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Exploring the Acoustic and Dynamic
Characteristics of Phase-Change Droplets
Ching-Hsiang Fan, Wei-Fu Kao, Shih-Tsung Kang, Yi-Ju Ho, Chih-Kuang Yeh, Senior Member,
IEEE
Abstract—Acoustic droplet vaporization (ADV) provides the
on-demand production of bubbles for use in ultrasound (US)-
based diagnostic and therapeutic applications. The droplet-to-
bubble transition process has been shown to involve localized
internal gas nucleation, followed by a volume expansion of three-
to fivefold and inertial bubble oscillation, all of which take place
within a few microseconds. Monitoring these ADV processes is
important in gauging the mechanical effects of phase-change
droplets in a biological environment, but this is difficult to achieve
using regular optical observations. In this study we utilized
acoustic characterization (i.e., simultaneous passive cavitation
detection [PCD] as well as active cavitation detection [ACD]) to
investigate the acoustic signatures emitted from phase-change
droplets ADV, and determined their correlations with the physical
behaviors observed using high-speed optical imaging. The
experimental results showed that activation with three-cycle 5-
MHz US pulse resulted in the droplets (diameter: 3.0–6.0 μm)
overexpanding and undergoing damped oscillation before settling
to bubbles with a final diameter. Meanwhile, a broadband shock
wave was observed at the beginning of the PCD signal. The intense
fluctuations of the ACD signal revealed that the shock wave arose
from the inertial cavitation of nucleated small gas pockets in the
droplets. It was particularly interesting that another shock-wave
signal with a much lower acoustic frequency (<2 MHz) was
observed at about 5 μs after the first half signal. This signal
coincided with the reduction of the ACD signal amplitude that
indicated the rebound of the transforming bubble. Since internal
gas nucleation is a crucial process of ADV, the first half signal may
indicate the occurrence of an ADV event, and the second half
signal may further reveal the degrees of expansion and oscillation
of the bubble. These acoustic signatures provide opportunities for
monitoring ADV dynamics based on the detection of acoustic
signals.
Index Terms—acoustic droplets, ultrasound, acoustic droplet
vaporization, acoustic signatures, high-speed microscopy
I. INTRODUCTION
T he phospholipid shell with a liquid perfluorocarbon core of
droplet can be converted into gas bubbles through
ultrasound (US) sonication—called acoustic droplet
vaporization (ADV) [1-3]—has provided a multifunction
platform for noninvasively addressing various diseases via
This paragraph of the first footnote will contain the date on which you
submitted your paper for review. This work was supported in part by support
by the Ministry of Science and Technology (MOST) of Taiwan, under grants
nos. 108-2221-E-007-041-MY3, 108-2221-E-007-040-MY3, and 106-2218-E-
007-022-MY3.
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1
methods including embolotherapy, drug delivery, enhancement
of the permeability of the blood–brain barrier (BBB), and the
destruction of tumor vessels [4-12] However, these desired
goals are only achievable by the successful occurrence of ADV.
Also, overdosing of ADV potentially results in side effects such
as irreversible cellular damage, cell death, or undesired gas
embolism [13, 14]. This situation means that developing a
technique for the real-time, high-sensitivity, and accurate
detection of ADV event occurrences could benefit its clinical
translation.
ADV events have mainly been monitored using US B-mode
imaging and optical observations. The bubbles generated from
the ADV process are capable of increasing the amplitude of
backscattered US by up to 20–30 dB, thereby serving as
effective US B-mode imaging contrast agents [10, 15].
Although this method can visualize ADV events in deep tissue,
its low sensitivity may result in it only been capable of detecting
large numbers of events. Optical observations allow the
occurrence of ADV events to be monitored with a high
temporal resolution, but it is only suitable for in vitro
applications or superficial regions [16, 17].
Interpreting acoustic signals emitted during the ADV process
by passive cavitation detection (PCD) probably be a feasible
tool for overcoming the limitations of the present detection
methods. PCD relies on the distinct Fourier spectra of acoustic
signals, in which the strengths of stable cavitation hallmarks
(harmonic signals, subharmonic signal and ultraharmonic
signal) and inertial cavitation markers (broadband signals) can
be measured in real time with high sensitivity and a good tissue
penetration depth. When utilizing microbubble-based contrast
agents in ultrasonography, the activity of the microbubbles (e.g.,
harmonic signals for steady-state oscillations or broadband
spectral content for destruction processes), the location of
microbubbles (nonlinear signals), the occurrence of brain
damage, and even the amount of delivered chemodrugs can be
characterized by analyzing the acoustic signals produced by
microbubbles during the onset of US sonication [18-21].
Previous studies have demonstrated that the violent physical
behaviors occurring in ADV result in pressure waves and
produce acoustic signals [22, 23]. Reznik et al. suggested that
Ching-Hsiang Fan is with Department of Biomedical Engineering, National
Cheng Kung University, Tainan, Taiwan, and Medical Device Innovation
Center, National Cheng Kung University, Tainan, Taiwan. Wei-Fu Kao, Shih-
