1. Introduction
Microbubbles are gas-filled bubbles encapsulated in a shell and are typically 1–10
m in diameter. Microbubbles are used as Ultrasound Contrast Agents (UCAs) because they oscillate in the presence of an acoustic field. This oscillation is due to the stiffness of the enclosed gas and the inertia of the liquid surrounding the microbubble [
1,
2]. Microbubbles act as resonant systems during insonification [
3]. The resonant frequency is dependent on the physical properties of the encapsulating shell, the gas core, and the surrounding medium [
2,
4,
5]. An oscillating microbubble dissipates energy through re-radiation, viscous dissipation, and thermal dissipation [
3,
6].
The behaviour of single microbubbles can be extended to populations provided that the concentration is sufficiently small [
1]. At low concentrations, multiple scattering and the interactions between oscillating microbubbles can be ignored. Thus, for a population, both scattering and attenuation can be measured acoustically. The total scattering measured is the summation of the scattering from individual microbubbles and the total attenuation measured is the summation of the scattering and absorption from individual microbubbles [
2]. Because the microbubble radius is inversely proportional to the resonant frequency [
2], the population size distribution has an impact on the acoustic response. By measuring the overall acoustic response of a microbubble population, as opposed to single microbubble measurements, the number of measurements required to obtain a result that is statistically significant is reduced [
7]. This can reduce the time needed to perform the characterisation experiment.
A common clinical application for UCAs is in contrast-enhanced echocardiography for Left Ventricular Opacification (LVO) [
8]. For this application, the contrast agent is required to generate strong backscattered signals in the presence of a pressure wave in the range of frequencies used for diagnostic cardiac ultrasound imaging. Typically, cardiac imaging has a frequency range of 2–8 MHz. The scattering power of oscillating microbubbles is the largest near to the resonant frequency. Another requirement for cardiac imaging is that microbubbles are sufficiently small (less than 8
m in diameter) to pass through the pulmonary microcirculation and stable enough to reach the left ventricle [
4]. The use of gases with a high molecular weight and the encapsulation of microbubbles in a shell have improved the stability of UCAs. These advances also cause an increase in both resonant frequency and viscosity, which increases the damping [
3]. Microbubbles less than 8
m in diameter have resonances within the range of frequencies of diagnostic ultrasound [
9].
The acoustic properties of UCAs can induce imaging artefacts unique to contrast-enhanced ultrasound imaging. In LVO, the concentration of microbubbles administered must be kept sufficiently low to limit the shadowing artefacts that can be introduced as a result of microbubble attenuation. At the Mechanical Index (MI) typically used in conventional echocardiography, destruction can occur by fragmentation of the microbubble or diffusion of the gas [
10]. For most contrast-enhanced imaging applications, the MI is typically below 0.5 or, to limit microbubble destruction further and alleviate the swirling artefact, an MI lower than 0.2 may be used [
8].
When driven at resonance, microbubble oscillation can be highly non-linear, resulting in the emission of sub-harmonics or harmonics of the incident frequency [
11]. This non-linear response can be exploited to improve the delineation of the endocardial border in contrast-enhanced echocardiography through contrast-specific imaging modalities. Second-harmonic imaging [
12] and sub-harmonic imaging [
13] are two such modalities that rely on separating the contrast signal from that of tissue based on frequency filtering. In pulse inversion imaging [
14], two pulses of opposite polarity are emitted and are subsequently compounded in the receive beamformer. Amplitude modulation is another multi-pulse technique, which results in a similar cancellation of the linear echoes [
15].
Contrast specific imaging modalities are exploited in perfusion imaging. A rapid succession of high pressure pulses can be transmitted to destroy the microbubbles in the region of interest. Destruction still occurs well within FDA guidelines that states for cardiac applications the MI should not exceed 1.9 [
16]. By suppressing the tissue response, quantitative assessment of reperfusion is possible. It may be possible to further improve quantitative perfusion imaging by using a monodisperse population of microbubbles, with higher sensitivity and more control over the acoustic response [
7]. To take full advantage of a monodisperse population for such applications, it is important to characterise the population acoustically and to tailor the insonification accordingly.
Stable cavitation and inertial cavitation can be exploited for therapeutics, either through attachment and delivery of a therapeutic agent [
17] or through sonoporation of cells and co-administration of a therapeutic agent [
18]. A number of mechanisms may contribute to sonoporation, including stable cavitation and micro-streaming [
19]. When streaming occurs close to a cell membrane, shear stress created by the moving fluid causes pores to appear. During more violent inertial cavitation, jet formations can occur, which can cause large pores to appear in the cell membrane [
19,
20]. The microbubbles experience their maximum radial response at resonance. For sonoporation, this translates into more forces exerted on the surface of the cell [
21]. Sonoporation efficiency may be increased by using a monodisperse population of microbubbles created through filtering of a polydisperse population [
7] or directly formed using microfluidic flow focusing techniques [
22]. Alternatively, the efficiency may be increased for a polydispersed population through a broadband excitation such as a linear-frequency-modulated pulse [
23].
