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An in vitro study of a microbubble contrast agent V Sboros, K V Ramnarine, C M Moran et
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INSTITUTE OF PHYSICS PUBLISHING PHYSICS IN MEDICINE AND BIOLOGY
Phys. Med. Biol. 49 (2004) 159–173 PII: S0031-9155(04)60751-2

An in vitro study of a microbubble contrast agent

using a clinical ultrasound imaging system
V Sboros, C M Moran, S D Pye and W N McDicken
Department of Medical Physics, University of Edinburgh, Royal Infirmary of Edinburgh,
1 Lauriston Place, Edinburgh EH3 9YW, UK

E-mail: Vassilis.Sboros@ed.ac.uk

Received 11 March 2003, in final form 20 August 2003

Published 15 December 2003
Online at stacks.iop.org/PMB/49/159 (DOI: 10.1088/0031-9155/49/1/011)

Abstract
Optimal insonation settings for contrast imaging are yet to be specified, mainly
due to the lack of good understanding of the behaviour of the microbubbles.
A satisfactory model that explains the behaviour of individual contrast agent
scatterers has not yet been reported in the literature. An in vitro system based
on a commercial scanner (ATL HDI3000) has been developed to investigate
the backscatter of such agents. Suspensions of Definity
R
were introduced
in an anechoic tank. The frequency of transmitted ultrasound varied from
1 to 5 MHz, pulse period from 2 to 10 periods and peak negative acoustic
pressure from 0.08 to 1.7 MPa. The backscatter at the fundamental and
second harmonic frequency windows from the agent was normalized in terms
of the corresponding components of backscatter from a blood mimicking fluid
suspension. The agent provided a dominant resonance effect at 1.6 MHz
transmit frequency. Second harmonic normalized backscatter averaged around
9 dB higher than the fundamental. The normalized fundamental backscatter
intensity was linear with peak negative pressure. The second harmonic at
resonance peaked at 0.5 MPa suggestive of bubble disruption above such
pressure. The system proved capable of illustrating the ultrasonic behaviour
of Definity
R
in vitro, and the investigation suggested particular insonation
conditions for optimal image enhancement using Definity
R
.
(Some figures in this article are in colour only in the electronic version)

1. Introduction

Contrast enhanced ultrasound is a growing field not only limited to diagnostic applications,
but also expanding to novel drug delivery approaches. New imaging techniques, introduced
in recent years and found in the new models of ultrasonic equipment, have increased the use
of contrast agents and introduced new areas of clinical diagnosis. However, this increase has

0031-9155/04/010159+15$30.00 © 2004 IOP Publishing Ltd Printed in the UK 159

160 V Sboros et al

not been followed by a sufficient expansion of knowledge on the behaviour of contrast agents
(UCAs) in the presence of ultrasound fields. The microbubble interaction with the incident
ultrasound field during a contrast imaging examination is only qualitatively understood. So far
experimental investigations have not led to a comprehensive microbubble model. Complicated
insonation protocols, such as those that use pulse sequences, make this inadequacy more
obvious. There is very little understanding of the microbubble behaviour with such protocols,
and also very little work that investigates the behaviour of microbubbles under a wide range
of beam settings. The combinations of pulse sequences that might be used in pulse inversion
or amplitude modulation protocols make the problem more elaborate. Ultrasound equipment
manufacturers mainly brought such novel techniques forward. It is understood that elaborate
experimentation aiming at maximization of sensitivity, preceded the launch of these new tools.
Very little however, is based on theoretical knowledge or fundamental understanding of the
microbubble behaviour.
The basic limitation of the majority of in vitro investigations originates in the small number
of field parameters used (transmit frequency, acoustic pressure, pulse period). This leads to
qualitative conclusions that offer little towards a thorough understanding of the behaviour of
the microbubbles. As a result an experiment that accurately assesses microbubble resonant
frequency is yet to be published. The main problem is the small number of transmit frequencies
used. Most studies use up to three (Schrope et al 1992, Schneider et al 1992, Schrope and
Newhouse 1993, de Jong et al 1994, Wei et al 1997, Wu and Tong 1998, Moran et al
1998, Morgan et al 1998, Dayton et al 1999, Krishna et al 1999) and do not attempt to
describe the effect of driving frequency on microbubble behaviour. Those that do attempt
such measurements (Frinking and de Jong 1998, Frinking et al 1999, Marsh et al 1998),
with even up to five different transmit frequencies (de Jong et al 1992, de Jong and Hoff
1993), mainly due to broadband transmission, provide results of microbubble scatter of a
rather qualitative nature. Interesting microbubble scatter results were obtained by Krishna
and Newhouse (1997) and Chang et al (1996), with narrowband transmission. In the case of
Krishna and Newhouse (1997), even though only four different frequencies were employed,
the effect of acoustic pressure to contrast scatter was shown to be dependent on transmit
frequency. Chang et al (1996) used 0.5 MHz increments in transmit frequency from 1.5 to
5 MHz and obtained Doppler shift spectra that provide reasonable quantitative information on
the behaviour of two contrast agents. Narrowband transmission is most suited to microbubble
scatter studies. The above transmit frequency increment (0.5 MHz) can be decreased to provide
good spectral resolution that is vital for the estimation of a resonant behaviour. Furthermore,
it is known that microbubble oscillations have an initial transient behaviour that takes
2–3 periods to settle (Parlitz et al 1990). As a result, the study of microbubble behaviour
requires long sampling, i.e. several periods of insonation. Ideally studies that include a range
of pulse periods could be conclusive for time domain changes of the microbubble behaviour.
Studies that investigate the impact of acoustic pressure on microbubble behaviour are
also not conclusive (Krishna and Newhouse 1997, Morgan et al 1998, Krishna et al 1999,
Shi et al 1999, Shi and Forsberg 2000, Sboros et al 2001b, 2002a, 2003). The use of larger
ranges of transmit frequency and pulse period could have assisted these studies to locate
frequency and acoustic pressure thresholds of microbubble destruction. The introduction of
optical systems to monitor such phenomena initiated a new era in microbubble investigations
(Dayton et al 1999, de Jong et al 2000, Morgan et al 2000). Microbubble destruction became
visible (Chomas et al 2000, 2001a, 2001b, May et al 2002). Visualization of the behaviour of
microbubbles under different beam settings (Morgan et al 2000, May et al 2002) or in different
surroundings became possible (Dayton et al 2001). The only limitation of optical microscopy
is that it compromises the quality of acoustical data due the tightness of the used set-ups.
In vitro contrast agents 161

