Ultrasound in Med. & Biol., Vol. 45, No. 12, pp.

32463260, 2019
Copyright © 2019 World Federation for Ultrasound in Medicine & Biology. All rights reserved.
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https://doi.org/10.1016/j.ultrasmedbio.2019.08.018

Original Contribution

ACOUSTIC DROPLET VAPORIZATION IN ACOUSTICALLY RESPONSIVE

SCAFFOLDS: EFFECTS OF FREQUENCY OF EXCITATION, VOLUME FRACTION
AND THRESHOLD DETERMINATION METHOD

TAGEDPMITRA ALIABOUZAR,* XIAOFANG LU,* OLIVER D. KRIPFGANS,*,y,z

J. BRIAN FOWLKES,*,y,z and MARIO L. FABIILLI*,y,zAGEDNTE
* Department of Radiology, University of Michigan, Ann Arbor, Michigan, USA; y Applied Physics Program, University of
Michigan, Ann Arbor, Michigan, USA; and z Department of Biomedical Engineering, University of Michigan,
Ann Arbor, Michigan, USA
(Received 26 April 2019; revised 11 August 2019; in final from 23 August 2019)

Abstract—Ultrasound-induced vaporization of liquid perfluorocarbon (PFC) droplets into microbubbles, termed

acoustic droplet vaporization (ADV), has potential therapeutic and diagnostic applications. Recently, we demon-
strated how ADV—a threshold-based phenomenon—can modulate the release of biomolecules from composite
hydrogels, thereby stimulating regenerative processes, such as angiogenesis. These composite hydrogels, called
acoustically responsive scaffolds (ARSs), consist of monodispersed, micron size PFC emulsions embedded within a
fibrin matrix. This study investigated the effects of frequency of excitation (2.25, 5, 7.5 and 10 MHz) and volume frac-
tion (0.05%, 0.2% and 1% [v/v]) of monodispersed, double emulsions in the ARSs on the ADV threshold. We deter-
mined and compared the ADV thresholds via acoustic methods, including active detection, passive detection and
attenuation, as well as an echogenicity-based method using B-mode imaging. The ADV threshold determined via
these four techniques showed an increasing trend with frequency of excitation. Further analysis of the wave propaga-
tion showed that the amplitudes of high frequency harmonics were diminished in ARSs with high volume fractions of
emulsion. The ADV threshold inversely correlated with the volume fraction of emulsion at the lowest excitation fre-
quency. However, at higher frequencies, possibly due to the high acoustic reflectivity of the PFC emulsions, the ADV
threshold correlated directly with the volume fraction of the emulsion. Additionally, the ADV efficiency correlated
with the supra-threshold acoustic pressure. Overall, these results elucidate fundamental acoustic properties of the
ARSs, which can be used in future applications. (E-mail: mfabiill@umich.edu) © 2019 World Federation for
Ultrasound in Medicine & Biology. All rights reserved.
Key Words: Acoustic droplet vaporization, Ultrasound, Perfluorocarbon, Fibrin hydrogel, Regenerative
medicine.

INTRODUCTION which the phase transition becomes energetically favor-

able (Kawabata et al. 2006).
Over the past years, phase shift droplets of volatile per-
Perfluoropentane (C5 F12) has frequently been used
fluorocarbon (PFC) liquids have demonstrated better sta-
as the PFC phase in phase shift emulsions because of its
bility than conventional ultrasound contrast agents (i.e.,
bulk boiling point (i.e., 29˚C), which is close to body
microbubbles) and the ability to be formulated in the
temperature. However, recent techniques have enabled
sub-micron size range (Mullin et al. 2011; Sheeran et al.
production of stable, sub-micron size perfluorobutane
2013). These liquid PFC droplets can be vaporized in
(C4F10) and octafluoropropane (C3F8) droplets (Sheeran
situ into highly echogenic microbubbles by external
et al. 2012; de Gracia Lux et al. 2017; Li et al. 2018).
application of ultrasound pulses. The vaporization of the
Under physiologic conditions, these droplets are meta-
liquid PFC core using acoustic waves, termed acoustic
stable in the liquid state, despite being superheated.
droplet vaporization (ADV), occurs beyond a critical
Researchers have pointed to the suppression of homoge-
peak negative pressure (i.e., the ADV threshold) at
nous nucleation, rather than elevated boiling point
through Laplace pressure, as the likely mechanism for
Address correspondence to: Mario L. Fabiilli, Ph.D., University
of Michigan, 1301 Catherine Street, 3226 A Medical Sciences Building the metastability of superheated droplets (Mountford and
I, Ann Arbor, MI 48109-5667. E-mail: mfabiill@umich.edu Borden 2016). Alternatively, PFCs with higher bulk