Tsung Kang, Yi-Ju Ho, and Chih-Kuang Yeh * are with Department of
Biomedical Engineering and Environmental Sciences, National Tsing Hua
University, Hsinchu, Taiwan (e-mail: ckyeh@mx.nthu.edu.tw).
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when dodecafluoropentane droplets were vaporizing into
bubbles, the changes in the amplitudes of the scattered
fundamental and harmonic acoustic signals over time could be
utilize to distinguish growing bubbles from stable bubbles [24].
Rapoport et al. found that the subharmonic frequencies and
broadband noise detected during the ADV of perfluoropentane
(PFP) droplets resulted from the oscillation and unstable growth
of ADV bubbles, respectively [25]. Sheeran et al. demonstrated
that US-activated decafluorobutane droplets and
octafluoropropane droplets would overexpand and suffer
unforced radial oscillations while settling to a stable bubble
diameter. These radial oscillations generated several
narrowband peaks of the spectral range from 0.1 to 1.3 MHz
[26].
The findings of these previous studies were heterogeneous
due to inconsistencies in the experimental conditions, including
acoustic parameters, droplet components, observation periods,
and numbers of observed droplets. This situation makes it
necessary to characterize the correlation between ADV
behaviors and acoustic signals before the acoustic signals can
be used as indicators of ADV events in vivo.
In this study we simultaneously used PCD and active
cavitation detection (ACD) approaches to investigate the
acoustic signals emitted from phase-change droplets during
ADV, and determined their correlations with the physical
behaviors observed using high-speed optical imaging. The
origin and categories of ADV acoustic signals were first
investigated under the setup of single-droplet ADV. The
correlation between ADV acoustic signals and the droplet
initial size were then revealed. Finally, we used the
characteristic signatures in groups of droplets to develop a
detection system based on M-mode imaging.
II. MATERIALS AND METHODS
A. Preparation of droplets
In order to prepare droplets, 5 mol% DSPE-PEG2000
(distearoylphosphatidylethanolamine–polyethylene glycol
2000, Avanti Polar Lipids, AL, USA) and 95 mol% DSPC
(distearoylphosphatidylcholine, Avanti Polar Lipids) were well
dissolved in chloroform [3]. The lipid mixture was then drained
and dissolved with 1 wt% glycerol-containing phosphate-
buffered saline. Subsequently, 100 μL of PFP (ABCR,
Karlsruhe, Germany) was added into the solution at 4 °C. The
droplets were formed by 40 kHz sonication (100 W, 2510,
Branson, CT, USA) at 20 ℃ for 5 min.
The size distribution and concentration of prepared droplets
were estimated using a Coulter counter (Multisizer 3, Beckman
Coulter, CA, USA). The morphology of the droplets was
visualized by a microscope (model IX71, Olympus Corporation,
Tokyo, Japan). Figure 1 demonstrated that the diameter and
concentration of droplets were 2.8±1.4 μm and (1.2±0.1) × 109
droplets/mL, respectively.
B. High-speed photography and cavitation detection
An integrated acousto-optical system comprising an US
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sonication system and a high-speed microscopy imaging system
was used to perform the experiments of this study [3]. The high-
speed microscopy imaging system was setup by interfacing a
high-speed camera (model FASTCAMSA4, Photron Ltd.,
Tokyo, Japan) to a microscope (model IX71, Olympus
Corporation) for obtain serial optical images of the droplets
ADV at 500,000 fps. The imaging system was attached a self-
made water tank. For US sonication system, a 5-MHz US
transducer (model SU-128, Sonic Concepts, WA, USA) (Table
1) was confocally positioned with a waterproof 40× objective
(Carl Zeiss, Tokyo, Japan). The vaporization pulses (5-MHz, 3
cycles, single pulse, and 7-8 MPa) were transmitted by the US
transducer. Our previous study has reported that the acoustic
vaporization threshold of droplet (1.5-3.0 µm) with 5-MHz
ultrasound sonication was approximately 6 MPa [27]. In order
to ensure the successfully ADV occurrence, we therefore chose
the acoustic pressure of 8 MPa to trigger ADV in this study.
The US transducer was driven by an arbitrary waveform
generator (model AWG 2005, Tektronix, CA, USA) and a
radiofrequency (RF) power amplifier (model A150, E&I, NY,
USA) to transmit the vaporization pulses. Note that the water
tank was filled with degassed as well as deionized water at 37°C
for mimicking the in vivo temperature throughout the
experiments. In order to avoid the occurrence of inertial
cavitation from medium surrounding the droplet during
experiment, all the solutions used in this study have been
degassed overnight. The acoustic pressure was measured by a
polyvinylidene difluoride type hydrophone (model HGL-0085,
ONDA Corporation, CA, USA) in with distilled and degassed
water at 25°C.
A droplet with diameter of 3.0, 4.0, 5.0, or 6.0 μm was
administrated into a self-made agar tube (diameter: 200 μm)
and settled at the focus of microscopy via a microinjector
(model IM-6, Narishige, Tokyo, Japan) to ensure that only one
droplet appeared at the optical image. The desired size of
droplet was optically selected before the ADV experiments by
microscopy’s software (Photron FASTCAM Viewer, Photron
Ltd.).
A 2.25-MHz or 25-MHz US transducer (model V305 or
model V324, Olympus NDT, MA, USA) was confocally
arranged with a mutual focus for simultaneous PCD and ACD,
as illustrated in Figure 2a and 2b. Note that the beam axes of
these two transducers were perpendicular to the agar tube in