In a previous communication an in vitro acoustical set-up for the characterization of

contrast agents was described (Sboros et al 2001a). This system accommodated a wide range
of beam settings as a result of the flexibility offered by the combination of a commercial
medical scanner and RF data acquisition. The investigation of the properties of the contrast
agent Definity
R
(Bristol-Myers Squibb Inc., MA, USA) with that system, is the subject of
the present paper. Definity
R
suspensions were subjected to different transmit frequencies,
acoustic pressures and pulse periods. In most in vitro contrast studies it is common to use
‘microbubble density less than that required to cause shadowing’ (Krishna and Newhouse
1997). The concentrations used are in general greater than 104 microbubbles per ml of
suspension. These concentrations are relatively high, and self-attenuation is expected to affect
measurements in a complex manner. But high microbubble concentrations involve further
complications such as multiple scatter, bubble–bubble interactions (Sboros et al 2002b). For
these reasons the microbubble concentration was chosen to be the lowest possible that would
still provide detectable echoes at all the used settings. The results are presented in terms
of normalized backscatter intensity using a blood mimicking fluid (BMF) suspension as the
reference (Ramnarine et al 1998). The suitability of the BMF for the present study has been
demonstrated in previous communications (Sboros et al 2000, 2001a). The present study
presents, in a quantitative and systematic way, the behaviour of a contrast agent in diagnostic
ultrasound fields.

2. Materials and methods

2.1. Imaging protocol

A full specification of the system was presented in a previous communication (Sboros et al
2001a). An HDI3000 ATL scanner was used for the experiments. The scanner was controlled
using a PC through a serial port connection. The combined transmit frequency range for
the two phased array probes (P3-2 and P5-3) was 1.2–4.3 MHz, each probe could provide
a mechanical index (MI) range of 0.1–0.7 (as displayed on the monitor), and a pulse period
range of 2–10 periods (pulse period refers to the number of periods per pulse throughout the
paper). The different ultrasound fields were controlled by the PC using Labview graphical
programming software (National Instruments, Austin, TX, USA). RF data were also acquired
using the same PC. The digitization rate of the scanner was 20 MHz and the number of data
points acquired was 256 for each line. The acquired ROI for a frame was 96 lines wide, and
fixed at an offset distance of 4 cm from the probe. While the calibration of transmit frequency
and pulse period was straightforward, the acoustic pressure is more complicated due to the
nature of the ROI which lies in three-dimensional space. The peak negative pressure (PNP) at
the centre of the ROI was measured (Sboros et al 2001a) and provided an axial average peak
negative pressure. However, peak negative pressure is the measurement of maximum pressure
in the other two spatial dimensions (width and thickness of beam). It is therefore understood
that the complex distribution of acoustic pressures, that a contrast suspension is subjected to,
is difficult to characterize. Conventionally, it is acceptable for scatter measurements, that the
greater part of echoes originates around the peak pressure, and as a result peak pressure is
most useful. But this should not necessarily be the case for nonlinear scatterers. However,
any other option would only complicate things. The calibration of peak negative pressure was
chosen as the most appropriate for the present study, but the reader should be aware of the
above facts when the peak negative pressure is mentioned.
A Perspex tank 10 cm deep, 15 cm long, 7 cm wide, was used in the experiments
and was lined with an acoustic absorber (CERAM AB, Lund, Sweden). The distance of
162 V Sboros et al