3246
 ADV in acoustically responsive scaffolds
M. ALIABOUZAR et al.
boiling points ultimately offer better thermal stability by
eliminating any potential spontaneous vaporization as
well as higher probability of repeated vaporization and
re-condensation, which are desired for extended diagnos-
tic and therapeutic applications (Asami and Kawabata
2012). In our recent paper, we investigated the use of
phase shift micro-emulsions with perfluorohexane (PFH)
(C6F14), perfluoroheptane (C7F16) and perfluorooctane
(C8F18) (Lu et al. 2019).
The ability to non-invasively and spatiotemporally
control ADV using focused ultrasound makes phase shift
droplets desirable for therapeutic applications (Kripfgans
et al. 2005; Fabiilli et al. 2010; Rapoport 2012; Moncion
et al. 2016a). For example, phase-shift emulsions can be
formulated to contain a payload (e.g., drug), thereby
enabling localized payload delivery following ADV
(Couture et al. 2012; Duncanson et al. 2014). We have
utilized ADV to control extravascular release of thera-
peutic agents by incorporating phase shift emulsions
with a double emulsion structure of water-in-PFC-in-
water (W1/PFC/W2) into implantable fibrin hydrogels.
These composite hydrogels, termed acoustically respon-
sive scaffolds (ARSs), have been used to stimulate
regenerative processes like angiogenesis via the ADV-
controlled release of growth factors. For tissue regenera-
tion, the ability to spatiotemporally control the release of
growth factors is desired given that regenerative pro-
cesses are driven by the spatiotemporally controlled pre-
sentation of growth factors.
The impact of the following parameters on the
ADV threshold within ARSs has been previously
reported: fibrin concentration, emulsion concentration,
PFC core, droplet size and pulse repetition frequency
(Fabiilli et al. 2013; Moncion et al. 2016a). However,
the effect of the frequency of excitation used to generate
ADV within ARSs is currently unknown. The ADV
threshold has been shown to increase (Sheeran et al.
2013; Aliabouzar et al. 2018b) or decrease (Kripfgans
et al. 2000; Schad and Hynynen 2010) with the fre-
quency of excitation within liquids. For homogenous
nucleation in liquids, the length of the negative pressure
half-cycle, which is longer at lower frequencies, directly
correlates with the probability of nucleation (Church
2002). Alternatively, the inverse correlation between the
ADV threshold and frequency has been shown to result
from non-linear propagation and super-harmonic focus-
ing, which is only effective for larger droplets (Li et al.
2014; Shpak et al. 2014). The use of higher frequency
ultrasound to generate ADV within ARSs would enable
more tightly focused ADV as well as minimize any cavi-
tation-related damage to large molecular payloads or
adjacent cells.
To measure the threshold of vaporization, different
methods of detection—such as passive detection
3247
(Giesecke and Hynynen 2003; Aliabouzar et al. 2019),
active detection (Schad and Hynynen 2010; Reznik et al.
2011), high speed optical microscopy (Kripfgans et al.
2004; Sheeran et al. 2013) and echogenicity-based tech-
niques (Kripfgans et al. 2000; Kawabata et al. 2006;
Fabiilli et al. 2009; Williams et al. 2013)—have been
employed. Because of the differences in threshold deter-
mination techniques, droplet size distributions and
experimental setups, it is difficult to directly compare
ADV thresholds among the previous studies. Although
each technique has its own merit, a systematic study
comparing different methods of ADV detection has not
been completed before.
In this work, acoustic properties of fibrin gels and
ARSs, including attenuation and sound speed, were stud-
ied prior to ADV. The ADV thresholds in ARSs were
determined as a function of frequency of excitation and
volume fraction of monodispersed, double emulsions. We
determined and compared the ADV thresholds using
acoustic methods, including active and passive detection,
attenuation measurements and an echogenicity-based
method using B-mode imaging. Knowing how the acous-
tic properties of ARSs, such as attenuation, change pre-
and post-ADV can be a valuable tool to monitor and
quantify the release of a therapeutic payload in situ. Fur-
thermore, wave propagation and generation of harmonics
in ARSs was investigated. The efficiency of vaporization,
which is relevant for drug delivery, was also measured in
ARSs. Overall, these fundamental acoustic properties can
be used to hone future therapeutic studies with ARSs as
well as applications of ADV within viscoelastic media
(e.g., tissue).
MATERIALS AND METHODS
Double emulsion preparation and characterization
Micron-sized, double emulsions with a W1/PFC/W2
structure were prepared using a microfluidic-based tech-
nique following a previously described method (Moncion
et al. 2017). PFH (C6 F14, CAS# 355-42-0, Strem Chemi-
cals, Newburyport, MA, USA), with a bulk boiling point
of 56˚C, was used as the PFC phase. The shell stabilizing
the primary emulsion (W1/PFC) was a fluorosurfactant
copolymer consisting of Krytox 157 FSH (CAS# 51798-
33-5, DuPont, Wilmington, DE, USA) and polyoxyethy-
lene glycol (molecular weight: 1000 g/mol, CAS# 24991-
53-5, Alfa Aesar, Ward Hill, MA, USA). The fluorosurfac-
tant was dissolved at 2% (w/w) in PFH. The PFH solution
was combined with phosphate buffered saline (PBS, Life
Technologies, Carlsbad, CA, USA) and then sonicated
(Q55 with a CL-188 immersion probe, QSonica, LLC,
Newton, CT, USA) for 30 s while on ice. The resulting pri-