order to reduce the backscattered energy of US. The 2.25 MHz
transducer was used to detect ADV (as usually subhamornics
and wide-band occurrence are characteristic features for ADV
detection), and the 25 MHz transducer was suited for
monitoring the bubble fluctuation during ADV. The frequency
response of 2.25 MHz transducer was shown in Fig. S1.
For PCD, the transducer was used to passively collect the
acoustic signatures emitted during ADV. The signal was then
digitized by an oscilloscope (model LT354, LeCroy
Corporation, NY, USA) The sampling rate of oscilloscope was
set at 1 Gsample/s. The digitized signal was sent to a computer
by a GPIB interface and then analyzed offline via MATLAB
software (MathWorks, MA, USA).
For ACD, the 25-MHz US transducer was operated in pulse-
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echo mode to interrogate the morphological evolution of
bubbles using a low-amplitude US pulse (200 cycles, single
pulse, and 1 MPa). A waveform generator (model AWG 2041,
Tektronix) was utilized to produce a detection pulse that was
amplified with a radiofrequency (RF) power amplifier (model
A150, E&I,) to trigger the transducer. The RF signal was firstly
obtained via a diode limiter/transformer diplexer circuit (model
DIP-3, Matec Instruments NDT, MA, USA) and amplified by a
pulser/receiver (model 5073PR, Olympus NDT). The signal
was then digitized by the oscilloscope and stored in the
computer. The signal was then processed offline by MATLAB
software.
C. Data acquisition and analysis
In PCD, the obtained signals were translated to the frequency
domain by the fast Fourier transform. Because inertial
cavitation would produce a wideband spectral distribution
among the fundamental and harmonic peaks, the inertial
cavitation dose could be estimated from the area under the
receiver operating characteristics curve within the -6 dB
bandwidth of the 2.25-MHz transducer, which did not contain
contributions from the fundamental and harmonic components.
In ACD, the amplitude of the received signals was positively
correlated with the size of the ADV bubbles. Therefore, the time
interval between the highest signal amplitude (ADV-bubbles
expansion) and lowest signal amplitude (ADV-bubbles
compression) could be estimated and then compared with the
spectra dose of PCD. The relative changes in the signal
amplitude after ADV (referred to as the compression ratio of
the rebound bubbles) was calculated as:
Compression ratio of rebound bubbles
𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸 𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑚𝑚𝑚𝑚𝑚𝑚 /𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑚𝑚𝑚𝑚𝑚𝑚
= respectively (Fig. 3C). No wideband signals were observed in
𝑇𝑇𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 − 𝑇𝑇𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸
where Expansion voltage max is the highest signal amplitude
(ADV-bubbles expansion), Compression voltage min is the
lowest signal amplitude (ADV-bubbles compression),
T Compression is the delay time of bubble compression, and
T Expansion is the delay time of bubble expansion.
D. US M-mode imaging of ADV
For M-mode imaging, the droplet concentration was set at
2×105 droplets/mL. Each M-mode image consisted of 100 RF
signals. In order to reduce stationary signals, a high-pass
Butterworth filter (cutoff frequency: 100 Hz) was performed
over a slow time index. A baseband demodulation was used to
yield the envelope-detected images demonstrating the acoustic
signatures of phase-change droplets during ADV and the
signal-to-noise ratio (SNR) within regions of interest was also
calculated.
E. Statistical analyses
All results are presented as mean and standard deviation (SD)
values from at least three independent measurements. All
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statistical analyses were performed by one-way ANOVA, with
the Bonferroni post-hoc test applied for multiple comparisons
(SPSS version 13.0, SPSS, IBM, NY, USA). A p value of less
than 0.05 was considered to be indicative of a significant
difference.
III. RESULTS
A. Two acoustic signatures appeared during droplet
vaporization by PCD observation
Figure 2C shows typical PCD and ACD signals for single 5-
μm droplets subjected to ADV upon exposure to US at 8 MPa.
A 0.5-μs sinusoidal signal was observed at the beginning of the
PCD signal. The intense fluctuations of the ACD signal reveal
dramatic size expansion of the nucleated droplet, denoted as the
gas nucleation signal. It was particularly interesting that another
sinusoidal signal was observed at about 5 μs after the first half
signal. That signal coincided with the reduction of the ACD
signal amplitude that indicated rebound of the transforming
bubble, as evident in high-speed microscopy images (Fig. 2C,
bottom), and it was denoted as the bubble rebound signal.
We then investigated the droplet-size-dependence properties
of the PCD signals for droplets with typical sizes ranging from
3.0 to 6.0 μm (US parameters: 8 MPa, three cycles, single pulse).
The time-domain signals demonstrated that increasing the size
of the droplets did not change the amplitude of the gas
nucleation signals (Fig. 3A). The spectral analyses revealed that
the gas nucleation signals spectra that strongly resembled the -
6 dB bandwidth of 2.25-MHz transducer for all sizes of droplets,
suggesting that this signal was a wideband signal (Fig. 3B). The
quantified spectral doses were also consistent in these groups,
at 230.8±8.8, 234.0±8.5, 233.7±5.4, and 230.3±5.5 a.u. for