the scanning surface of the ultrasound imaging probe from the bottom of the tank was
fixed at 6 cm. The contrast agent Definity
R
was used, which is a Perflutren lipid coated
microbubble with perfluoropropane gas and average diameter between 1.1 and 1.3 µm. The
concentration in the tank was approximately 23 microbubbles per ml of suspension and
the volume of the suspension was 300 ml. The concentration was as low as possible to
observe individual scattering events in the reconstructed images at the lowest sensitivity. This
ensured the presence of echoes at all settings. The risks of performing measurements using
high microbubble concentrations have been previously discussed (Sboros et al 2002b). Here
the microbubble concentration remained at much lower levels than commonly used. Individual
microbubbles and particles were distinguishable at most transmit frequencies, but at peak
transducer sensitivity the scattering suspension looked more dense. As a result, a slight
variation in the attenuation of both contrast and BMF suspensions was expected. However, it
has already been shown that the spectral behaviour of the normalizing suspension used provided
good agreement with the transmit spectrum of the transducers (Sboros et al 2001a), and the
variation of scatter and attenuation did not affect the results significantly. The measurements
were performed at room temperature (∼18 ◦ C, variation < 10%).
The ECG lead of the scanner was connected to a pulse generator (Phillips PM5705) in
order to trigger frames at different time intervals. The suspension was stirred for 30 s using a
magnetic stirrer, and the first triggered frame was captured and stored in the PC.

2.2. Data analysis

IDL 5.0.2 programming software (Research Systems, Boulder, CO, USA) was used to
reconstruct the image data. Visualization of all data was necessary in order to search for
artefacts or impurities that would corrupt the results. While artefacts are generally multiple
reflections and reverberations and were easy to avoid, echoes from impurities were slightly
more difficult. The appearance and real time behaviour of echoes, which were present at the
suspensions before the introduction of contrast, were used for guidance to reject data. This
process was empirical and is explained in Sboros et al (2003).
After acquisition the RF data from each frame were signal processed by means of an elliptic
filter to extract the fundamental and second harmonic components using Matlab software
(Mathworks, Natick, MA, USA). The average component intensity was calculated and the
ratio of the component intensities of contrast to that of the BMF formed the component’s
normalized backscatter. Figure 1 illustrates the fundamental and second harmonic backscatter
amplitudes in the frequency domain. The data in this figure were acquired at the maximum
MI (0.7), 1.45 MHz transmit frequency and a pulse period of 8 periods. The filter for
both frequencies provided a 1 MHz passband. The fundamental component was centred
on 1.45 MHz, and the harmonic component at 2.9 MHz. The backscatter intensity of the
fundamental frequency component for Definity
R
was equivalent to the area with margins


R
under the Definity FFT curve, the 0.95 and 1.95 MHz gridlines, and the frequency axis
(the light grey and BMF areas centred on 1.45 MHz). The backscatter intensity of the
fundamental frequency component for the BMF was equivalent to the area under the BMF
FFT curve, the 0.95 and 1.95 MHz gridlines, and the frequency axis (the black area centred on
1.45 MHz). The ratio of the two intensities gives the fundamental normalized backscatter
intensity. Likewise, the second harmonic component of the backscatter intensity of Definity
R

was the light grey area centred on 2.9 MHz. For the BMF, the second harmonic was the
black area centred on the same frequency. The ratio of these two intensities gives the second
harmonic normalized backscatter intensity. The normalized backscatter of different spectral
In vitro contrast agents 163

4
x 10 FFT
14

12

10
Amplitude
8

0
0.95 1.45 1.95 2.4 2.9 3.4
Frequency in Hz 6
x 10

◦
Figure 1. Amplitude of the signal of the Definity R and BMF suspensions in the frequency domain.
The principle of the calculation of normalized backscatter at different parts of the spectrum is
illustrated. Acquisition was performed at MI = 0.7, 1.45 MHz transmit frequency and 8-period
pulse. The filter for both fundamental and second harmonic frequencies provided a 1 MHz
passband. The fundamental component is centred at 1.45 MHz, and the harmonic component at
2.9 MHz. The backscatter intensity of the component of the fundamental frequency for Definity R ◦
was equivalent to the light grey and black areas centred at 1.45 MHz. The backscatter intensity of
the component of the fundamental frequency for the suspension of BMF was equivalent to only the
black area centred at 1.45 MHz. The ratio of the two intensities gives the fundamental normalized
backscatter intensity. Likewise, the second harmonic component of the backscatter intensity of
◦
Definity R was the light grey and the black areas centred at 2.9 MHz, and for the BMF only the black
area centred at the same frequency. The ratio of those two intensities gives the second harmonic
normalized backscatter intensity.

parts of the signal are the physical quantities used to assess the enhancement of the agent with
respect to a reference material.