mary emulsion had a W1/PFC ratio of 1:2 (v/v).
3248 Ultrasound in Medicine & Biology
The primary emulsion was pumped at 0.5 mL/min
through an in-line filter (0.5 mm stainless steel frit, Cat#
24993, Restek, Bellefonte, PA, USA) and into the inner
channel of a quartz microfluidic chip (Cat# 3200146,
Dolomite, Royston, United Kingdom) using a syringe
pump (KDS-410, KD Scientific, Holliston, MA, USA).
Simultaneously, 50 mg/mL Pluronic F68 (CAS# 9003-
11-6, Sigma-Aldrich, St. Louis, MO, USA) in PBS was
pumped at 2.5 mL/min through an in-line filter and into
the outer channels of the chip using a second syringe
pump (78-0388, KD Scientific). The chip was mounted
on an inverted microscope (DMIL, Leica Microsystems,
Buffalo Grove, IL, USA), which enabled visualization of
emulsion production. Emulsions were stored at 4˚C until
use. The size distribution, coefficient of variation and
concentration of the double emulsion (n = 5), analyzed
using a Coulter Counter (Multisizer 4, Beckman Coulter,
Brea, CA, USA) with a 50 mm aperture tube, were 13.2
§ 0.8 mm, 2.6% § 0.2% and 3.44 £ 108 § 0.57 £ 108
droplets per mL, respectively.
ARS preparation
ARSs were prepared by first dissolving bovine fibrin-
ogen (Sigma-Aldrich) in PBS at 20 mg/mL clottable pro-
tein while under gentle vortex mixing for 30 s. Tubes
containing the fibrinogen solution and PBS were degassed
in a vacuum chamber (Isotemp vacuum oven, Model 282
A, Fisher Scientific, Dubuque, IA, USA) at approximately
6 kPa for 60 min to minimize the amount of dissolved gas,
which could act as cavitation nuclei. ARSs were made by
combining the prepared fibrinogen, PBS, varying volume
fraction of double emulsions (0.05%, 0.2% and 1% [v/v])
and bovine thrombin (Thrombin-JMI, King Pharmaceuti-
cals, Bristol, TN, USA). The final concentration of fibrino-
gen and thrombin in the ARSs were 10 mg/mL and
2 U/mL, respectively. The ARS mixture was aliquoted
into custom-made cylindrical sample chambers (outer
diameter, 27 mm; inner diameter, 20 mm; length, 12 mm)
made of polyvinyl chloride pipe. The open ends of the
cylindrical chamber were sealed by Tegaderm membrane
(3 M Health Care, St. Paul, MN, USA), as shown in
Figure 1a, which permitted polymerization of the ARS
within the chamber. During polymerization, the ARSs
were stored at 4˚C for 3 h before use. Fibrin-only gels (i.e.,
without double emulsions) were made in a similar manner
as a control. Fibrin-only gels and ARSs were used on the
same day of preparation.
Ultrasound exposure setup and parameters
All ADV threshold measurement experiments were
conducted in an acrylic tank (32 cm £ 13 cm £ 13 cm)
filled with circulating, deionized (DI) water at 37˚C. Four
single-element, spherically focused, immersion trans-
ducers (diameter = 19.05 mm, focal length = 38.1 mm,
Volume 45, Number 12, 2019
Panametrics, Olympus NDT Corporation, Waltham, MA,
USA) were used to generate ADV within the ARSs. The
specifications of the transducers, which operated as trans-
mitters at their central frequencies, were as follows:
2.25 MHz (Model A305 S; 6 dB: 1.463.2 MHz),
5 MHz (Model V308; 6 dB: 3.066.52 MHz), 7.5 MHz
(Model A321 S; 6 dB: 5.569.92 MHz) and 10 MHz
(Model A315 S; 6 dB: 6.7513.03 MHz).
All transducers were calibrated in the free field at
the focus using an in-house built fiber optic hydrophone
with a fiber diameter of 100 mm. Each ADV transducer
was driven by a pulsed waveform (pulse duration, 5.4
ms; pulse repetition frequency, 100 Hz) generated by a
dual-channel function generator (33500 B, Agilent Tech-
nologies, Santa Clara, CA, USA) and amplified by a
gated radiofrequency (RF) amplifier (GA-2500 A Ritec
Inc., Warwick, RI, USA). The amplified signals were
viewed in real-time on an oscilloscope (HDO4034, Tele-
dyne LeCroy, Chestnut Ridge, NY, USA).
Acoustic characterization of ARSs and fibrin-only gels
prior to ADV
Acoustic properties of ARSs containing 1% (v/v)
double emulsion, prior to ADV studies, as well as fibrin-
only gels were determined using a substitution technique
(Selfridge 1985; Zell et al. 2007; Parker et al. 2010). We
measured ultrasound attenuation coefficient and sound
speed in the above mentioned gels in the water tank
with focused, single-element transducers centered at
2.25 MHz, 5 MHz and 10 MHz at room temperature
(21BC). Transducers were driven with a pulser-receiver
(5077 PR, Olympus, Center Valley, PA, USA) operating
in pulse echo mode at low pressures to avoid undesirable
non-linear propagation and ADV. Attenuation and sound
speed were calculated as described previously (Fabiilli
et al. 2016; Aliabouzar et al. 2018c).
Determination of ADV thresholds in ARSs
Active detection technique. For active vaporization
detection, a spherically focused immersion transducer with
a center frequency of 2.25 MHz (Model A305 S; 6 dB:
1.463.2 MHz), the same diameter and geometric focus as
the transmitters, was used as a detector. The detecting
transducer was connected to the pulser-receiver operating
in pulse-echo mode. The ADV and detecting transducers
were placed at twice their focal lengths apart and confo-
cally aligned. Each ARS and fibrin-only gel was positioned
between the two transducers, such that the axial focus was
positioned approximately in the middle of the ARSs and
fibrin-only gels (approximately 32 mm from the surface of
the transmitter) and allowed to thermally equilibrate in the
37˚C water bath for 15 min before the ADV experiments.
A schematic representation of the active detection method
 ADV in acoustically responsive scaffolds
M. ALIABOUZAR et al. 3249

