Activation and 3D Imaging of Phase-change Nanodroplet Contrast Agents with a 2D Ultrasound Probe

Nanodroplets are on-demand ultrasound contrast agents. They can remain in circulation longer in vivo than mi-crobubbles and can be spatiotemporally and selectively activated to provide contrast when required. Perfluorocarbon nanodroplets have been used for conventional 2D ultrasound imaging and different types of nanodroplets have been studied and characterized by researchers regarding their suitability for imaging. However, the use of nanodroplets for 3D imaging and their activation using a 2D ultrasound probe has not been reported. In this study, we investigate the nanodroplet activation using a 2D ultrasound imaging probe within clinical safety limits.


I. INTRODUCTION
Super-resolution ultrasound (SR-US) imaging techniques are capable of breaking the diffraction limit and imaging microvasculature. Localization-based SR-US imaging relies on detection of ultrasound contrast agents' positions with high precision. Locations of spatially isolated point sources from multiple frames are combined to generate a super-resolved image. Most of the existing super-resolution methods use microbubbles [1]- [17]. Microbubbles follow the slow physiological flow inside microvessels and therefore super-resolution requires long data acquisition time, which together with the motion during data acquisition make it challenging for clinical use [10], [18], [19]. The choice of microbubble concentration can also affect the quality of the SR-US image. Multiple overlapping microbubble echoes within the resolution limit can cause position errors, therefore isolated microbubble echoes should be localized with the best possible method [11], [20]- [23].
Nanodroplets, also called phase-change contrast agents, have been widely investigated for ultrasound imaging [24]- [26]. They offer potential solutions to aforementioned issues of SR-US imaging with microbubbles. Nanodroplets can be selectively activated on demand to provide an ultrasound contrast signal and can be imaged at high frame rates. Low-boilingpoint nanodroplets have been demonstrated for reducing the SR-US acquisition time as they can be activated, destroyed and imaged on a sub-second time scale [27]. Thanks to these advantages, the use of nanodroplets as an echogenic point source for super-resolution imaging is increasing [28]- [30].
Researchers demonstrated the use of nanodroplets for 2D ultrasound imaging, including 2D SR-US imaging. However, the use of nanodroplets for 3D ultrasound imaging has not been demonstrated yet. In this study, we investigate the activation process of nanodroplets using a 2D ultrasound imaging probe within clinical safety limits. The main purpose of this study is to investigate the data acquisition time for nanodropletbased 3D SR-US imaging and minimize the acquisition time by tuning the activation parameters. A range of activation waveforms with varying acoustic pressures are employed to investigate the activation efficiency of nanodroplets. Feasibility of activation and 3D ultrasound imaging of nanodroplets is demonstrated using high frame rate imaging.

A. Nanodroplet Preparation
Nanodroplets were fabricated with a Definity-like lipid shell and decafluorobutane (DFB) core with as previously described in [24], [26]. Briefly, a lipid colloid was generated by dissolving a 9:1 molar ratio of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG-2000) into a solution of phosphate-buffered saline (PBS), propylene glycol, and glycerol (16:3:1, V: V) to achieve a total lipid concentration of 1 mg/mL. All the lipids described above were purchased from Avanti Polar Lipids, Inc., USA. A volume of 1 mL of lipid solution was added to a 2 mL glass vial. The headspace of the vial was filled with decafluorobutane gas (Fluoromed, USA) via an inlet needle along with a vent needle. Mechanical agitation was applied using a bubble shaker (Vialmix, USA) to produce the stable and microscale bubbles composed of a gaseous decafluorobutane core encapsulated by a lipid shell.
In order to condense microbubbles into nanodroplets, the headspace of the vial was pressurized according to the previously described methods. The vial of microbubbles was immersed in an ice-salt bath (−5 to −8 • C) followed by pressurization with ambient air into the vial septum while the vial contents remained in the ice-salt bath.
Size and concentration of the generated nanodroplets were measured by nanoparticle tracking analysis (NTA) using a NanoSight NS300 (Malvern Instruments Ltd, UK). Nanodroplets has a mean diameter of 119 ± 7 nm and a mean concentration of 3.5 × 10 10 nanodroplets/mL. For the ultrasound experiments, nanodroplets were diluted at a volumevolume ratio of 1:30000 (Native nanodroplet solution: Water) in a beaker at 37 • C.

B. Experimental Setup
A 512-element 2D sparse array was used for nanodroplet activation and imaging with a center frequency of 3.7 MHz and a bandwidth of 60%. Two synchronized ULA-OP 256 systems (MSD Lab, University of Florence, Italy) were used to activate nanodroplets and acquire data using the 2D ultrasound probe [31], [32].
Nanodroplet activation was performed using focused beams with 2, 4, 6 and 10 cycle 4 MHz sinusoidal waveforms focused at a depth of 30 mm, as illustrated in Figure 1. Activated nanodroplets were imaged in 3D at a center frequency of 4 MHz using plane waves. After every activation pulse, 100 imaging frames were acquired at an MI of 0.1 and frame rate of 1000 Hz.
Experiments were repeated 5 times for a range of activation pulses with varying amplitudes between 40%-100%, where 100% corresponds to the maximum excitation voltage of 40 Volts, with the MI values given in Table I. Activated nanodroplets, which are microbubbles, were imaged using the 2D sparse array probe. Figure 2 shows the 3D images (-20 dB isosurfaces) of the activated nanodroplets for an MI range of 0.75 (40% amplitude) to 1.65 (100% amplitude) using a 2 cycle wave. The same experiments were also performed for 4, 6 and 8 cycle sinusoidal waves (not shown here). For all activation pulses, the experiment performed at a higher pressure resulted in the activation of more nanodroplets. The results show that increasing the activation MI increased the ultrasound contrast generated by nanodroplets in and around the focal region.
The contrast enhancement was measured around the activation region within a volume of 10×10×10 mm 3 . The measured average intensity values in this region for different activation amplitudes and waveforms are plotted in Figure 3. All activations generated higher intensity values than the reference experiments shown in Figure 3 (left) without any nanodroplets. The activation amplitude, which changed between an MI of 0.75 and 1.9 as given in Table I, had a positive correlation with image intensity. Increasing the number of cycles in the activation waveform also increased the image intensity.
This study combined the recent advances in high-frame-rate 3D ultrasound imaging and ultrasound contrast agents with a motivation of reducing the imaging acquisition duration for 3D super-resolution ultrasound imaging. The achieved results in this study agree with the previous research performed using 2D ultrasound imaging. Changing the number of cycles and acoustic pressure of the activation pulses affects the image contrast, which is proportional to nanodroplet activation.

IV. CONCLUSION
This study presents an in vitro demonstration of nanodroplet activation using 3D ultrasound imaging. Results show that increasing the number of transmit cycles and acoustic pressure can increase the number of activations, which was measured as a change in the image contrast over the imaging volume. The activation of nanodroplets can be controlled by tuning the activation pulses for specific applications. Controlling the nanodroplet activation events may be important in droplet-based super-resolution imaging for minimizing the data acquisition time.
The advantage of using 3D imaging and activation is that the experiments can be performed without confining the nanodroplets. By measuring all activation events in the imaging Fig. 2. 3D image of the activated nanodroplets using a 2 cycle sinusoidal excitation waveform focused at 30 mm (shown in Figure 1) with a varying MI between 0.75 and 1.65.  Table I for the corresponding MI values. volume, signals are less likely to be lost due to out of plane motion in 2D imaging.