3. Results

The dependence of the second harmonic normalized backscatter (SHNB) of Definity
R

on
transmit frequency, is shown in figure 2. The measurements using two different pulse periods
of 3 and 6 periods are displayed. Each point in these plots referred to a measurement performed
at one setting (the unique combination of transmit frequency, acoustic pressure and pulse
period). Figure 2 is a cumulative display of the results from all different acoustic pressures
used at each frequency. Because different acoustic pressures were used at different frequencies
and also because of the large variability of the measurements, the results are impossible to
group here and a scatter plot was considered as the only option for display. The SHNB was
only displayed, at a particular setting, if both the contrast agent and normalizing suspension
provided echoes above the noise level. There were distinct peaks of the harmonic component
in both graphs around 1.6 MHz that are strongly suggestive of resonant behaviour. At all the
other used pulse periods, the fundamental and second harmonic normalized backscatter also
displayed resonant behaviour around the same transmit frequency.
Figure 3 displays the fundamental and second harmonic normalized backscatter versus
transmit frequency for the lower pulse periods (2-, 3- and 4-period pulses). The data in the
graphs are from selected pressure ranges. A very good overlap between the normalized data
164 V Sboros et al

12000

10000

Normalized backscatter
8000

6000

4000 3 periods

2000

0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
Transmit frequency (MHz)
12000

10000
Normalized backscatter

8000

6000

4000 6 periods

2000

0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
Transmit frequency (MHz)

Figure 2. Second harmonic normalized backscatter. All the data acquired for 3- and 6-period
pulses are displayed. The normalized backscatter peak lies between 1.5 and 2.0 MHz for both 3
and 6 periods.

from the two probes is displayed in all eight plots (2.7–3.2 MHz transmit frequency), and
demonstrates the effectiveness of the normalization approach used in the analysis. Figure 3
shows that the SHNB lies generally at tenfold higher levels than the fundamental normalized
backscatter (FNB). The SHNB was 0.2–17.2 dB higher than the FNB (mean 9.0 dB). The
SHNB displays a slight resonant peak at the lowest displayed acoustic pressure window,
while the FNB does not. There is a general amplification of resonant behaviour with the
increase of acoustic pressure for both SHNB and FNB. This behaviour seems to achieve a
peak above 0.6 MPa. The difference between the levels of SHNB and FNB is maximum
below 0.6 MPa. This is in agreement with Morgan et al (1998) who found that at acoustic
pressures higher than 0.5 MPa the ratio of the second harmonic to fundamental did not further
increase. However, it must be noted that due to the transducer bandwidth limitation, at high
acoustic pressures the data are not sufficient at the low transmit frequencies (<1.6 MHz). Off
resonance, according to figure 3, the SHNB and FNB appear generally independent of transmit
frequency. Because of the shortness of pulses the bandwidths of both fundamental and second
harmonic were large enough to cause overlap between the two components. In other words,
the filtering of the data by using a spectral windowing is inadequate to fully separate a spectral
In vitro contrast agents 165

1000 Fundamental 12000

900
Second Harmonic
800 10000
0.2-0.4 MPa 0.2-0.4 MPa
Normalized Backscatter

Normalized backscatter
700
8000
600

500
6000
400

300 4000

200
2000
100

0
1 1.5 2 2.5 3 3.5 4 4.5 0
1 1.5 2 2.5 3 3.5 4 4.5
Frequency (MHz)
Frequency (MHz)
1000
12000
900 0.4-0.6 MPa
0.4-0.6 MPa
800 10000
Normalized Backscatter

Normalized backscatter
700
8000
600

500
6000
400

300 4000

200
2000
100

0
1 1.5 2 2.5 3 3.5 4 4.5 0
1 1.5 2 2.5 3 3.5 4 4.5
Frequency (MHz)
Frequency (MHz)
1000
12000
900 0.6-0.9 MPa
800 10000 0.6-0.9 MPa
Normalized Backscatter

Normalized backscatter

700
8000
600

500
6000
400

300 4000

200
2000
100

0
1 1.5 2 2.5 3 3.5 4 4.5 0
1 1.5 2 2.5 3 3.5 4 4.5
Frequency (MHz)
Frequency (MHz)
1000
12000
900 0.9-1.2 MPa
800 10000 0.9-1.2 MPa
Normalized Backscatter