droplets with typical diameters of 3.0, 4.0, 5.0, and 6.0 μm,
PBS only group (50.3±3.8 a.u.) (Fig. 3b), suggesting that no
inertial cavitation event was involved in these observations.
B. The amplitudes of the ADV-bubble rebound signal is
associated with droplet size
The amplitudes of the bubble rebound signals decreased as
the droplet size increased for 3.0-, 4.0-, and 5.0-μm droplets,
respectively. The amplitude of the bubble rebound signal for
6.0-μm droplets approached that of the PBS signal with a
similar amount of noise. Furthermore, we noticed that
enlargement of the droplets also delayed the onset time of the
bubble rebound signals, being 4.6, 5.0, 5.5, and 5.9 μs for 3.0-,
4.0-, 5.0-, and 6.0-μm droplets, respectively. The distribution of
the frequency components of the bubble rebound signals
decreased as the droplets increased in size. The frequency
response of the signals for 3.0-μm droplets spanned the spectral
range from 0 to 3 MHz (Fig. 3d), whereas a lower frequency
ranges from 0 to 2 MHz appeared for 6.0-μm droplets. The
narrowed frequency distribution was also associated with a
decreasing spectral dose of the signals, being 160.5±3.7,
152.0±13.7, 144.0±23.1, and 91.8±26.1 a.u. for 3.0-, 4.0-, 5.0-,
and 6.0-μm droplets, respectively (Fig. 3e). Because this signal
appeared following the vaporization pulse had passed, it was
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independent of the US pulse, but potentially was contributed to
by radial oscillations of droplet-formed bubbles. Besides, the
extremely low intensity of spectral dose (45.5±4.0 a.u.) in PBS
group suggested that oscillations of droplet-formed bubbles
were not caused by the occurrence of inertial cavitation from
medium surrounding the droplet.
C. ADV-bubble rebound signal was resulted from the
rebound of ADV bubble by ACD observation
Further investigation of the morphological evolution during
droplet ADV with the ACD setup helped to understand the
origin of the bubble rebound signals. Figure 4A demonstrates
the superimposition of ACD and PCD signals in the time
domain for droplets with typical sizes of 3.0, 4.0, 5.0, and
6.0 μm (US parameters: 8 MPa, three cycles, single pulse).
Upon US activation, the amplitude of ACD signals started
increasing over time (from 0 to 3 μs) in each case, suggesting
that droplets were produced during the expansion phase. It was
apparent that the ACD signals steadily decayed from 3 to 5 μs,
indicating that the ADV bubbles were being compressed. The
amplitude of the ACD signals subsequently increased again,
suggesting re-expansion of the ADV bubbles, which was called
ADV-bubble rebound. It is notable that the rebound of ADV
bubbles appeared right after the vaporization pulse had passed
and was independent of the pulse, suggesting that the rebound
phenomenon was a natural oscillation instead of representing
forced or driven oscillations.
Combining with the PCD data, the time point at which the
second signals appeared strongly overlapped the transition of
the ADV-bubbles compression and re-expansion. The strong
correlation between the PCD delay times (3.2±0.3, 3.6±0.2,
4.0±0.3, and 4.3±0.2 μs for 3.0-, 4.0-, 5.0-, and 6.0-μm droplets,
respectively) and the ACD delay times (2.3±0.7, 2.7±0.4,
2.7±0.4, and 3.8±0.2 μs, respectively) also supported this
observation (R2 = 0.872) (Fig. 4B). Moreover, Fig. 4C
demonstrates that the amplitude of the second signal detected
by PCD (0.10±0.01, 0.08±0.02, 0.06±0.02, and 0.02±0.00 V for
3.0-, 4.0-, 5.0-, and 6.0-μm droplets, respectively) and the
ADV-bubbles compression ratio (2.3±0.9, 1.6±0.1, 1.4±0.1,
and 0.9±0.2 μm–1, respectively) decreased with the droplet size
(R2 = 0.921). From these data it can be concluded that the ADV
bubbles underwent a cycle of oscillation and simultaneously
emitted acoustic signals. Therefore, we considered that the
observed second acoustic signal for PCD resulted from the
rebound of ADV bubbles.
D. Volumetric overexpansion and constriction of ADV-
bubble observed from high-speed microscopy imaging
We also utilized high-speed microscopy imaging to visualize
the above-mentioned ADV bubble behaviors for 3.0-, 4.0-, 5.0-
μm, and 6.0-μm droplets under US sonication. The image
sequence and analysis of the diameter–time curve revealed that
the droplets showed volumetric overexpansion from 0 to 2 μs
to form a bubble, with the formed bubble constricting from 2 to
4 μs to settle to a stable diameter that was smaller than the
maximum size reached during the initial expansion phase (Fig.
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5). This type of activity was observed for droplets of all sizes
and increased as the size of droplet decreased. For the 3.0-µm
droplets, the contraction phase seemed to induce an additional
momentum in the formed bubble that resulted in it re-expanding
from 4 to 6 μs and then returning to the final bubble size beyond
6 μs. These results were consistent with the data of previous
studies which demonstrated that the volumetric dampening of
droplet during ADV as a function of droplet size [26]. Besides,
these results also indicated that bubble rebound resulted from
the overexpansion of droplet ADV.
E. Acoustic signatures of droplet vaporization-based US M-
mode imaging
Next, we acquired the acoustic signals of groups of
droplets (concentration: 2×105 droplets/mL) to verify if the
acoustic signals would be distorted by the simultaneous
occurrence of multiple ADV events. We found that the gas
nucleation signal was higher for groups of droplets than for a
single droplet, probably due to the occurrence of signal