Fig. 1. Schematic representation of three setups used to determine the ADV threshold in ARSs: (a) active detection, (b)
passive detection and (c) B-mode imaging methods. For ADV threshold determination via attenuation measurements,
the setup in panel (a) was used, and the signal from the back wall of the chamber was time gated and analyzed. The trans-
mitting and detecting transducers were confocally aligned, and the ARSs, having a thickness of 12 mm, were positioned
32 mm from the surface of the transmitter such that the axial focus was positioned approximately in the middle of the
gel. ADV = acoustic droplet vaporization; ARS = acoustically responsive scaffold; PVC = polyvinyl chloride.

is shown in Figure 1a. The ADV threshold was determined
by exposing each ARS to pulses of increasing acoustic
pressure. Following exposure at each pressure level, the
pulse-echo signal from the detecting transducer was
acquired directly from the oscilloscope (sampling rate of
100 MHz) and stored for offline processing.
Passive detection technique. For the passive detec-
tion method, ADV was generated in the ARSs using the
aforementioned 2.25 MHz and 10 MHz transducers. A cap-
sule hydrophone (HGL-1000, frequency range 120 MHz,
Onda, Sunnyvale, CA, USA) was placed 5 cm away from
the focus of the transducer and used as a broadband
receiver to detect backscattered signals from the ARSs dur-
ing ultrasound exposure as shown in Figure 1b. The RF sig-
nals, collected with the hydrophone and digitized by the
oscilloscope, were stored for further analysis.
Attenuation measurements. For the ADV threshold
measurements, the droplet to bubble transition was deter-
mined by monitoring the effective attenuation of ultrasound
propagating through the ARSs containing varying volume
fractions of emulsions. For this study, we employed the
active detection setup illustrated in Figure 1a. The ARSs
were exposed to ultrasound at 2.25 MHz, 5 MHz or
10 MHz at 37˚C. A second transducer with a center fre-
quency of 2.25 MHz was used as an active detector in the
pulse-echo mode. Effective attenuation in ARSs was mea-
sured by time-gating the echo from the back wall of the
Tegaderm window and monitoring the change in its ampli-
tude as the peak negative pressure was incrementally
increased. The signal amplitude at each peak negative pres-
sure was compared with the signal received by the active
detector from the ARS for a sham exposure (i.e., 0 MPa,
ADV transducer off). Thus, no correction for reflection
from the Tegaderm membrane was required.
B-mode imaging. In an acrylic tank (30 cm £
60 cm £ 30 cm) filled with 37˚C DI water, echogenic-
ity-based studies were conducted at two excitation fre-
quencies of 2.25 MHz and 10 MHz for ARSs containing
0.05% (v/v) emulsion. In this method, a diagnostic ultra-
sound imaging system (ZS3, Mindray North America,
Mountain View, CA, USA) with a 4 MHz linear array
(Mindray, C4-1) was used to record B-mode images for
detection of bright echoes from bubbles created by
ADV. The mechanical index (MI) of the array was set to
the lowest value of 0.31, which did not generate any
apparent ADV within the ARSs. Based on a MI of 0.31
at 4 MHz, the maximum peak rarefactional pressure in
3250 Ultrasound in Medicine & Biology
the imaging plane was 0.6 MPa. B-mode images were
analyzed using ImageJ software (http://imagej.nih.gov).
Detection for ADV bubbles was evaluated in terms of
pixel intensity in a given region of interest (ROI). ROIs,
consistent in size for a given frequency, were selected at
the ADV location in each B-mode image to measure the
mean pixel intensity (Lo et al. 2006).
Vaporization efficiency measurements
The efficiency of ADV, which was the ratio of the
number of droplets remaining after ADV to the number
of droplets before ADV, was assessed by modifying pre-
viously described methods (Fabiilli et al. 2010; Moncion
et al. 2016b; Moncion et al. 2017). ARSs containing
0.05% and 1% (v/v) emulsion were exposed to acoustic
pressures at and above the ADV threshold at an excita-
tion frequency of 2.25 MHz. The focal region (i.e., loca-
tion of ADV) in each ARS was removed using a 6 mm
biopsy punch (Miltex, Inc. York, PA, USA) and then
digested in 0.25% trypsinethylenediaminetetraacetic
acid (Life Technologies) for 3 h in the fridge. As a con-
trol, this procedure was repeated for ARSs that were not
exposed to ultrasound. The number of droplets in the
digested samples was counted using a Coulter counter
and compared with the control to find the percentage of
vaporized emulsions at each supra-threshold pressure.
Criteria for determining ADV thresholds
Upon vaporization, a PFC droplet undergoes a volu-
metric expansion of about 125-fold, as shown by Kripf-
gans et al. (2005), Rapoport et al. (2009) and Sheeran
et al. (2011), resulting in a sudden increase in scattering.
For the active and passive detection methods, the peak-to-
peak RF voltage and the fundamental frequency compo-
nent of the scattered response from the ARSs were plotted
as a function of peak negative pressure. Similarly, for
attenuation measurements and B-mode imaging following
Fig. 2. (a) Frequency-dependent attenuation and (b) speed of sound in fibrin gels and ARSs with 1% emulsions (n = 6).
Speed of sound did not significantly change in either scaffold as a function of frequency. Speed of sound was signifi-
cantly (p < 0.05) higher in fibrin than ARS with 1% emulsion. ARS = acoustically responsive scaffold.
Volume 45, Number 12, 2019
ADV, the effective attenuation of the medium and mean
pixel intensity of the ARSs were plotted, respectively, as a
function of increasing peak negative pressure. As in other
studies, we found that the peak-to-peak RF voltage, funda-
mental response, effective attenuation and the mean pixel
intensity in the ARSs remained relatively constant at
acoustic pressures below the ADV threshold (i.e., sub-
threshold values). At pressures above the ADV threshold,
the metrics all increased from the sub-threshold values and
remained at elevated levels. Data were then fit with a
piece-wise linear function with two pieces using GraphPad
Prism software (GraphPad Software, Inc., La Jolla, CA,
USA). The ADV threshold was defined as the peak nega-
tive pressure corresponding to the intersection of the two
linear fits (Radhakrishnan et al. 2013).
Statistical analysis
All statistical analyses were performed using Graph-
Pad Prism software. All data are presented as the mean
and corresponding standard deviations. ADV thresholds
are listed as peak negative pressure. For each frequency
and concentration, measurements were repeated twice per
gel in three independently produced ARSs (n = 6) at each
excitation amplitude unless otherwise stated. The 95%
confidence intervals of the calculated parameters are listed
in the format of [SL, SH], where SL is the lower bound
value and SH is the upper bound value. Significant differ-
ences between groups were determined using a one-way
analysis of variance followed by Tukey’s multiple com-
parisons test, with a significance level of 0.05.
RESULTS AND DISCUSSION
Acoustic characterization of fibrin-only gels and ARSs
prior to ADV measurements
The frequency-dependent attenuation and speed of
sound in fibrin-only gels and ARSs with 1% (v/v)
 ADV in acoustically responsive scaffolds
M. ALIABOUZAR et al. 3251

emulsion are shown in Figure 2. The attenuation data
were compiled from all three transducers (i.e., 2.25, 5
and 10 MHz), thus covering the range of 112 MHz.
Linear fits of the attenuation data yielded slopes of 0.08
§ 0.005 dB cm1 MHz1 (R2 = 0.91, [0.07, 0.09]) and
0.14 § 0.006 dB cm1 MHz1 (R2 = 0.94, [0.13, 0.15])
for fibrin gels and ARSs, respectively. The measured
attenuation of the Tegaderm membrane, which consti-
tuted the window of the sample chamber, with respect to
free field water was 0.12 § 0.07 dB cm1 MHz1,
which is close to the value of 0.2 dB/MHz reported by
Deng et al. (1996).
Note that the frequency-dependent attenuation val-
ues are with respect to a water-filled sample chamber to
account for attenuation by the Tegaderm windows. The
speed of sound in water at room temperature (21˚C) was
taken to be 1483 m s1 (Greenspan and Tschiegg 1959;
Duck 2013). The attenuation coefficients of fibrin gel
and ARSs show a linear dependence on frequency, while
the speed of sound has negligible frequency-dependence
within the range studied here. Attenuation coefficient
has been reported to be proportional to the nth power of
frequency, where n is approximately between 1 and 2
for soft tissues over a broad frequency range from
150 MHz (Madsen et al. 1998; Chaffaı et al. 2000;
Rabell-Montiel et al. 2018).
ADV threshold determination in ARSs
Active detection method. Figure 3 shows the peak-
to-peak voltage and the fundamental component of the
scattered response, acquired using the active technique,
from ARSs containing varying volume fractions of dou-
ble emulsion as well as fibrin-only gels (i.e., control) at
excitation frequencies of 2.25 MHz and 10 MHz. Scat-
tered responses from ARSs remained constant before
ADV (Fig. 3). However, once the bubbles were formed
as a result of ADV, the scattered signal increased sharply
because of the higher impedance mismatch and com-
pressibility of gas-filled bubbles compared with liquid
droplets. Using piece-wise linear fitting (as described
above), the ADV thresholds were 2.55 § 0.11 MPa, 3.15
§ 0.13 MPa and 3.67 § 0.04 MPa for ARSs with 1%,
0.2% and 0.05% (v/v) emulsions, respectively, at