Normalized backscatter

700
8000
600

500
6000
400

300 4000

200
2000
100

0
1 1.5 2 2.5 3 3.5 4 4.5 0
1 1.5 2 2.5 3 3.5 4 4.5
Frequency (MHz)
Frequency (MHz)

Figure 3. Normalized backscatter of the fundamental (first column), and of the second harmonic
(second column) versus transmit frequency. The data were captured using 2-, 3- and 4-period
pulses using both probes. Both FNB and SHNB provided a resonant peak around 1.6 MHz,
which also increased with acoustic pressure. Data from the two probes overlap, suggestive of a
successful normalization approach. Acquisition of harmonic data below 1.5 MHz was not possible
for acoustic pressures higher than 0.6 MPa.
166 V Sboros et al

1200
Fundamental 14000
Second Harmonic
1000 12000
Normalized backscatter

Normalized backscatter
10000
800

8000 0.2-0.4 MPa

600 0.2-0.4 MPa
6000
400
4000

200
2000

0 0
1 1.5 2 2.5 3 3.5 4 4.5 1 1.5 2 2.5 3 3.5 4 4.5
Frequency (MHz) Frequency (MHz)

1200 14000

12000
1000
Normalized backscatter

Normalized backscatter
10000
800

8000
0.4-0.6 MPa
0.4-0.6 MPa
600
6000

400
4000

200
2000

0 0
1 1.5 2 2.5 3 3.5 4 4.5 1 1.5 2 2.5 3 3.5 4 4.5
Frequency (MHz) Frequency (MHz)

1200 14000

12000
1000
Normalized backscatter

Normalized backscatter

10000
800

8000 0.6-0.9 MPa

0.6-0.9 MPa
600
6000

400
4000

200
2000

0 0
1 1.5 2 2.5 3 3.5 4 4.5 1 1.5 2 2.5 3 3.5 4 4.5
Frequency (MHz) Frequency (MHz)

1200 14000

12000
1000
Normalized backscatter

Normalized backscatter

10000
800
0.9-1.2 MPa
0.9-1.2 MPa 8000
600
6000

400
4000

200
2000

0 0
1 1.5 2 2.5 3 3.5 4 4.5 1 1.5 2 2.5 3 3.5 4 4.5
Frequency (MHz) Frequency (MHz)

Figure 4. Normalized backscatter of the fundamental (first column), and of the second harmonic
(second column) versus transmit frequency. The data were captured using 6-, 8- and 10-period
pulses using both probes. A resonance found at 1.6 MHz was maximized for the second harmonic
between 0.4 and 0.6 MPa. Above these acoustic pressures the intensity of SHNB drops. The
FNB, at resonance, increases with acoustic pressure. A secondary resonant peak is also detectable
from the SHNB below 0.6 MPa. Data from the two probes overlap, suggestive of a successful
normalization approach. Acquisition of harmonic data below 1.5 MHz was not possible for acoustic
pressures higher than 0.6 MPa.
In vitro contrast agents 167

800
fundamental
(a)
1.7<MHz<2.0

Normalized backscatter
600 2.0<MHz<2.3

1.4<MHz<1.7
400
2.3<MHz<2.6

200
1.1<MHz<1.4 2.6<MHz<2.9

0
0 0.4 0.8 1.2 1.6
Peak negative pressure (MPa)

8000
2nd harmonic
(b)
Normalized backscatter

6000
1.7<MHz<2.0

4000 1.4<MHz<1.7
2.0<MHz<2.3

2000
1.1<MHz<1.4 2.3<MHz<2.6

0
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
Peak negative pressure (MPa)

Figure 5. Normalized backscatter (a) for the fundamental, and (b) for the second harmonic, plotted
versus peak negative pressure and grouped in frequency (pulse period of 6 to 10 periods). The figure
presents fitted trendlines to the data. The fundamental normalized backscatter increased linearly
with increasing peak negative pressure, and the gradient was maximized at the peak frequency. The
second harmonic normalized backscatter did not follow similar behaviour. There was a distinct
peak at around 0.5 MPa, also associated with the peak frequency.