For delivery of a therapeutic agent, the agent can be attached to the shell in liposomal form [
24] or can be contained in a thin oil layer in the microbubble [
21]. The therapeutic agent can be released through inertial cavitation or through controlled lipid shedding [
25]. Once again, exciting the microbubbles at the resonant frequency increases the efficiency of the payload delivery [
21]. The changes in the shell parameters of the liposome-loaded microbubbles have been shown to alter their acoustic characteristics through optical [
26] and acoustic measurements [
27]. The addition of an oil layer in the shell also leads to changes in the microbubbles’ acoustic characteristics [
28]. Because the efficiency of the drug delivery is dependent on the insonification parameters, it is important to characterise these modified microbubbles when trialing their use for therapeutics applications.
Examples of acoustic characterisation experiments that have been performed previously are included in
Table 1. Despite the clear benefit to both imaging and therapeutics applications, there is very little consistency in the experimental setup or protocol used to investigate the acoustic characteristics of microbubbles. Variations in the parameters of the transmitters and receivers, vessel geometry, and the interrogating acoustic wave can make comparison between different research groups challenging.
For attenuation measurements, the transmitter and receiver are usually placed in line with each other. A hydrophone is often used as the receiver to obtain the response from the sample over a broad range of frequencies [
27,
29]. An alternative approach uses the transmit transducer as the receiver and a strong reflector positioned behind the sample [
4]. Scattering can be measured by the transmit transducer [
30] or by another transducer, or hydrophone, placed in a position orthogonal to the transmit transducer [
27,
29]. In the case where the transmit transducer is used to measure backscatter, the limitations of the transducer bandwidth may prevent the measurement of sub-harmonics or second harmonics. Measurements are taken with deionised water to obtain a reference. Measurements are then taken with a known dilution of microbubbles. The experiment is usually performed with only one transmitter and one receiver, and the different acoustic measurements are normally taken separately [
27,
29].
Generally, a sample vessel with acoustically transparent windows is used to constrain the solution of microbubbles in the far-field of the transmitter and receiver. The vessel dimensions used vary considerably. In [
29], the volume of the vessel was 500 mL, and in [
27], the vessel had a total volume of 100 mL. The dimensions of the vessel used is a trade-off between limiting the size of the microbubble sample required and minimising the interactions between the chamber walls and the ultrasound beam. The sample volume is made larger than the receiver beam-width to ensure as many microbubbles are measured in a single insonification as possible. In procedures using a needle hydrophone, the beam-width is small and the sensitivity is low compared to a transducer. Only a small subset of the microbubbles in the sample vessel are in the path of the receiver and a large percentage of the sample goes unmeasured. A magnetic stirrer is used to keep the microbubbles uniformly distributed in the ultrasound beam during the experiment. The sample vessel must be refilled with a fresh sample regularly for new measurements and measurement repetitions. This can lead to the requirement of a large quantity of microbubbles to perform the complete acoustic characterisation.
The transmitter is typically excited with a short duration pulse [
29] to excite the broadband response of the transducer or a linear frequency-modulated pulse [
27] to obtain a broadband frequency response. By using a broadband transmission, the response of the microbubbles over a range of frequencies can be obtained in a short time. In other works, narrowband excitation is used [
7,
31]. Although some of this variation can be accounted for due to the different objectives of the research, it remains to be determined which approach is the most robust in characterising a microbubble population for direct comparison.
The large amount of variation in the experimental setup and protocol used to characterise microbubbles makes it difficult to compare results across multiple research groups. This variability also makes it difficult to attribute any changes in measured characteristics to changes made to the microbubble population alone. An open access design for the acoustic characterisation of populations of microbubbles is presented. This design consists of 3D-printed parts that can be reproduced with ease. The Microbubble Acoustic Characterisation Chamber (MACC) aims to facilitate the characterisation of established and novel microbubbles. The designs are freely available to download (
https://github.com/UARPGitHub/MACC) to enable a direct comparison across research groups. The latest information can be found in the corresponding knowledge base (
https://github.com/UARPGitHub/MACC/wiki). An experiment in which the scattering characteristics of SonoVue
®, a UCA with widely reported acoustic characteristics, was performed for the purposes of validating the presented design.
3. Results
For every acoustic scattering measurement presented in this section, a two-tailed t-test was performed between the average RMS amplitude component of the received data corresponding to a scattering measurement and a reference measurement in which no microbubbles were present. The null hypothesis that “the introduction of microbubbles into the acoustic path has no effect on the amplitude component at frequency ” was rejected at the 5% significance level for all of the amplitude components used to determine the scattering characteristics in the remainder of this section. This gives confidence that the MACC is suitable for performing microbubble scattering measurements.