Fig. 3. Peak-to-peak voltage and fundamental components of the scattered response for ARSs containing varying vol-
ume fractions (0.05%, 0.2% and 1% [v/v]) of double emulsions at the excitation frequencies of (a, b) 2.25 MHz and (c,
d) 10 MHz, recorded using active method. A linear piece-wise function was fit to the data points. The ADV threshold
was defined as the pressure where the first two lines of the piece-wise function intersected (indicated by arrows in
Fig. 3a). ADV = acoustic droplet vaporization; ARS = acoustically responsive scaffold.
3252 Ultrasound in Medicine & Biology
2.25 MHz. A similar trend in the threshold was observed
for the fundamental component of the scattered response
for ARSs at this excitation frequency (Fig. 3b). Note that
the scattered fundamental response from PFH double
emulsions seemed to saturate at higher excitation pres-
sures at 2.25 MHz. Similar saturation behavior can also
be seen in ADV studies of PFC liquids (Reznik et al.
2011; Moncion et al. 2017) as well as numerical predic-
tions of scattered responses from microbubbles (Sarkar
et al. 2005). At an excitation frequency of 10 MHz, the
ADV thresholds of ARSs with 0.2% and 0.05% (v/v)
emulsions were 5.16 § 0.07 MPa and 4.76 § 0.09 MPa,
respectively (Fig. 3c and 3d). For ARSs with 1% (v/v)
emulsions, no ADV was detected at an excitation fre-
quency of 10 MHz for the maximum peak negative pres-
sure of 6 MPa studied here.
A comparison of the slope (a) of each line above
the ADV threshold in Figure 3a shows a three-fold
increase for ARSs with lower emulsion volume fractions
of 0.2% (a = 0.11, R2 = 0.96, [0.08, 0.13]) and 0.05%
(a = 0.1, R2 = 0.98, [0.08, 0.11]) compared with 1%
emulsion (a = 0.03, R2 = 0.95, [0.02, 0.04]). The steeper
slopes in ARSs with lower volume fraction of emulsions
could be an indication of a higher vaporization effi-
ciency. It has been shown in previous studies that higher
volume fractions of vaporized droplets within the focus
could shield the acoustic energy from propagating fur-
ther, therefore hindering additional ADV (Xin et al.
2017). Similarly, in Figure 3c, slopes of 0.29 (R2 = 0.99,
[0.28, 0.32]) and 0.21 (R2 = 0.96, [0.16, 0.25]) were mea-
sured for ARSs with 0.2% (v/v) and 0.05% (v/v) emul-
sions, respectively.
Additionally, we studied the effect of emulsion vol-
ume fraction on the fundamental response of the ARSs
for sub-threshold pressures (i.e., 02.5 MPa) as shown
in Figure 4. In this region, the slope of the fundamental
response was not significantly different than zero. For
Fig. 4. At sub-threshold peak negative pressures (i.e., 02.5 MPa), the fundamental response of ARSs at 2.25 MHz (a)
and 10 MHz (b) correlated with emulsion volume fractions. Statistically significant differences (p < 0.05) are denoted as
follows: a: versus 1%; b: versus 0.2%; g: versus 0.05%. ARS = acoustically responsive scaffold.
Volume 45, Number 12, 2019
sub-threshold pressures, the fundamental response from
ARSs with 1% (v/v) and 0.2 % (v/v) double emulsions
was significantly greater than fibrin alone, as shown in
Figure 4a at 2.25 MHz. The fundamental response of an
ARS with 1% (v/v) double emulsion was 7.1 and 16.6-
fold higher compared with ARSs with 0.2% (v/v) and
0.05% (v/v) emulsions, respectively. This was because
of a lower speed of sound in PFC liquids, which resulted
in larger acoustic impedance mismatch between PFH
emulsions (approximately 0.8 MRayl) and water
(approximately 1.48 MRayl). The echogenic properties
of PFC droplets have been investigated in previous stud-
ies (Couture et al. 2006; Rapoport et al. 2009). Note that
Couture et al. (2006) reported a linear correlation
between the scattered response and volume fraction of
(0.03%0.5%) sub-micron PFH particles. Such linear
behavior also demonstrates that the measurements are
not affected by multiple scattering.
Similarly, at 10 MHz, the slope of the sub-threshold
region was not significantly different than zero for all
ARSs. The fundamental response of all ARSs with vary-
ing volume fraction of emulsions was higher than fibrin-
only gels (Fig. 4b). At 10 MHz, the average fundamental
response of ARSs with 1% (v/v) emulsion was 7.8- and
17.5-fold higher than ARSs with 0.2% (v/v) and 0.05%
(v/v) emulsion, respectively.
A summary of the average ADV thresholds in ARSs
of varying emulsion volume fraction at different excita-
tion frequencies, determined using the active method, is
displayed in Figure 5. Overall, the ADV threshold corre-
lated with the excitation frequency, with the dynamic
range of the threshold at each frequency correlating with
the emulsion concentration. At 2.25 MHz, the ADV
threshold correlated inversely with emulsion concentra-
tion because of the enhanced probability of nucleation
(Reznik et al. 2011; Lu et al. 2019). However, at higher
frequencies (i.e., 7.5 MHz and 10 MHz), an inverse
 ADV in acoustically responsive scaffolds
M. ALIABOUZAR et al.
Fig. 5. Average ADV thresholds in ARSs with varying volume fractions (0.05%, 0.2% and 1% [v/v]) of double emulsion
as a function of frequency of excitation, determined using the (a) active and (b) passive detection methods.
ADV = acoustic droplet vaporization; ARS = acoustically responsive scaffold.
correlation was observed between the ADV threshold
and emulsion concentration in the ARS. At each fre-
quency, there were significant differences in the ADV
thresholds of ARSs with varying volume fraction of
emulsions, with the exception of ARSs with 0.2% (v/v)
and 0.05% (v/v) emulsions at 5 MHz. In addition, when
comparing the same concentration across different fre-
quencies, no significant differences in the threshold were
observed between ARSs with 0.05% (v/v) emulsion at
2.25 MHz, 5 MHz and 7.5 MHz, as well as ARSs with
0.2% (v/v) emulsion at 5 MHz and 7.5 MHz.
Passive detection method. With the passive detec-
tion technique, the profiles of peak-peak voltage and fun-
damental responses displayed similar trends as in
Figure 3 (data not shown). Figure 5b shows the ADV
threshold of ARSs, with varying emulsion volume frac-
tion, at excitation frequencies of 2.25 MHz and 10 MHz
obtained using the passive detection method. There was
no significant difference in thresholds for ARSs with
0.2% (v/v) and 0.05% (v/v) emulsions at 10 MHz. Pas-
sive thresholds displayed a similar trend with respect to
frequency and volume fraction, as was observed in
Figure 5a, using active detection. Active and passive
techniques have been previously used as classic methods
for detection of inertial cavitation in vitro (Roy et al.
1990; Holland et al. 1996). While active methods have
higher sensitivity to detect weak or single bubble oscilla-
tions, emissions detected by passive techniques are more
associated with resonance-sized bubbles or clouds of
bubbles (Holland et al. 1992; Ding et al. 2015). The
major drawback of active detection is production of
acoustic bursts that might interfere with the transmitting
signal. However, unlike passive detection, active meth-
ods can be used to determine where and when vaporiza-
tion is taking place, allowing both spatial and temporal
3253
resolution. Note that in the context of transient cavita-
tion, Roy et al. (1990) showed that thresholds measured
by the active method were lower than or equal to those
detected by passive methods.
Attenuation measurements. Droplet-to-bubble tran-
sition was also studied by measuring the effective attenu-
ation through the ARSs at sub- and supra-threshold
pressures at 2.25 MHz. The results are shown in Figure 6.
The effective attenuation of the ARSs increased sharply
above the ADV threshold because of the presence of
vaporized droplets in the acoustic path and was higher at
an excitation frequency of 2.25 MHz (Fig. 6a) compared
with 5 MHz (Fig. 6b) and 10 MHz (Fig. 6c). The gener-
ated bubble cloud correlated inversely with frequency,
which was consistent with the size of the focal volume.
The focal volume, calculated from the transducer specifi-
cations, at 2.25 MHz was approximately 30 mm3 and 0.3
mm3 at 10 MHz (Olympus 2006).
Similar to Figure 5, at each excitation frequency,
there was a significant difference in ADV thresholds
between ARSs with varying volume fraction of emul-
sions, except for ARSs with 0.2% (v/v) and 0.05% (v/v)
emulsion at 5 MHz. There were significant differences
among ADV thresholds when comparing the same vol-
ume fraction of emulsions across frequencies, with the
exception of ARSs with 0.05% (v/v) at 2.25 MHz and
5 MHz.
The measured attenuation was due to absorption
and scattering of bubbles generated by ADV. As men-
tioned earlier, upon vaporization, the PFC phase under-
goes a volumetric expansion, which is approximately
125-fold in water (Rapoport et al. 2009; Sheeran et al.
2011). The presence of a viscoelastic medium such as
gels will reduce this expansion factor (Qamar et al.
2010; Sheeran et al. 2011). Since the produced bubbles
3254 Ultrasound in Medicine & Biology Volume 45, Number 12, 2019