component from its neighbours. As can be seen in figure 1, even at 8-period pulse, there is
some overlap between the second harmonic and the fundamental. However, pulses that were
longer than 6 periods were found to provide significant separation between fundamental and
second harmonic components.
The data for figure 4 are from the three highest pulse periods used in this study (6, 8
and 10 periods). The SHNB is 2.2–17.1 dB higher than the FNB (average 9.3 dB). The FNB
graphs display similarities with figure 3. There is an amplification of the resonant behaviour
with the increase of acoustic pressure. The resonant frequency domain was also the same.
The SHNB is still at much higher levels compared to the FNB, but its behaviour is different
here. There is a pronounced resonance at the lowest acoustic pressures (0.2–0.4 MPa), which
was further enhanced and peaked between 0.4 and 0.6 MPa. The resonance at this acoustic
pressure range provides maximum difference between the SHNB and FNB levels. Above
0.6 MPa there is a decline of this behaviour. Furthermore, at low acoustic pressures the SHNB
provided a possible secondary resonance around 2.8 MHz, which peaks from 0.4 to 0.6 MPa
and is not detectable above 0.6 MPa.
Figure 5 shows the data displayed in figure 4 plotted versus incident acoustic pressure
grouped in transmit frequency, with (a) being the FNB, and (b) the SHNB. The FNB followed
168 V Sboros et al

Resonance 250 Off-resonance

700 > 0.6 MPa
> 0.6 MPa

Average Normalized
Average Normalized

< 0.4 MPa < 0.4 MPa

600 200

Backscatter
Backscatter

500
150
400
300 100
200
50
100
fundamental fundamental
0 0
0 2 4 6 8 10 0 2 4 6 8 10
Pulse period (periods) Pulse period (periods)

2400
10000

> 0.6 MPa 2000 > 0.6 MPa

Average Normalized
8000
Average Normalized

< 0.4 MPa < 0.4 MPa

1600

Backscatter
Backscatter

6000
1200
4000
800
2000 400
second harmonic second harmonic
0 0
0 2 4 6 8 10 0 2 4 6 8 10
Pulse period (periods) Pulse period (periods)

Figure 6. Average fundamental (top two graphs) and second harmonic (bottom two graphs)
normalized backscatter plotted versus pulse period. The graphs on the left belong to resonance
data and those on the right to off-resonance data. The average data were grouped in low <0.4 MPa
(empty points) and high >0.6 MPa (filled points) peak negative pressure (PNP).

a positive linear relationship to acoustic pressure below 2.75 MHz. For frequencies above
2.75 MHz there was no correlation between FNB and acoustic pressure. The slope of the
linear fits was maximum at resonance (figure 3). The behaviour of the SHNB was different.
There was a definite maximum at acoustic pressures around 0.5 MPa, which was also more
pronounced at resonance. Above 0.5 MPa the SHNB followed a rather monotonic increase
with acoustic pressure.
This peak harmonic behaviour at 0.5 MPa suggested a physical threshold for the
microbubble behaviour. The behaviour seems to be generally different at low acoustic
pressures (below 0.4 MPa) compared to high acoustic pressure (above 0.6 MPa). Figure 6
displays the average FNB and SHNB, on and off resonance and over the acoustic pressure
range at each pulse period. This averaging aimed at illustrating the level of normalized
backscatter at different pulse periods. Lines were fitted to the data to make reading of figure 6
easier. At resonance the average FNB and SHNB display a similar pattern of behaviour. Above
0.6 MPa there is maximum behaviour for the short pulses, which declines for longer pulses.
This decline is steeper for the SHNB compared to the FNB. Below 0.4 MPa the FNB provided
a small peak for 3-period pulses, but its behaviour was generally smoother than that above
0.6 MPa. On the other hand, the average SHNB increased with pulse period to reach saturation
at 4 periods. The per cent standard deviation for the FNB and SHNB ranged from 25 to 58%
and 14 to 103%, respectively. Off resonance the respective per cent standard deviations were
26–63% and 19–91%, which is a very similar result and probably could be attributed to the
arbitrary choice of resonant and off-resonant ranges of frequencies at each pulse period. Off
resonance, above 0.6 MPa, up to 8-period pulses there was no decline of FNB or SHNB with
the increase of pulse period (figure 6), as was the case at resonance. The SHNB showed an
increase with pulse period and peak for 8-period pulses. Off resonance and below 0.4 MPa
the SHNB was nearly independent of pulse period, while the FNB peaked at 6 periods.
In vitro contrast agents 169