Figure 6 shows the fundamental backscatter calculated over the frequency range of 1.6–6.4 MHz using Equation (
1). The mean backscatter values from
Figure 6a are included in Appendix
Table A1 and for
Figure 6b in Appendix
Table A2. From
Figure 6, it can be seen that the scattering from SonoVue
® is highest at the lowest frequencies investigated. This is in general agreement with previous studies [
31]. As is expected, because the measured scattering is the summation of the scattering from individual microbubbles, the lower dilution sample resulted in a lower scattering value. Both dilutions show the same general trend with frequency, and this gives confidence that the chamber is suitable for the measurement of dilutions of SonoVue
® in the range of clinical doses. The measurements from the various transducers also overlap and are in general agreement with regards to the overall trend. For the pairs of transducers that are used to investigate the same frequency range, the scattering measurements are in agreement. This is further validation that the measurements are reliable. There is some discrepancy between the scattering values at the frequencies that overlap between different transducer pairs. This is likely due to mismatches in the transducer sensitivity compounded by the limits of the usable bandwidth for a transducer. This highlights the importance of performing the experiment within reasonable frequency limits for a given transducer and in the robust calibration of transducer receive-sensitivity.
Figure 7 shows the average RMS pressure spectrum obtained using various transducers from the first acquisition and for a transmit frequency of 1.6 MHz transmitted using a 2.25 MHz transducer (T1 in
Figure 4). The spectrum measured using the inline 2.25 MHz transducer (T2 in
Figure 4) for the reference measurement is included in
Figure 7a.
Figure 7b shows the backscattered data received by the transmit transducer (T1 in
Figure 4) at the fundamental frequency and second harmonic. The second harmonic response is very low. This can be accounted for due to the limited bandwidth of the transducer.
Figure 7c shows the data received by a 3.5 MHz transducer (T3 in
Figure 4). In this case, the fundamental response is low, but the second harmonic is more pronounced.
Figure 7d shows the combination of the fundamental response recorded by the transmit transducer and the second harmonic recorded by the 3.5 MHz transducer. It highlights how the signal received on multiple transducers can be combined to obtain the frequency response over a broader range of frequencies than would be possible with just a single receive transducer. It should be noted that the fusion of data from different receivers relies on the ability to obtain an accurate RMS pressure value for the receiver amplitude.
Figure 8 shows the second harmonic scattering calculated over the frequency range of 3.2–6.4 MHz using various receive transducers and Equation (
1). The mean backscatter values from
Figure 8a are included in Appendix
Table A3 and for
Figure 8b in Appendix
Table A4. The scattering at the second harmonic shows the same trend as the scattering at the fundamental frequencies. The insonifications that provided the maximum scattering response at the fundamental frequency also provide the maximum at the second harmonic. Once again, the measurements from the various transducers overlap, and the lower dilution measurement resulted in lower scattering values.
4. Discussion
A statistically significant difference was found for all the reported acoustic measurements between the measured control sample, containing only deionised water, and the measured sample for both dilutions of contrast agent. Furthermore, the scattering characteristics measured are in agreement with previous studies [
31] for the ultrasound contrast agent SonoVue
®. By performing the measurements in both directions across the chamber, a great degree of confidence can be taken in the measured characteristics. For all the measurements taken, the lower dilution sample resulted in a lower scattering value. This validates the efficacy of the MACC for performing scattering measurements at both the fundamental frequency and the second harmonic.
The motivation behind the MACC is to provide a standardised piece of equipment for characterising microbubbles acoustically. In this work, narrowband waveforms were used to interrogate the sample at fixed frequency intervals. An alternative approach might have been to use broadband waveforms to obtain the response over a range of frequencies. The transmission parameters and digital signal processing steps required to obtain a robust acoustic characterisation methodology that can be used for differing research objectives and across research groups are yet to be determined. In this work, it was necessary to pre-distort the transmitted waveforms to obtain uniform pressures across a range of frequencies and transducers. It was also necessary to account for the sensitivity of the different transducers at different frequencies in receive. Without these steps, it would not have been possible to fuse the data from different transducers.
In providing a unified piece of equipment, it is possible for researchers in different locations to reproduce results. Researchers are able to share the parameters of the interrogating acoustic waves and flow rates used with the chamber alongside results. It is envisaged that, with a more standardised apparatus, a consistent protocol can be developed and direct comparison across research groups will become possible.
Due to the vast range of research objectives that may be encountered when characterising microbubbles, it is difficult to create a single piece of equipment to cover all possible scenarios. The MACC has been designed to allow a flexible configuration. The chamber, in its current form, allows the incorporation of absorbers and reflectors. These can be used at the expense of removing one or more of the transducers. Whilst the transducers chosen in this study offer the ability to investigate a broad range of frequencies around those typically required for microbubble characterisation, they are interchangeable, and transducers covering different frequency ranges may be used. For instance, if research objectives require the measurement of a lower fundamental frequency or sub-harmonic measurements, a pair of transducers with a central frequency of 1 MHz could be incorporated. Other research objectives may require attenuation measurements. Typically, attenuation measurements are made through a microbubble screen that is wider than the beam width. At the expense of performing the measurements in both directions across the chamber, the unfocused transducers on one side of the chamber could be replaced with focused transducers. By ensuring these focused transducers have a fixed focal depth coinciding with the sample stream, they could be used for the sole purpose of measuring attenuation.
The MACC has been released to allow other researchers the ability and freedom to modify and extend the design. Future modifications might include the incorporation of optics to provide optical characterisation capabilities. Another possible modification is the incorporation of an acoustic window housing to allow a phased array cardiac probe to be used to image the flowing microbubbles. Further modification and extensions to the MACC are welcomed.