Low-Pressure Burst-Mode Focused Ultrasound Wave Reconstruction and Mapping for Blood-Brain Barrier Opening: A Preclinical Examination

Burst-mode focused ultrasound (FUS) exposure has been shown to induce transient blood-brain barrier (BBB) opening for potential CNS drug delivery. FUS-BBB opening requires imaging guidance during the intervention, yet current imaging technology only enables postoperative outcome confirmation. In this study, we propose an approach to visualize short-burst low-pressure focal beam distribution that allows to be applied in FUS-BBB opening intervention on small animals. A backscattered acoustic-wave reconstruction method based on synchronization among focused ultrasound emission, diagnostic ultrasound receiving and passively beamformed processing were developed. We observed that focal beam could be successfully visualized for in vitro FUS exposure with 0.5–2 MHz without involvement of microbubbles. The detectable level of FUS exposure was 0.467 MPa in pressure and 0.05 ms in burst length. The signal intensity (SI) of the reconstructions was linearly correlated with the FUS exposure level both in-vitro (r2 = 0.9878) and in-vivo (r2 = 0.9943), and SI level of the reconstructed focal beam also correlated with the success and level of BBB-opening. The proposed approach provides a feasible way to perform real-time and closed-loop control of FUS-based brain drug delivery.


Results
Backscattered wave reconstruction allows short-burst low-pressure focused ultrasound visualization. First, we investigated the effect of exposure level by testing the acoustic wave reconstruction at different exposure levels with a default tested transducer frequency of 1.5 MHz (theoretical deviations of the proposed backscattered acoustic-wave reconstruction are shown in the Supplementary method). To show the proposed approach was suitable for capturing short-period ultrasound exposure, we compared the acoustic wave reconstructed maps of ultrasound exposures with various burst length. Figure 1 shows an experiment using a 1.5-MHz focused ultrasound transducer to deliver excitation with the pressure ranging from 0.391 to 1.194 MPa and a burst length ranging from 0.01-ms to 10-ms. On one hand, the wave front can be identified when the pressure value exceeded 0.467 MPa, but not when the pressure was lower than 0.391 MPa. Further, the signal level of the reconstructed maps seemed to be increased with increasing pressure levels. Besides, this experiment also showed that the reconstructed maps from 0.05-ms to 10-ms excitation can identify the wave front of the propagated ultrasonic waves, but the wave fronts cannot be identified for excitation lengths equal to or less than 0.01-ms. In summary, the map quality was not obviously effected by the exposure burst length, but was dominated by exposure level. For comparison, we also showed that it was failed to reconstruct the focal beam patterns with the asynchronous data (with exposure condition of 1.194 MPa and 10 ms of burst length), indicating the necessity of synchronization between FUS exposure and backscattered acoustic wave receiving for short-burst ultrasound beam visualization ( Fig. 1; bottom-right subplot).
The relationships between the peak signal intensity (SI) of the estimated beam patterns with various exposure burst lengths as well as exposure pressure levels were also investigated (Fig. 2). We defined SI as the average of the square root of the data acquired from ROI (size: 1.5 mm × 1.5 mm) covering beam pattern. Beam patterns were detectable for all tested pressures under the burst length reaching 0.05 ms. As exposure level increased to greater than 0.467 MPa, the peak SI showed a linear correlation with the increased exposure level, but was nearly independent to burst lengths (The p values among 0.05-to 10-ms exposures were all > 0.05). When excluding those failed low-exposure cases, there was a high linear regression (y = 138.09x + 8.50, R 2 = 0.9878), indicating that the reconstruction map intensity was highly correlated with exposure level. Figure 3 shows the corresponding signal-to-noise ratio (SNR) of acoustic wave reconstruction maps. SNR level was 16 ± 4.75 dB at 0.467-MPa exposure (for all detectable burst length), and increased to 23.48 ± 7.49 dB at 0.705-MPa exposure (for all detectable burst length) and then remained plateau as the pressure further increased. Statistical analysis showed no significant difference in SNR change among various detectable burst-length groups, indicating that SNR level was nearly independent to the burst length when exposure pressure reaching 0.705 MPa. SNR seemed to increase as the exposure level increased, but not as the exposure burst length. Together with Figs 2 and 3 illustrated that both the reconstructed acoustic wave level and the acoustic wave reconstruction map quality depend heavily on the ultrasound exposure level, implying that the peak SI level of the reconstructed beam patterns accurately reflects the exposure pressure in in vitro phantoms. Figure 4 shows representative examples of using the proposed approach to reconstruct the focal beam patterns from a 10-ms exposure. We demonstrated that the focal beam pattern delivered from a 0.55 to 2 MHz FUS transducer can be successfully reconstructed. These results showed that the proposed algorithm is capable of visualizing the acoustic maps with single short-burst FUS exposure. It was noted that the algorithm successfully reconstruct the acoustic emission maps although the exposure signals (locates at 0.55-2 MHz) did not well match the receiving probe been employed (i.e., 5-10 MHz).  Short-burst FUS wave can be captured and reconstructed using single-frame backscattered wave reconstruction. The above results (Figs 2 and 4) demonstrated that the proposed acoustic wave construction algorithm can successfully provide FUS beam patterns with low-pressure short-burst exposure. Next, we investigated the effect of construction frame-averaging. Figure 5(A) provides typical examples of a reconstructed acoustic wave map conducted with different frame-averaging numbers (1, 5, 10 and 20 captures of single 10-ms bursts of FUS excitation in (A)-(D), respectively). The reconstructed beam patterns could be identified under different amounts of frame-averaging; yet, the wave front could be clearly identified in the low frame-averaging setting, but the time errors at each synchronization time point began to accumulate and wave front synchronization between frames gradually lost alignment and moving averaging effect was introduced. The SNR corresponding to the number of averaged frames is shown in Fig. 5(B). The acoustic wave maps showed a 5-dB SNR improvement with the number of averaged frames reached 20, but the SNR level were saturated or even decreased when average number of frames was increased further. The SNR increase due to frame averaging did not present a monotonic increase as the frame number increased, implying that even a single map reconstruction still provides good SNR, and maximally a < 5-dB SNR loss when compared with the optimized frame-averaging case. The above supported the capability of capturing short-burst FUS waves via using single-frame capture from the proposed algorithm and system setup.
