximized so as to counterbalance the effect of bubble flotation. A trial analysis for static single bubbles with diameters of 8–65 mm yielded standard deviations below 14% of the estimated diameters in different depths within the tube. The presence of smaller bubbles
Fig. 3. Processing of optical and acoustic data. (a) Bright-field microscopy image of vaporized droplets. (b) Binary picture of (a). (c) Bubble counts and diameters. (d) Radiofrequency M-mode image. (e) Filtered and envelope-detected Mmode image. (f) Integrated echo power (IEP) over the time interval 0–1 s in (e).
Acoustic droplet vaporization for control of bubble generation d S.-T. KANG et al.
produced a higher standard deviation in the diameter estimation. Each M-mode image comprised 2000 RF signals. Ten M-mode images were acquired for each experimental test to allow statistical analysis of the echogenicity of ADV-generated bubbles. The two sets of prominent horizontal lines in Figure 3(d) represent the stationary signals arising from the primary and reverberation echoes from the tube walls. A high-pass Butterworth filter with a cutoff frequency of 100 Hz was applied over a slow time index to minimize these stationary signals. Further processing with baseband demodulation yielded the envelope-detected images representing the bubble contrast, an example of which is provided in Figure 3(e). The echogenicity of bubbles was assessed by integrating the power of the envelope-detected signals. The integration was first performed over the time interval of a single M-mode image (i.e., 1 s), yielding a curve of the integrated echo power versus depth, as illustrated in Figure 3(f). The integration was then performed over a suitable region of interest at a depth clear of the reverberation echoes from the tube walls. The integrated echo power obtained was positively correlated with the total bubble volume within the tube. Experimental parameters Initial experimental conditions were as follows: The droplet dilution ratio and flow velocity were set to be 1:6000 and 21.4 mm/s, respectively. The acoustic parameters—acoustic pressure (peak negative pressure), pulse duration and PRF—were set to be 10 MPa, 3 cycles and 18 Hz, respectively. This fairly high acoustic pressure was used to ensure that ADV was induced in most droplets in the diluted emulsions. These conditions were separately varied in each experimental case to investigate their independent effects on the population of ADVgenerated bubbles. When flow velocity was varied to imitate different hemodynamic conditions, the PRF was accordingly regulated to ensure that droplets were insonated once within the focal volume of the HIFU sonication. The droplet concentrations used in all experimental tests were at least 10 times lower than the critical doses reported to produce adverse bio-effects such as pulmonary hyperinflation (Kripfgans et al. 2005). Such low concentrations also helped to reduce the probability of bubbles overlapping in the acquired optical images. To evaluate the effect of fluid viscosity on the population of ADV-generated bubbles, droplets were diluted in either PBS or porcine blood plasma (with viscosities of 0.7 and 1.4–2.0 mPa$s, respectively) in some experimental cases (Windberger et al. 2003). The porcine blood plasma was prepared according to the Muskiet et al. (1981). Oxygen content of the PBS and blood plasma after flowing through the semipermeable tube were
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measured to be 6.89 6 0.21 and 5.72 6 0.32 ppm (N 5 3), respectively. RESULTS Flow velocity was varied to assess the effects of ADV under different conditions of arteriole blood flow (Wang et al. 2007). Figure 4(a) illustrates the temporal evolution of mean bubble diameter at different flow velocities after the onset of ADV for droplets diluted in PBS. Owing to the short length of the cellulose tube, the results for higher flow velocities are presented over shorter periods with the same amount of data points. The mean bubble diameter at t 5 0 s was approximately 9 mm for all flow velocities. Because the mean diameter of the vaporized droplets was measured to be about 1.2 mm, the expansion ratios of droplets to bubbles at t 5 0 s are slightly higher than those (i.e., an increase in diameter of at most 5- to 5.5-fold) reported for single droplets vaporized under static conditions (Kripfgans et al. 2000; Sheeran et al. 2011b; Wong et al. 2011). After ADV, mean bubble diameter slowly increased before reaching plateaus around 16–18 mm at t 5 0.7 s. The independence of growth rate from flow velocity suggests