superimposition in group-droplet ADV (Fig. 6A–D). In the time
domain, more than one rebound bubble signal could be
observed for groups of droplets, which is probably due to the
oscillations of ADV bubbles of multiple sizes.
Finally, the acoustic signals of group-droplet ADV
(concentration: 2×105 droplets/mL) were collected to develop
M-mode imaging. Figure 6E shows that the SNR was higher for
gas nucleation signals than for rebound bubble signals (16.6 dB
vs. 9.3 dB). Also, the appearance time of rebound bubble
signals was highly inconsistent compared with the gas
nucleation signals, suggesting that the latter is more suitable as
an indicator for the occurrence of ADV.
IV. DISCUSSION
This study successfully combined the detection of acoustic
signals and high-speed optical imaging to determine the
physical behaviors of phase-change droplets during ADV. Our
results demonstrate a strategy for monitoring the transient
physical dynamics of phase-change droplets based on the
detection of acoustic signals. This strategy potentially provides
benefits over existing techniques for use in medical applications.
Compared to US B-mode imaging for detecting ADV events,
which require a certain amount of bubbles to produce adequate
contrast enhancement, our proposed approach can detect single-
droplet ADV. Besides, the low frequency components of
acoustic signatures after droplet vaporization probably
potentially can be used to deep tissue imaging and translated to
larger animals and humans.
Although Fig. 6 demonstrated that the acoustic signatures of
droplet vaporization could be as an indicator of droplet
vaporization, the current droplet-based sequences may tend to
have lower resolution than a CEUS or B-mode sequence. For
instance, we found that a two-part droplet response playing out
over time that would either look like poor axial resolution or
clutter artifacts below the vaporization region (Fig. 6e).
However, in the CEUS or B-mode sequence the axial resolution
will be dictated by the point spread function alone, whereas the
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droplet sequence will likely stretch on for longer as the time
response evolves.
One previous published article observed that with shorter
ADV pulses, droplets would over-expand and undergo
unforced radial oscillation while settling to a final bubble
diameter [26]. The phenomenon of size dependent oscillation
generated a unique acoustic signal and the magnitude of the
acoustic signal increases as the boiling point of droplet core
decreasing. However, a confirmation between the amplitude of
oscillation and the frequency of oscillation for a single droplet
may not be apparent even using highly dilute samples of
droplets (lowest concentration of 0.05% in their study).
To characterize the acoustic signals emitted from droplets
during ADV, we conducted this study under the setup of single-
droplet ADV. Then, we revealed the correlations between ADV
acoustic signals and the different droplet sizes. Finally, we
further used the characteristic signatures in groups of droplets
to develop a detection system based on ultrasound M-mode
imaging. Besides, one previous study observed that the residual
oscillation after droplet vaporization but that did not appear in
our study. One of the reasons is potentially responsible for this
difference probably because the boiling point of
perfluorocarbon used in our study (PFP, 28 ℃) is largely higher
than that used in the referenced study (decafluorobutane, -2 ℃;
octafluoropropane: -36.7 ℃) [26]. Such high boiling point of
PFP potentially produced less momentum on expansion and did
not produce residual oscillation after droplet vaporization.
Previous studies have developed the approach that could be
viewed as the acoustic signatures during droplet vaporization to
generate high-sensitivity, high-contrast images of droplet
vaporization events. As the droplet was vaporized, it
overexpanded and oscillated down to its final resting diameter,
producing low frequency echo between 0.25 and 2.5 MHz
regardless of the excitation frequency (5.0 or 8.0 MHz) [28, 29].
In 2019, Rojas et al. demonstrated that this approach still could
provide a high contrast-to-tissue ratio image in vivo [30].
However, a long tail signal appeared in their received time
domain signal suggested that the wideband signal during
droplet vaporization and the un-forced ADV-bubble oscillation
signal probably mixed together [28]. In this study, we
successfully separated these two signals via increasing the size
of droplet (0.1 µm vs. 3.0-6.0 µm) because larger droplets
would generate larger microbubbles that oscillated at low
frequencies when compared with small droplets.
ADV is receiving increasing attention due to its ability to
produce microbubbles on demand in a noninvasive and
localized manner. A novel application is the utilization of laser-
activated nanometer-sized phase-change droplets undergoing
repeatedly vaporization and recondensation to their native
liquid nanodroplet state. This process can transiently produce
sonography-detectable microbubbles for time periods ranging
from several milliseconds to hundreds of milliseconds,
potentially providing a new methodology for high-resolution
US molecular imaging in deep tissue [31]. Moreover, this
repeated behavior also produces mechanical forces that can
disrupt the endothelial lining of the BBB, inducing BBB
opening and allowing agents to be delivered to brain tissue [32].
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However, a noninvasive imaging tool with good spatial and