Preclinical in-vivo experiments support the feasibility to guide ultrasound-induced blood-brain barrier opening. Next, we aimed to employ the proposed acoustic wave reconstruction algorithm to identify focal beam patterns and to test the potential use in guiding the FUS-BBB opening procedure on small animals. Figure S3 illustrates the concept to co-localize ultrasound B-mode imaging, backscattered acoustic emission reconstruction mapping and MRI together to perform FUS-BBB opening. While the a-prior obtained MRI  provides a high-resolution anatomical reference, ultrasound imaging provides a high temporal resolution anatomical position orientation to allow coordination of the reconstructed focal beam pattern distribution. Figure 6 describes the in vivo treatment showing the use of intermediate FUS exposure level to perform FUS-BBB opening (frequency = 1.5 MHz, acoustic power = 4.54 W, acoustic pressure = 0.467 MPa after skull penetration; rat skull causes about 10% of pressure loss). The focal beam maps can be easily generated and monitored, and with the co-localization of diagnostic ultrasound and MRI, the focal beam deposition can be clearly identified (peak SI = 168.41). The BBB-opening was successfully induced (confirmed in brain sections), and  In comparison with the first example in Fig. 6, in a second round of experiments ( Fig. 7), we employed an identical exposure level (i.e., 0.467 MPa) with the previous case, but intentionally introduce imperfect wave coupling by not perfectly conducting fur shaving of animal scalp. In this case, the BBB-opening was not successfully induced as brain sections showed no EB dye leakage or staining (Fig. 7D), and the focal beam cannot be reconstructed by co-localizing acoustic wave reconstruction maps with MRI and B-mode ultrasound imaging ( Fig. 7A,B). The failure of treatment was successfully reflected by the failure of focal beam reconstruction obtained during the exposure (peak SI = 61.16).
In the third animal treatment example (Fig. 8), the FUS exposure level was increased for comparison with former in vivo experiments (frequency = 1.5 MHz, acoustic power = 9.12 W, acoustic pressure = 0.705 MPa). A reconstructed focal beam pattern with an increased SI signal (peak SI = 271.14) obtained during exposure was observed. The increase of the peak SI level was observed to be ~160% higher than the first treatment case (compare Fig. 8A with Fig. 6A), which corresponds well with a MI increase (~150% higher than the first treatment case), implying that SI change in the reconstruction maps can well reflect the exposure level in the animal brain and outcome of the treatment. Histological examination also confirmed the successful treatment, which was more profound than that with the 0.467-MPa exposure (compare Fig. 8D with Fig. 6D). In 0.705-MPa exposure (group D), erythrocyte extravasations was also observed accompanying with BBB opening, while the extravasations was not observed in the 0.467-MPa exposure (Group C; the corresponding HE stains are shown in Fig. S4). The BBB-opened regions matched with the co-localized MRI/ultrasound/beam mapping imaging and the peak SI showed a good correspondence to the exposure energy level (compare Fig. 8B,D). Figure 9 summarized the dependence of the exposure level with the peak SI level during exposure. The experiments were divided into four groups based on the FUS-BBB sonication levels and FUS-BBB opening outcome: low-level exposure (Group A; pressure = 0.11 ± 0.01 MPa, corresponding SI = 49.69 ± 1.16), intermediate-level exposure with strong scalp interference (Group B; pressure = 0.44 ± 0.01 MPa, corresponding SI = 69.53 ± 11.99), intermediate-level exposure (Group C; pressure = 0.42 ± 0.01 MPa, corresponding SI = 160.20 ± 16.14) and high-level exposure (Group D; pressure = 0.703 ± 0.004 MPa, corresponding SI = 279.42 ± 15.59). Besides of group B, the acoustic wave level of groups A, C, and D was observed to be linearly correlated with the acoustic exposure level (y = 386.59x + 2.46, r 2 = 0.9943) and with signal intensity all statistically distinguishable among groups (all p < 0.0001), implying that acoustic wave SI level can effectively serve as an indicator to estimate the exposure level deposited into the brain. When comparing the group C with group B (identical exposure level but C/B are FUS energy with perfectly/poorly penetrated), the acoustic wave SI level provide high distinguished detectability (p < 0.0001) to identify the success/failure focal beam energy penetration. This implies that the acoustic wave reconstruction map provide effective index to well consider focal beam penetration, diffraction, and attenuation effects during the exposure. Putting these in vivo experimental tests together, it may be feasible to use backscattered acoustic wave level to predict the exact ultrasound deposition in rat brains, therefore may provide a strategy to conduct feedback control for FUS exposure fine adjustment and may have potential to be applied for guidance during FUS-BBB opening on rat brain.