that the post-ADV bubble expansion is a consequence of diffusion of dissolved gas from the host medium (Kripfgans et al. 2000; Reznik et al. 2012; Shpak et al. 2013). Moreover, the similarity in plateau levels suggests the presence of an equilibrium size after this growth phase, which corresponds to about twice the mean diameter relative to that observed at t 5 0 s. On the other hand, varying the droplet concentration (within the range tested in this study) did not markedly affect temporal evolution of the mean bubble diameter, as illustrated in Figure 4(b). Mean bubble diameter at t 5 0 s for a dilution ratio of 1:2000 was slightly larger than those of the other cases (with a p-value of 0.02 between 1:2000 and 1:10000). The slight difference might be due to the shorter distance between adjacent droplets, which increases the probability of bubble coalescence at the onset of ADV, and/or the shielding of ultrasound energy, which hinders the ADV of small droplets. In contrast, the mean diameter after t 5 0.7 s for a dilution ratio of 1:2000 was smaller than for the other cases, which might be attributable to a reduction of dissolved gas within the tube as a result of its rapid uptake by the produced bubbles. In Figure 4(a and b), the droplets diluted in blood plasma exhibit a slower growth rate and a lower plateau relative to those diluted in PBS. Because the diffusion constant is inversely proportional to fluid viscosity according to the Stokes-Einstein equation, the gas diffusion rate is two to three times slower in blood plasma than in PBS (Cussler 1984; Windberger et al. 2003). The initially lower gas content
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Fig. 4. Temporal evolution of mean bubble diameter after the onset of acoustic droplet vaporization (ADV) for perfluoropentane droplets in phosphate-buffered saline solution (a) under different flow velocities and (b) with different dilution ratios (N 5 10). The case marked with an asterisk was diluted in blood plasma. The acoustic pressure, pulse duration and pulse repetition frequency were set to 10 MPa, 3 cycles and 18 Hz, respectively.
and the suppressed rate of gas exchange across the wall of the semipermeable tube might be responsible for the reduced equilibrium bubble size in blood plasma. The results presented below are derived from detailed investigations of the effects of three acoustic parameters: acoustic pressure, pulse duration and PRF. Varying these parameters may exert various effects, such as size-selective ADV caused by the threshold effect, bubble disruption caused by unstable radial oscillation, bubble dissolution caused by rectified gas diffusion and bubble coalescence assisted by acoustic radiation forces, all of which can greatly influence the characteristics of the initial population of ADV-generated bubbles (Chomas
et al. 2001; Eller 1965; Fabiilli et al. 2009; Rapoport et al. 2009a). The effect of each acoustic parameter on temporal evolution of mean bubble diameter was first evaluated. Acoustic and detailed optical observations were then performed for analysis of integrated echo power, diameter distribution (in percentage terms) and total number and volume of the counted bubbles at t 5 0.93 s. This time point was chosen to ensure that the resulting bubble populations, in most cases, had reached their equilibrium sizes (corresponding to the plateau regions of the growth curves). Because of the polydispersive nature of the droplet population and the size-selective ADV, the
Fig. 5. (a) Temporal evolution of mean bubble diameter after acoustic droplet vaporization (ADV) for perfluoropentane droplets insonated at different acoustic pressures (N 5 10). (b–e) Bubble diameter distribution (in percentage terms), total bubble number (TBN), total bubble volume (TBV) and integrated echo power (IEP), respectively, as functions of the acoustic pressure at t 5 0.93 s after ADV. Each diameter distribution in (b) consists of only one typical data set, and the mean value is denoted by an open circle.
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Fig. 6. (a) Temporal evolution of mean bubble diameter after acoustic droplet vaporization (ADV) for perfluoropentane droplets insonated under different pulse durations (N 5 10). (b–e) Bubble diameter distribution (in percentage terms), total bubble number (TBN), total bubble volume (TBV) and integrated echo power (IEP), respectively, as functions of the pulse duration at t 5 0.93 s after ADV. Each diameter distribution in (b) consists of only one typical data set, and the mean value is denoted by an open circle.