temporal resolutions capable of detecting the vaporization and
recondensation of phase-changing nanodroplets is still lacking.
Given the properties of rebound bubble signals detected in this
study, we consider that rebound bubble signals can be used as
an acoustic signature of the recondensating droplets for the real-
time detection of the physical behaviors of phase-change
droplets in a biological environment.
In this study, the smallest droplets for which phase changes
were observed had diameters of around 3.0 μm, since it is
difficult to precisely estimate the sizes of smaller particles using
optical observations. Excessive US sonication (i.e., high
acoustic pressures and long exposure times) will cause the
collapse or oscillation of ADV bubbles [17]. In the present
study, we determined the suitable US sonication pressure at the
acoustic threshold for ADV and a pulse length of three cycles.
Applying these optimized sonication parameters resulted in no
disruption of ADV bubbles being observed. Moreover, the
damping of ADV bubbles also was not affected by the US
sonication.
Our data showed that increasing the diameter of the droplets
from 3.0 to 6.0 μm did not increase the amplitude of gas
nucleation signals, but it did decrease the amplitude of the
rebound signals. The gas nucleation signal was generated from
the rapid transition of PFP. Since these droplets were
insonicated at the same acoustic pressure (8 MPa), the
amplitudes of these signals should be similar. On the other hand,
the rebound signals resulted from the damped oscillation of
ADV bubbles. The degree of the oscillations was negatively
dependent on the droplet diameter. Together these observations
indicate that the gas nucleation signals and rebound signals can
be used to detect the occurrence of droplet ADV and the
formation of ADV bubbles, respectively.
While we have demonstrated that the physical behavior of
acoustic droplets during ADV can be characterized by
measuring acoustic signals, three major limitations still remain
in this study. First, the low frame rate of optical imaging.
Further increases in the frame rate of optical imaging are needed
to assess the origin of acoustic signals in detail for developing
ADV-specific detection techniques. Second, the current study
was the absence of in vivo tests. The ADV emitted acoustic
signals might be distorted by heterogenous tissues within the
body. Third, the detection setup is hard to present in vivo
because it is required to separate two frequency detectors co-
aligned within body. Forth, the acoustic pressure used in this
study might induce inertial cavitation in a non-degassed
medium within the body. A previous study demonstrated that
the inertial cavitation threshold of 4.7-MHz plused US in
human blood was higher than 6.3 MPa [33]. The next goal of
this project will focus on: (1) combining with low-boiling-point
phase-change contrast agents to reduce the acoustic energy of
droplet vaporization [18]; (2) developing novel acoustic
droplets that can precise tunability in vaporization behavior; (3)
integrating the transmitter with the detector into a single
transducer; (4) combing above two strategies for in vivo droplet
vaporization detection. Designing an algorithm and PCD setup
for obtaining the ADV emitted acoustic signals inside body also
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would be benefit to improve the in vivo application. One of the
challenges presented by ADV approaches is the ability to
successfully generate vaporization in the deep tissue (>10 cm
range) within human body when significant patient-to-patient
variability exists. Future attempts to reduce vaporization energy
or develop novel droplet may address this issue. Recently,
Shakya et al. proposed a tune-able droplet vaporization system
via encapsulating a fluorocarbon (FC) or hydrocarbon (HC)
endoskeleton into C 5 F 12 droplets [34]. FC skeletons could
inhibit vaporization, whereas HC skeletons trigger vaporization
near the rotator melting transition, potentially precisely
controlling vaporization behavior of droplets. On the other hand,
several studies have demonstrated biomedical applications of
drug-loaded phase-change droplets, but they have lacked a real-
time method for assessing the released cargos. In the field of
microbubble-assisted drug delivery, the acoustic signals
produced by microbubbles could be utilized to estimate the
amount of chemodrug that is delivered. Therefore, future
applications of our findings should be useful in classifying the
extent of drug-loaded phase-change droplets.
V. CONCLUSION
In this study we observed that a broadband shock wave
appeared at the beginning of the PCD signal. The intense
fluctuations of the ACD signal revealed that the shock wave
arose from the inertial cavitation of the nucleated small gas
pockets in the droplet. Another shock wave signal with a much
lower acoustic frequency (<2 MHz) was observed at about 5 μs
after the first half signal. That signal coincided with the
reduction of the ACD signal amplitude that indicated the
rebound of the transforming bubble. Since internal gas
nucleation is a crucial process of ADV, the first half signal may
indicate the occurrence of an ADV event, and the second half
signal may further reveal the degrees of expansion and
oscillation of the bubble. These acoustic signatures provide
opportunities for the real-time monitoring of ADV dynamics in
vivo based on the detection of acoustic signals.
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List of Table and Figure Captions