4. Discussion

4.1. The study of Definity

R

The SHNB results from the longer pulses, at 6- to 10-period pulses, demonstrated a resonant
behaviour that peaked around 0.5 MPa (figure 5). Visual inspection of the behaviour of
encapsulated bubbles, of similar structure to Definity
R
, demonstrated a nonlinear oscillation
at 0.4 MPa acoustic pressure (Dayton et al 1999). At such acoustic pressures the pulse drives
the oscillation of most of the encapsulated bubbles. According to visual evidence the shell
provides restriction in the oscillatory motion. Dayton et al (1999) recorded a higher contraction
than expansion of oscillating phospholipid encapsulated bubbles, which was suggestive of the
contribution of the shell to the nonlinear motion. The shell restricts the bubble motion twice
during one period of oscillation, on expansion and on contraction. This motion restriction
can be witnessed in the frequency domain at twice the driving frequency, hence the second
harmonic. In other words, motion asymmetry that is specific to the peak expansion and peak
compression of a bubble oscillation, at the time that the bubble is driven by an ultrasound
pulse, should provide a second harmonic signature. It seems therefore reasonable, when the
shell collapses at higher acoustic pressures, for such a movement restriction to be cancelled
before the bubble’s disappearance. This would be compatible with a destruction mechanism
occurring during a pulse for higher acoustic pressures above 0.6 MPa at resonance (figure 4)
where no further increase of SHNB is taking place.
Short pulse driving provides peak resonance above 0.6 MPa for both FNB and SHNB
(figure 3). Figure 6 also shows that, at resonance and above 0.6 MPa, the levels of FNB and
SHNB are higher compared to those below 0.4 MPa. FNB and SHNB are pulse normalized
physical quantities, and as a consequence figure 6 can be interpreted as the average behaviour
of the fundamental and the harmonic responses across the duration of the incident wave. The
first two to three periods of microbubble response is the duration of transient response of the
microbubble (Parlitz et al 1990). During these periods the microbubble oscillator is adapting
to the driving pulse and after this initial adaptation period it is expected that the amplitude
of oscillation becomes maximum. If the microbubble structure can endure this maximum
then the microbubble is likely to repeat an oscillation. If not, then the microbubble collapses.
(By collapse the impossibility of returning to the original stable microbubble at rest is meant.
Of course if the driving amplitude is very large, microbubble collapse can occur before the
transient oscillation has settled.) The decline of FNB and particularly SHNB for longer pulses,
above 3 periods, also suggests the collapse of the resonating microbubbles. The high values of
SHNB at short pulses and its fast drop at larger pulses (faster than that observed for the FNB)
are compatible with the above suggestion that second harmonic behaviour might be due to
motion restriction. Furthermore, the growth of SHNB with acoustic pressure, at resonance and
for short pulses, (figure 3) should also occur in the initial periods of the longer pulses. However,
at longer pulses the SHNB is not further increased above 0.6 MPa (figures 4 and 5), which
means that harmonic generation after the third period of oscillation is probably negligible
and confirms that the microbubbles are destroyed. Microbubble fragmentation after the first
period of oscillation, at 1.5 MHz and 0.8 MPa, for the agent MP1950 (Mallinckrodt Inc.,
St Louis, MO, USA) was confirmed by streak images (Chomas et al 2001b) and in general
most fragmentation occurs after the initial transient microbubble oscillation. Fragmented
microbubbles subsequently undergo oscillations too (Chomas et al 2000, 2001b). These
oscillations will be registered as FNB measurements, but from the present study it is suggested
that they might not be registered as SHNB. Figure 5 shows that the FNB follows a monotonic
increase with acoustic pressure at resonance, while the SHNB drops after a peak value around
170 V Sboros et al

0.5 MPa. Figure 6 shows that above 0.6 MPa, at resonance, the average FNB drops after the
sixth period of oscillation, while the SHNB drops after the third period. As discussed above
reduced SHNB could be attached to free unencapsulated bubble oscillations, which is likely
to be the case for the fragmented microbubbles.
Below 0.4 MPa the FNB remains constant with pulse period, while the SHNB increases
up to a constant value above 4 periods of oscillation (figure 6). Both these results indicate
that the microbubble oscillation is driven by the incident wave at such acoustic pressures.
The initial transient response from the bubbles seems to be poor in nonlinear content, which
picks up as these transients settle. After the transients have settled the different spectral
components remain constant (figure 6). The spectrum of the simulated motion of a driven
(and undestroyed) microbubble, subjected to 0◦ phase pulses, provided a spectral average
frequency that increased with pulse period and also seems to saturate for values higher than 4
periods (Morgan et al 2000). The increase of second harmonic with pulse period, measured in
the present study, can provide the contribution for the increase of spectral average frequency
and therefore is compatible with the above model.
There are interesting similarities between resonance data below 0.4 MPa and those off
resonance above 0.6 MPa. The FNB does not correlate with pulse period and the SHNB
increases with pulse period up to 8-period pulses (figure 6). There is no decline of FNB
and SHNB here as was the case for high acoustic pressure at resonance. All this suggests
that off resonance the acoustic pressures that cause microbubble collapse are at higher levels
than 0.6 MPa.