mean diameter of ADV-generated bubbles decreased with increasing acoustic pressure at t 5 0 s, as illustrated in Figure 5(a). Insonation at 7 MPa was only able to vaporize large droplets, meaning that the resulting bubble population contained a relatively large proportion of larger bubbles at all periods. However, the small size differences caused by size-selective ADV were smoothed during bubble growth when the pressures were higher than 8 MPa, which should have exceeded the ADV thresholds of most of the droplet population. The simultaneous acoustic and optical observations performed at t 5 0.93 s indicated strong correlations (R2 . 0.99) between the integrated echo power, total bubble number and total bubble volume, but not mean bubble diameter, as depicted in Figure 5(b–e). The values of integrated echo power, total bubble number and total bubble volume increased as acoustic pressure increased up to 8 MPa; at higher acoustic pressures, the characteristics of the bubble population and integrated echo power remained approximately constant. These results suggest that insonation with a three-cycle pulse at pressures far above ADV threshold levels does not affect the formation of intact bubbles. For droplets insonated under varying pulse durations, the mean diameter of bubbles formed at t 5 0 s after ADV decreased slightly with increasing pulse duration, presumably because of the decreased ADV thresholds and the presence of slight bubble disruption (Fig. 6a). The prolonged insonation might also have enhanced rectified gas diffusion to reduce the bubbles toward a resonance size, which is suggested to be about 1.5–2.5 mm in diameter for the given acoustic frequency
(Chatterjee and Sarkar 2003; Eller 1965). However, this correlation became negative after t 5 0.93 s, when the mean bubble diameter for longer pulse durations was much greater than the stable equilibrium size observed in the previous experiments. At t 5 0.93 s, the diameters of bubbles for pulse durations shorter than five cycles exhibited an intact unimodal distribution, whereas those for longer pulse durations exhibited a bimodal distribution, as illustrated in Figure 6(b). Most of the bubbles were larger than twice (40– 50 mm) the stable equilibrium diameter. Integrated echo power, total bubble number and total bubble volume, but not mean bubble diameter, were found to be negatively correlated with pulse duration, as illustrated in Figure 6(b–e). The absence of a stable diameter and the considerable decrease in total bubble number and total bubble volume for different pulse durations suggest that prolonged insonation has an adverse effect on the stability of ADV bubbles, making them more susceptible to gas transfer (both inward and outward), which causes bubble growth and dissolution. Increasing the pulse duration favors the production of fewer but larger bubbles. Varying the PRF had a much greater impact than varying the pulse duration on the population of ADV bubbles. The mean diameter of bubbles formed at t 5 0 s after ADV was independent of PRF, as illustrated in Figure 7(a). A positive correlation between mean bubble diameter and PRF was observed only when PRF was in the range 18–36 Hz. At PRFs , 18 Hz, droplets were insonated once only within the beam width of the HIFU, producing bubbles with a unimodal diameter distribution at t 5 0.93 s, as illustrated in Figure 7(b). In contrast, at
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Fig. 7. (a) Temporal evolution of mean bubble diameter after acoustic droplet vaporization (ADV) for perfluoropentane droplets insonated at different pulse repetition frequencies (N 5 10). (b–e) Bubble diameter distribution (in percentage terms), total bubble number (TBN), total bubble volume (TBV) and integrated echo power (IEP), respectively, as functions of the pulse repetition frequency (PRF) at t 5 0.93 s after ADV. Each diameter distribution in (b) consists of only one typical data set, and the mean value is denoted by an open circle.
PRFs . 36 Hz, droplets were insonated twice and more within the beam width of the HIFU, in which case the produced bubbles presented a multimodal diameter distribution at t 5 0.93 s with large fluctuations in their mean diameters for different PRFs. As illustrated in Figure 7(c–e), integrated echo power, total bubble number and total bubble volume decreased rapidly with increasing PRF at a rate that was significantly faster than when increasing the pulse duration for a similar total pulse energy. The small number of bubbles accounts for the large standard deviations of the growth curves in Figure 7(a). These results suggest that increasing the PRF provides superior performance in disrupting ADV-generated bubbles. DISCUSSION This study investigated the effects of varying droplet concentration, flow velocity and various acoustic parameters on the ADVof polydisperse droplets under clinically relevant flow conditions. Acoustic and optical observations were conducted to evaluate this dependence and the temporal evolution of bubble populations after the onset of ADV. Because the HIFU and optical microscopy systems were not synchronized, observation delays of up to 16 ms might have resulted in calculated expansion ratios at t 5 0 s slightly higher than the reported upper limits of 5.2 to 5.4 caused by gas diffusion (Sheeran et al. 2011b). Nevertheless, during t 5 0–0.7 s, the bubbles absorbed ambient dissolved gases and grew to an equilibrium diameter that was as much as twice their initial diameter. The expansion ratio is consistent with previous studies (Reznik et al. 2012; Sheeran et al.