TABLE Ⅰ. Transducer characteristics.

Fig. 1. Size distribution and concentration of the phase-change droplets used in this study. Inset: bright-field microscopy image of
the phase-change droplets.

Fig. 2. (a) Experimental setup of passive cavitation detection (PCD). (b) Experimental setup of active cavitation detection (ACD).
(c) Typical examples of a PCD signal, an ACD signal, and high-speed bright field images for a 5-μm droplet exposed to a three-
cycle ultrasound (US) signal at 8 MPa.

Fig. 3. (a) Individual voltage traces for the signals received from 3.0-, 4.0-, 5.0-, and 6.0-μm droplets produced by acoustic droplet
vaporization (ADV) at a US pressure of 8.0 MPa. (b) Spectral analysis and the (c) corresponding spectral dose for the first half of
the PCD signal (N=6). (d) Spectral analysis and the (e) corresponding spectral dose for the second half of the PCD signal (N=6).
*, p<0.05. PBS, phosphate-buffered saline. Data are mean and SD values.

Fig. 4. (a) Superimposition of voltage traces of droplet ADV received for PCD and ACD with different parameters. (b) Comparison
of delay times measured for PCD and ACD (N=6). (c) Comparison of voltage amplitudes and rebound bubble compression ratios
measured for PCD and ACD (N=6). *, p<0.05. Data are mean and SD values.