Fig. 6. Effective acoustic attenuation of ARSs containing varying volume fractions of double emulsions as a function of
peak negative pressure at excitation frequencies of (a) 2.25 MHz, (b) 5 MHz and (c) 10 MHz. The attenuation was mea-
sured at 2.25 MHz using a pulse echo technique. ARS = acoustically responsive scaffold.

due to ADV are expected to be around 4050 mm in
diameter, which accounts that each double emulsion
droplet contains 67% (v/v) PFH, attenuation due to scat-
tering will be more prominent since the product of the
wave number and scatterer size approaches one.
At the frequencies studied here, we did not observe
any change in the speed of sound in the ARSs as ADV
bubbles were generated. Note that speed of sound in a
mixure of bubbles will not depend on frequency at exci-
tation frequencies much higher or lower than the reso-
nance frequency of the bubbles at low volume fractions
of bubbles (Commander and Prosperetti 1989; Povey
1997; Wilson and Roy 2008). Bubbles with a diameter
of 4050 mm will have a resonance frequency in the
hundred-kilohertz range and therefore will be non-reso-
nant at frequencies studied here.
B-mode imaging. Acoustic methods used here to
study ADV are based on scattering emission and are 1-D
detection techniques, with the inability to spatially map
the distribution of vaporization. However, echogenicity
based techniques, such as conventional B-mode imaging,
can effectively reflect the spatial distribution of ADV. B-
mode images from ARSs with 0.05% (v/v) emulsion are
shown in Figure 7 (af) at 2.25 MHz and (gl) at
10 MHz at increasing peak negative pressures. Specular
reflection from the two windows of the sample chamber
can be seen in these figures. Based on mean pixel inten-
sity measurements, the ADV threshold in ARSs with
0.05% (v/v) emulsions was 3.58 § 0.26 MPa (n = 5) at
2.25 MHz and 4.65 § 0.23 MPa at 10 MHz (n = 5).
The change in the shape of the bubble cloud from
ellipsoidal (Fig. 7d) to a tadpole (Fig. 7f) due to acoustic
shadowing was consistent with the previously published
theoretical predictions (Xin et al. 2017). Variabilty in
cloud shape and size, in a backward propagating direc-
tion toward the transducer, with increasing amplitude
and pulse number, was also observed by Lo et al. (2006).
Comparison of ADV threshold detection methods
Figure 8 is a comparison of the ADV thresholds
measured using four different techniques of active, pas-
sive and attenuation along with B-mode imaging. For
the different techniques, thresholds were not statistically
different, except for ARSs with 0.05% (v/v) and 0.2%
(v/v) emulsion at 10 MHz. With the attenuation method,
ADV thresholds in 0.05% and 0.2% (v/v) ARSs were
5.14 MPa (5.2% higher) and 5.59 MPa (8% higher)
 ADV in acoustically responsive scaffolds
M. ALIABOUZAR et al. 3255

Fig. 7. B-mode images of ARSs with 0.05% (v/v) double emulsion exposed to ultrasound at increasing peak negative
pressures of (a) 1.1 MPa, (b) 3.2 MPa, (c) 4.2 MPa, (d) 4.8 MPa, (e) 5.8 MPa and (f) 6.1 MPa at 2.25 MHz frequency.
Exposures were repeated at 10 MHz at peak negative pressures of (g) 0.9 MPa, (h) 2.7 MPa, (i) 4.1 MPa, (j) 5 MPa, (k)
5.8 MPa and (l) 6.1 MPa. Direction of the sound wave is shown by the arrow. Scale bar shows 5 mm. ARS = acoustically
responsive scaffold.