2011b), and the duration is of the same order as those (2 s) predicted for uncoated PFC bubbles (Kabalnov et al. 1998a). Post-ADV bubble growth is inevitable because ADV-generated bubbles initially contain pure PFP vapor that is maintained against multiple inward-acting pressures, including atmospheric pressure, blood pressure and Laplace pressure (Kabalnov et al. 1998a; Schutt et al. 2003). The high purity of PFP vapor establishes a high concentration gradient for both PFP and air at the water–bubble interface. The rapid decrease in surface density of lipid molecules during ADV increases the permeability of the protective shell to gas (Reznik et al. 2012). For instance, assume that a PFP bubble with a diameter of 10 mm stays in a 100% air-saturated medium. The low Ostwald coefficient (6.6 3 1025) of PFP contributes to an extremely low diffusional flux (1.3 3 10218 mol/s) that maintains the content of PFP inside the bubble (Kabalnov et al. 1998a, 1998b). However, the high Ostwald coefficients of air components (1.6 3 1022 for nitrogen and 3.1 3 1022 for oxygen) contribute to high diffusional fluxes (1.5 3 10213 mol/s for nitrogen and 8.0 3 10214 mol/s for oxygen) inward to the bubble. The intake of dissolved air from the host medium gradually increases the bubble size until the diffusional flux of air decreases to zero when the air content reaches equilibrium between the host medium and the bubble. The temporary cessation of air influx and the weak protection provided by dilute lipids in the shell result in an osmotically stabilized bubble population. A simple theoretical calculation can be performed to estimate the equilibrium bubble size. The
Acoustic droplet vaporization for control of bubble generation d S.-T. KANG et al.
pressure equilibrium before and after air permeation into a pure PFP bubble produced by ADV can be written as Pair 1PPFP 1Pvapor 2PL 5 Patm 1Pblood 5 P0air 1P0PFP 1P0vapor 2P0L ;
(1)
where Patm is atmospheric pressure; Pblood is blood pressure; Pair, PPFP and Pvapor are the partial pressures of air, PFP and vapor, respectively, in the bubble; and PL is the Laplace pressure of the bubble. The final pressures after bubble growth are denoted with primes. When the bubble expands by a factor of E in diameter, it is reasonable to assume that the variation in surface tension of the dilute lipid molecules (near 50 mN/m) is negligible (Kabalnov et al. 1998a; Sheeran et al. 2011b). The decrease in Laplace pressure and partial pressure of PFP during bubble expansion can be calculated as PPFP’ 5 PPFP/E3 and PL’ 5 PL/E. Rearranging the left and right parts of eqn (1) gives 1 1 PPFP 12 3 5 P0air 1P0vapor 1PL 12 : (2) E E Pair and Pvapor approach zero as the bubble initially contains only PFP, so that PPFP equals the sum of Patm, Pblood and PL. Rearranging eqn (2) and replacing PPFP such that only environmental and droplet conditions are involved yield Pblood 1Patm 2P0air 2P0vapor E3 (3) 1PL E2 2ðPblood 1Patm 1PL Þ 5 0 Solving the real and positive root of this cubic equation yields the expansion ratio. According to Henry’s law, 0 Pair is proportional to the concentration of dissolved 0 air. This means that Pair finally reaches 1 atm (101.325 kPa) for a 100% air-saturated medium. For bubbles with initial diameters of 5–20 mm subject to 0 Patm, Pblood and Pvapor of 101.325, 12.36 and 6.15 kPa, respectively, the expansion ratios fall within the range 1.74–2.25 and are very close to the experimental results. This equation suggests that bubble expansion caused by air permeation depends on initial bubble size, ambient pressures and the concentration of dissolved air. A decrease in the concentration of dissolved air by 10% can reduce the post-ADV expansion ratio of a 10-mm bubble by about 12%. The volume growth trend is expected to be independent of flow velocity as long as the bubbles remain in the same medium for the same period, because the whole process is associated with classic gas diffusion (Kabalnov et al. 1998a). However, the volume growth trend is dependent on droplet concentration and fluid
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viscosity, which directly influence the rate of gas uptake by bubbles and the rate of gas exchange across the wall of the tube. Insufficient replenishment of dissolved gas in the tube, which causes local gas depletion, may also reduce the rate of gas uptake by bubbles and hence retard the volume growth of bubbles. Note that the presence of osmotic equilibrium does not guarantee a permanently maintained bubble population, as the lack of sufficient protection means that the bubbles can experience further air permeation enhanced by flow disturbance to grow more than expected because of a tendency to decrease Laplace pressure. Large bubbles are barely subject to rapid dissolution presumably because of the decrease in Laplace pressure after diffusion-assisted volume growth and the low solubility (0.77 mg/L at 25 C) of PFP in water (Kabalnov et al. 1998a). Moreover, it has been found that the ADV of albumin-stabilized droplets results in fragmented shells that are no longer able to stabilize the produced bubbles (Reznik et al. 2012). These bubbles are more susceptible to external disturbances than bubbles having dilute but uniform shells, resulting in the absence of a temporarily stable size during post-ADV bubble growth. The role of surfactants in post-ADV bubble growth behavior may need to be considered in clinical applications. Other results from this study, as described below, provide information that is valuable