4.2. Limitations
Nearly all the limitations encountered in this in vitro investigation are induced by the transducer
characteristics. Attenuation and multiple scatter were negligible over the whole range of
settings as discussed above. On the other hand, not all transmit frequencies provided the same
range of acoustic pressures. It was impossible to use acoustic pressures greater than 0.6 MPa
at frequencies that belong to the low sensitivity band of the transducers. Though this does not
impose a systematic error on the results, it creates a considerable imbalance on the range of
investigated settings, as can be seen at the high acoustic pressure graphs of figures 3 and 4,
where results for frequencies below 1.5 MHz do not exist.
The shape variability of the transmitted pulses is also closely linked to the spectral
behaviour of the transducers. Transmitted pulses are specified in terms of frequency, peak
negative acoustic pressure and number of periods. However, at the low ends of transducer
sensitivity the shape of the pulses is different from that encountered at peak sensitivity, and
the three parameters mentioned above (frequency, pulse period and peak acoustic pressure)
are not enough to describe adequately a transmitted pulse. Pulse shape changes are related
to the mechanics of the transducer construction, the materials used and the electronics that
support them. Experiments with real pulses however, will always face such limitations. The
overlap in the results from the two transducers (figure 3) strongly suggests that the problem
is insignificant at the frequency range employed (around 3 MHz). The assessment of the
behaviour of the contrast agent is more dubious below 1.35 MHz, where the pulses include
several harmonics.
Finally the variable noise levels at different settings, along with transducer sensitivity
affect the level of detectability of contrast microbubbles. It is difficult to assess accurately the
size range of detectable microbubbles at each setting in the present study. This will always
be a problem for ultrasonic transducers that have a limited bandwidth. However, the spectral
characteristics of Definity
R
, at the low sensitivity transmit frequencies (<1.8 MHz), that
In vitro contrast agents 171

were apparent at very low acoustic pressures, proved consistent with those at higher acoustic
pressures. Moreover, the second harmonic that was registered at twice the above frequencies
(i.e. more sensitive frequency band) showed identical spectral patterns, which suggests that
the variable detectability of microbubble echoes did not affect significantly the quality of data.

4.3. General critique

Despite the limitations, the system proved capable of accurately describing the behaviour of
a contrast agent and can be used to determine the regime of optimal settings for contrast
imaging when used in in vivo investigations. The investigation of Definity
R
using this system
provided a wide range of results. It gave an account for the combination of 26 different
transmit frequencies, seven acoustic pressures and six different pulse periods, overall 1092
settings. The range of frequencies, acoustic pressures and pulse period could be wider, but
overall an investigation with a high number of settings has not been previously published.
In particular, the resolution of the settings is unprecedented in the literature. Technological
advancement can widen the range of settings, but the present capability proved useful for the
characterization of Definity
R
in vitro. As a first step, resonances have been detected, the
range of acoustic pressures that give optimal fundamental and second harmonic signatures
has been specified, and the impact of the period of a pulse on the acoustic behaviour of the
microbubbles has been quantified and discussed. As discussed in the introduction, previous
in vitro experiments involved limited use of settings and in particular transmit frequency. The
experimental assessment of resonant frequency should support studies of optical observation
of the microbubble behaviour. Chomas et al (2001b) studied the microbubble destruction
mechanisms but the use of only eight different transmit frequencies is a major limitation
to the understanding that their observations can offer. As a result, the threshold frequency
of microbubble destruction was found to have a linear relationship with microbubble size,
which is not compatible with the enhanced destruction that might occur at resonance. The
experimental location of resonant frequency cannot be exchanged with theoretical calculations,
as there is no published theoretical model that accurately predicts microbubble behaviour. The
present system can supply data for model validation.
Finally, such a study can be directly implemented in vivo. The changes of different
physical environment such as viscosity, ambient pressure and temperature are expected to
account for different physical behaviour of the microbubbles. The in vivo resonant frequency
of Definity
R
might be shifted from that measured in the present study and the response to
acoustic pressure changes as well as pulse period might be different. The temperature change
in particular might physically influence the shell elasticity and friction more than viscosity
and ambient pressure, as lipids are expected to physically respond differently at different
temperatures. However, the present study should create the platform for such experiments and
provide guidance for optimal use of this or any other contrast agent.

5. Conclusion

A system for the study of the interaction of low density microbubble suspensions with an
ultrasonic beam was evaluated using Definity
R
. A detailed analysis of the behaviour of the
fundamental and second harmonic components demonstrated that in vitro experiments are
necessary in order to assess the optimal imaging settings for a contrast agent. Definity
R

displayed resonant behaviour around 1.6 MHz. A peak for the harmonic signals was also
found at acoustic pressures of around 0.5 MPa, and was suggested to be a threshold acoustic
pressure for the Definity

R
microbubble destruction at resonance. The increase of harmonic
172 V Sboros et al

response with the increase of pulse period at lower acoustic pressures provided further evidence
for microbubble driving at resonance. Despite some limitations, this system provides a novel
and efficient tool in the investigation of the physical properties of the contrast agents.

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