Fig. 8. Comparison of ADV thresholds determined using different techniques including active, passive, attenuation and
echogenicity methods at (a) 2.25 MHz and (b) 10 MHz. Statistically significant differences (p < 0.05) are denoted as
follows: a: versus 0.05% (v/v) attenuation and b: versus 0.2% (v/v) attenuation. ADV = acoustic droplet vaporization.

compared with thresholds measured using other techni-
ques. This difference was possibly because of the small
size of the generated bubble cloud having a negligible
effect on attenuation of the propagating signal.
ADV efficiency in ARSs
In Figure 9, we determined the vaporization effi-
ciency at 0 MPa (i.e., sub-threshold), at the ADV thresh-
old and at supra-threshold pressures for ARSs containing
0.05% and 1% (v/v) double emulsions at an excitation
frequency of 2.25 MHz. At higher applied pressures, the
supra-threshold focal volume increased, along with the
ADV efficiency (Fig. 9a). This has also been previously
observed (Radhakrishnan et al. 2016). ADV efficiency
increased linearly with the applied peak negative pres-
sure for ARSs with 0.05% (v/v) emulsion while it satu-
rated, possibly due to acoustic shadowing, at higher
pressures for the ARSs with 1% (v/v) emulsion. This is
also consistent with the calculated slopes of lines above
the ADV threshold shown earlier in Figure 3. Recently,
3256 Ultrasound in Medicine & Biology
Fig. 9. (a) Efficiency of vaporization in ARSs containing 1% (n = 5) and 0.05% (v/v) (n = 6) double emulsions as a func-
tion of peak negative pressure at the excitation frequency of 2.25 MHz. (b) Corresponding attenuation in the ARSs as
the ADV efficiency increased at an excitation frequency of 2.25 MHz. The effective attenuation of the gel was measured
at 2.25 MHz. ADV = acoustic droplet vaporization; ARS = acoustically responsive scaffold.
we demonstrated that the ADV efficiency for ARSs with
PFH emulsion was less dependent on ultrasound expo-
sure parameters, such as lateral raster spacing and num-
ber of axial planes, compared with ARSs with higher
boiling point PFCs. In general, a high ADV efficiency is
desired in applications where ADV is used to trigger
release of a therapeutic agent from an ARS (Lu et al.
2019).
As seen in Figure 9b, the effective acoustic attenua-
tion correlated with the ADV efficiency. Monitoring the
droplet-to-bubble transition by measuring the change in
the acoustic properties of the medium, such as attenua-
tion, could enable new methods of monitoring the dose
of a therapeutic payload released by ADV within an
ARS in situ. Previously, acoustic mapping has been used
to co-localize cavitation activity with drug delivery
(Choi et al. 2014). Further studies are required to investi-
gate the growth mechanism of ADV bubbles in the vis-
coelastic fibrin gels over the physiologically relevant
time points and their potential impact on the payload
release. Parameters such as PFC boiling point, volume
fraction of the emulsions, dissolved gas concentration,
ambient pressure, temperature and fibrin concentration
will impact the physical stability of an ARS post-ADV.
We have previously shown that the volumetric expan-
sion of ARSs following ADV correlated inversely with
the bulk boiling point of the PFC liquid, with a 233%
volumetric increase for ARSs containing perfluoropen-
tane emulsions (Lu et al. 2019). For ARSs implanted
in vivo, the percent of fibrin remaining in ARSs contain-
ing PFH emulsions was 39% § 9.1% at day 14 post-
ADV (Moncion et al., 2016b).
Free field and in situ wave propagation
Earlier studies of ADV have shown a decreasing
trend in threshold with increasing frequency (Kripfgans
Volume 45, Number 12, 2019
et al. 2000; Kawabata et al. 2006; Schad and Hynynen
2010), which is opposite to the current results. The
potential mechanisms proposed for this inverse correla-
tion have included droplet deformation (Kripfgans et al.
2004), inertial cavitation (Fabiilli et al. 2009), non-linear
propagation and super-harmonic focusing due to the low
speed of sound in PFCs, as well as the presence of higher
harmonics at the focus (Li et al. 2014; Shpak et al. 2014;
Miles et al. 2016). However, all these studies were done
in water, which is a non-attenuating, non-linear medium
(Khelladi 2012). The Goldberg number, defined as the
ratio of non-linear distortion over attenuation, is much
higher for water than tissue (Szabo et al. 1999). For
example, the Goldberg ratio for water at 5 MPa and
5 MHz is 266 and for fibrin gel is 12 (i.e., similar to soft
tissues such as breast and liver). This indicates that non-
linear distortion is easier to achieve in water than tissue.
To further investigate this, we studied wave propa-
gation and generation of harmonics in ARSs with 1%
(v/v) emulsion, fibrin gels and free-field water using a
fiber optic hydrophone as a passive receiver. Waveforms
in the time domain, recorded by the fiber optic hydro-
phone at each excitation amplitude and frequency, were
converted to the frequency domain using the fast Fourier
transform (FFT) to obtain the harmonics. Figures 10a
and 10b show the fundamental and third harmonic
responses as ultrasound at 2.25 MHz propagated through
the three different media. Analogous data at 10 MHz is
displayed in Figures 10c and 10d. Attenuation dimin-
ished the generation of harmonics by decreasing their
amplitudes, with a greater impact at 10 MHz versus
2.25 MHz. Because of the finite active element size of
the fiber optic hydrophone (100 mm) and associated spa-
tial averaging errors (wavelength of 50 mm at 30 MHz),
harmonics above the third were not analyzed. However,
higher harmonics, which are more effective for the
 ADV in acoustically responsive scaffolds
M. ALIABOUZAR et al. 3257

Fig. 10. Effect of the propagating media on generation of harmonics at (a, b) 2.25 MHz and (c, d) 10 MHz. Waveforms
were recorded in the free field and in the presence of a fibrin gel and an ARS containing 1% (v/v) double emulsion using
a fiber optic hydrophone. Recorded waveforms were Fourier transformed using the FFT to obtain the harmonics. For
ARSs containing 1% (v/v) double emulsions, measurements were done up to 4 MPa at 2.25 MHz and 5.5 MPa at
10 MHz to avoid ADV at room temperature. ADV = acoustic droplet vaporization; ARS = acoustically responsive scaf-
fold; FFT = fast Fourier transform.