Fig. 5. (a) High-speed microscopy images (500,000 fps) and (b) changes in the diameters of droplets acquired during ADV for
typical droplet sizes of 3.0 and 6.0 μm (US parameters: 8 MPa, three cycles, single pulse) (N=6).

Fig. 6. (a) Individual voltage traces, (b) time–frequency analysis, (c) spectra of gas nucleation signals, and (d) spectra of rebound
bubble signals received from PCD for single droplets and groups of droplets. (e) M-mode images produced via droplet ADV-
emitted acoustic signals of groups of droplets. Red dot rectangular: region of gas nucleation signals. Blue dot rectangular: region
of rebound bubble signals.

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TABLE Ⅰ
TRANSDUCER CHARACTERISTICS.
Transducer model Sonic Concepts SU- Olympus NDT V305 Olympus NDT V324
128
Center frequency (MHz) 5 2.25 25
Focal length (mm) 2.3 5.6 1.3
Focal width (mm) 0.3 1.9 0.2
Focal depth (mm) 21.6 35.2 13.2
Outer diameter (mm) 25.4 19.1 6.4
–6 dB bandwidth (%) 53 75.6 45.1

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Fig. 1. Size distribution and concentration of the phase-change droplets used in this study. Inset: bright-field microscopy image
of the phase-change droplets.

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Fig. 2. (a) Experimental setup of passive cavitation detection (PCD). (b) Experimental setup of active cavitation detection (ACD).
(c) Typical examples of a PCD signal, an ACD signal, and high-speed bright field images for a 5-μm droplet exposed to a three-
cycle ultrasound (US) signal at 8 MPa.

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Fig. 3. (a) Individual voltage traces for the signals received from 3.0-, 4.0-, 5.0-, and 6.0-μm droplets produced by acoustic
droplet vaporization (ADV) at a US pressure of 8.0 MPa. (b) Spectral analysis and the (c) corresponding spectral dose for the
first half of the PCD signal (N=6). (d) Spectral analysis and the (E) corresponding spectral dose for the second half of the PCD
signal (N=6). *, p<0.05. PBS, phosphate-buffered saline. Data are mean and SD values.

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Fig. 4. (a) Superimposition of voltage traces of droplet ADV received for PCD and ACD with different parameters. (b) Comparison
of delay times measured for PCD and ACD (N=6). (c) Comparison of voltage amplitudes and rebound bubble compression ratios
measured for PCD and ACD (N=6). *, p<0.05. Data are mean and SD values.

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Fig. 5. (a) High-speed microscopy images (500,000 fps) and (b) changes in the diameters of droplets acquired during ADV for
typical droplet sizes of 3.0 and 6.0 μm (US parameters: 8 MPa, three cycles, single pulse) (N=6).

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Fig. 6. (a) Individual voltage traces, (b) time–frequency analysis, (c) spectra of gas nucleation signals, and (d) spectra of rebound
bubble signals received from PCD for single droplets and groups of droplets. (e) M-mode images produced via droplet ADV-
emitted acoustic signals of groups of droplets. Red dot rectangular: region of gas nucleation signals. Blue dot rectangular: region
of rebound bubble signals.

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