for manipulating the population of ADV-generated bubbles by varying acoustic parameters. Applying an acoustic pressure that exceeds the threshold level will maintain the bubble population, but varying both pulse duration and PRF has a much greater impact on the bubble population. Lengthening the pulse duration favors the production of large bubbles, presumably driven by enhanced gas exchange rather than bubble coalescence. The evidence is that the bubbles exhibited a unimodal diameter distribution irrespective of the pulse duration at t 5 0 s after ADV, but subsequently exhibited a bimodal diameter distribution at t 5 0.93 s for pulse durations longer than 10 cycles (Fig. 8). The concurrent growth and dissolution of bubbles are indicative of an enhanced two-way gas diffusion. We speculated that the prolonged insonation might partly result in the exclusion of surfactant molecules from the bubble surfaces (Yount 1982). On the other hand, varying the PRF might affect the stability of produced bubbles in a similar manner, but is much more effective for bubble disruption than varying the pulse duration for a similar transmitted acoustic energy. A sufficiently high PRF will result in insonation of bubbles more than once within the focal volume of the HIFU. Insonation at low acoustic pressures will disrupt bubbles and also hinder their ability to aid the ADV of remaining droplets that have not yet been vaporized (Lo et al. 2007). This speculation
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long pulse duration and high PRF leads to a low integrated echo power, which may reduce the accuracy and sensitivity of the obtained results. The results also suggest that integrated echo power does not adequately reflect the characteristics of the bubble population. The reported strategies for reducing the ADV threshold by increasing pulse duration and PRF need to be carefully considered in terms of their impact on the bubble population produced in a particular application scenario. CONCLUSIONS
Fig. 8. Bubble diameter distributions (in percentage terms) at (a, c) t 5 0 s and (b, d) t 5 0.93 s after acoustic droplet vaporization (ADV) for perfluoropentane droplets insonated under pulse durations of (a, b) 3 cycles and (c, d) 20 cycles. The transition from unimodal distribution to bimodal distribution is an indication of enhanced gas exchange.
implies that applying a subsequent acoustic pulse at pressures lower than ADV thresholds may be advantageous to bubble clearance. Moreover, the intervals between successive acoustic pulses allow bubbles to grow larger than their initial sizes. Any change in the effects of cavitation, disruption and coalescence of bubbles in response to the size difference should also be considered to account for the enhanced bubble disruption. Elucidation of a detailed mechanism requires further investigation. Accordingly, determining the optimal acoustic parameters is of great importance for different application scenarios. For example, ultrasound contrast agentenhanced imaging requires high concentrations of small bubbles, which may be achieved by using high acoustic pressures, low pulse durations and matching the PRF with the velocity of the blood flow. Lengthening the pulse duration at a sufficient acoustic pressure and a matched PRF may produce the larger bubbles that are beneficial for gas embolotherapy. Moreover, several studies have monitored the extent of ADV and measured the threshold pressure based on an increase in imaging contrast caused by the presence of bubbles (Fabiilli et al. 2009; Kripfgans et al. 2000; Lo et al. 2007; Reznik et al. 2011). However, our results suggest that insufficient bubble generation at a
The main aim of this study was to extend our understanding of how ADV-generated bubbles can be optimally manipulated under flow conditions. For droplets with concentrations below 1 3 107 droplets/mL insonated under short pulse durations (,5 cycles) above their threshold pressures (.8 MPa), the principal aspect that affects the characteristics of the bubble population is the post-ADV bubble growth driven by air permeation, and not size-selective ADVor coalescence and disruption of bubbles. The results obtained suggest that there is a stable equilibrium size during the growth phase for vaporized droplets with phospholipid shells. The growth trend may be influenced by parameters that affect the gas content of the host medium, including droplet concentration (affecting the rate of gas uptake) and fluid viscosity (affecting the rate of gas exchange across the wall of the semipermeable tube). Varying pulse duration and PRF, but not acoustic pressure, markedly reduces the number of bubbles. Lengthening pulse duration favors the production of a smaller number of larger bubbles, whereas increasing PRF interestingly provides superior bubble disruption. These results suggest that the ADV bubble population may not be assessable simply on the basis of initial droplet sizes or their enhancement of imaging contrast. Determining the optimal acoustic parameters requires careful consideration of their impact on the bubble population produced for different application scenarios. Future studies should be conducted to provide in vivo validation of the effects observed in the present work. Acknowledgments—The authors acknowledge the support of the National Science Council of Taiwan under Grants 101-2628-E-007-001 and 101-2221-E-007-035-MY3 and National Tsing Hua University under Grant 102 N2046 E1.
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