focusing effect (Li et al. 2014; Shpak et al. 2014), are
expected to be even more attenuated. In addition to the
effect of the viscoelastic fibrin matrix on ultrasound
propagation, the effect of dissolved gas concentration
should be considered as well. All the presented experi-
ments were conducted in DI water (i.e., non-degassed
medium), in which small changes in pressure could
impact the dissolved gas content. This could affect sound
propagation, with a greater impact at higher frequencies
(Povey 1997). It is also noteworthy to mention that the
patterns of homogenous nucleation in pure PFC liquid
droplets, studied by previous authors using high speed
cameras (Kripfgans et al. 2004; Shpak et al. 2013), may
not necessarily be the same for double emulsions, where
heterogeneous nucleation may occur. Further studies
using high speed cameras are required to further eluci-
date the vaporization dynamics of double emulsions.
The other factor that could impact the trend of ADV
threshold with frequency is the constant pulse length of
5.4 ms that was used throughout the ADV studies, which
yields an increasing number of cycles for higher
frequencies. Although several authors have shown negli-
gible dependence of ADV threshold on the number of
cycles and microsecond pulse lengths (Lo et al. 2007;
Reznik et al. 2011; Aliabouzar et al. 2018a), the nucle-
ated vapor bubble may be disrupted when exposed to
more cycles at high pressures because of inertial cavita-
tion or re-condensation (Reznik et al. 2013).
The effect of droplet concentration in the ARS on
wave propagation was investigated in a similar manner.
Figures 11a and 11b compare the frequency spectrum of
an acoustic wave with a peak negative pressure of 3.5
MPa as it propagated through water and in an ARS con-
taining 1% (v/v) double emulsion at 2.25 MHz and
10 MHz, respectively.
As shown in Figure 11a, the frequency spectrum of the
signal from an ARS containing 1% (v/v) emulsion was sim-
ilar to that of water at 2.25 MHz at this high-pressure ampli-
tude. However, the higher order harmonics were highly
damped in the presence of an ARS with 1% (v/v) emulsion
at 10 MHz compared with the free field (see Fig. 11b).
Thus, the double emulsion in the ARS can act as a low pass
3258 Ultrasound in Medicine & Biology
Fig. 11. Frequency spectrum of the pressure waveform, normalized to the amplitude of the fundamental component, in
free field water and in the presence of an ARS containing 1% (v/v) double emulsion at a peak negative pressure of 3.5
MPa at (a) 2.25 MHz and (b) 10 MHz. ARS = acoustically responsive scaffold.
filter at higher frequencies of excitation, decreasing the
amplitudes of higher harmonics. Although droplets are
weak, linear scatterers of ultrasound, compared with gas-
filled microbubbles, the reflection due to the acoustic
impedance mismatch can be strong (Marsh et al. 2002; Cou-
ture et al. 2006; Rapoport et al. 2009). Reflection due to the
impedance mismatch of micron-size PFH emulsions was
shown earlier in Figure 3. Therefore, as the volume fraction
of PFH emulsions in the ARSs increased, the scattering
cross section and attenuation due to scattering also
increased.
Note that in the long wavelength limit (i.e., Ray-
leigh scattering), scattering intensity is proportional to
the fourth power of frequency and sixth power of particle
size (Povey 1997), which could explain the trend
observed with the ADV threshold and volume fraction at
higher frequencies. Couture et al. (2006) showed a linear
increase in the overall scattering from sub-micron PFH
droplets with concentration as well as frequency. Fur-
thermore, several studies reported significant enhance-
ment (up to 32 dB) in surface reflectivity of substrates
when targeted with sub-micron PFH emulsions at high
frequencies, while less enhancement was observed when
the same particles were in suspension (Lanza et al. 1998;
Marsh et al. 2002).
Previous studies have reported a decreasing trend of
ADV threshold with concentration due to the enhanced
probability of nucleation at the focus (Reznik et al.
2011; Arena et al. 2015). In these aforementioned stud-
ies, a single excitation frequency was used to phase-tran-
sition polydispersed droplets at concentrations up to
0.3% (v/v). Because of the polydispersed droplet popula-
tion, an increase in concentration also increased the like-
lihood that larger droplets traversed the region of
excitation and therefore reduced the ADV threshold. At
10 MHz, Williams et al. (2013) observed no significant
Volume 45, Number 12, 2019
dependence of ADV on droplet concentrations over the
range 0.002%0.1% (v/v). The inverse correlation
between ADV threshold and volume fraction of emul-
sions, as a result of increased number of vaporization
events at the focus, was only observed here at the lowest
frequency (2.25 MHz), where the focal volume was
almost 100 times larger than that at 10 MHz. At higher
frequencies, because of the combined effects of focal
volume and acoustic impedance mismatch, lower vol-
ume fractions have lower ADV thresholds.
CONCLUSION
In this work, we systematically investigated the
effect of frequency of excitation on the ADV thresholds
in ARSs containing varying volume fractions of mono-
dispersed, double emulsions. We studied the ADV phe-
nomenon through acoustic means, including active and
passive detection techniques, attenuation measurements
and enhanced echogenicity of B-mode images. The
ADV thresholds determined via these four techniques
were consistent with respect to the frequency of excita-
tion and emulsion volume fraction. Results showed that
ADV threshold increased with the frequency of excita-
tion. We showed that the propagating media affected the
generation of non-linearly distorted waves, diminishing
the amplitude of harmonics at higher frequencies and
higher volume fractions of double emulsions and there-
fore potentially inhibiting super-harmonic focusing. The
ADV threshold decreased with increasing volume frac-
tion of double emulsions in the focal volume at the low-
est excitation frequency studied here. However, at
higher frequencies, as a result of high acoustic reflectiv-
ity of PFH emulsions, the ADV threshold correlated
directly with the volume fraction of emulsions. Future
 ADV in acoustically responsive scaffolds
M. ALIABOUZAR et al.
studies will employ the presented findings to optimize
regenerative studies using ARSs.
Acknowledgments—This work was supported by NIH grant R01
HL139656 (MLF).
Conflict of interest disclosure—The authors declare no competing
interests.
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