Discussion
In this study, we investigated a synchronized backscattered acoustic-wave reconstruction approach in order to visualize short-burst low-pressure focused ultrasound beams. By implementing this approach in a commercialized diagnostic ultrasound system, the focal beam patterns can be successfully visualized with a wide FUS exposure frequency range that typically been employed for BBB-opening application (0.5-2 MHz). To our knowledge, the proposed scheme is the first reconstruction method that enables focal beam pattern visualization with a burst length down to 0.01 ms and exposure level down to 0.39 MPa (equivalents to MI of 0.3), with the imaging process without the involvement of microbubbles or acoustic cavitations (it should be noted that current threshold to open the BBB in microbubble-presented FUS exposure is about 1-10 ms of burst length with MI level reaching 0.46 30 ). We also proposed to co-localize the focal beam visualization with diagnostic ultrasound imaging as well  as pre-acquired MRI, so that the focal beam can be successfully guided to a specific brain target and perform precise FUS exposure control during the intervention. The proposed approach will be valuable when attempting to perform FUS beam pattern visualization and monitoring, and can be beneficiary when using the proposed approach for FUS exposure guidance for future CNS drug delivery application.
Previously, intense (typically 5-10 MPa in pressure in CW excitation) and continuous-wave (CW) FUS energy exposure were typically applied for thermal ablation purpose to induce sufficient energy/viscous heating accumulation (acoustic cavitation is typically involved during this process). It was shown that the reconstruction of backscattered acoustic waves enabled the identification of CW-mode FUS exposure, but the approach still required bubble cloud generation as a strong backscattered source 13,31 . Since a bubble cloud can only be generated when acoustic cavitation occurs, intense pressure from the therapeutic ultrasound is required. More recently, a number of reports have introduced microbubbles as an effective cavitation nuclei substitute, and therefore the ultrasound exposure level can be significantly reduced to a sub-MPa level similar to reported level reported from this study 13,28,31 . It may also possible to employ the non-synchronized reconstructed strategy to perform beam pattern visualization and then concurrently to implement real-time BBB-opening control 28 . However, it should be noted that, with the administration of microbubbles, FUS exposure under such exposure level concurrently induces capillary effects at the targeted area. The synchronization-based reconstruction strategy therefore is advantageous in providing extra flexibility to allow focal beam pattern visualization for pre-treatment evaluation and without inducing CNS capillary effect (since no microbubbles are presented).
For traditional beamformed engines designed for diagnostic ultrasound imaging, B-mode imaging is typically reconstructed based on the collection via a series of A-line acquisitions (64 or 128), thus requires transmission and reception processing times to intrinsically hamper the implementation of short-burst FUS exposure detection (for example, frame rate of 50-100 Hz in B-mode imaging equates to the required time slot of 10-20 ms). In this study, a diagnostic ultrasound engine with beamforming was designed and implemented in a broad-beam "zone"-like plane-wave approach that can complete the single B-mode frame in an extremely short period. These zones are much broader than conventional line-per-line acquisitions and this method typically increases the frame rate by more than 100-fold (completes a frame in tens of microseconds, and the B-mode frame rate is up to 1 kHz) 32 , which makes it possible to track transient activities of short-burst ultrasonic beam propagations. Moreover, synchronization between FUS and the plane wave-based beamforming allows the capturing of backscattered emission events from short-burst FUS exposures. Another advantage is, traditional asynchronization approach can detect cavitation event only when FR reaches 400 frames/s 31 and in our synchronization method, beam patterns can be reconstructed down to a very low data-capture dynamics (2 frames/s). This not only raises data acquisition efficiency but also reduces workload of the machine memory. Bridging the concepts of ultrafast plane wave-imaging and synchronized acquisition, we anticipated that only short-burst low-pressure FUS exposure can be achieved under this system structure.
Our current approach can obtain FUS beam visualization/monitoring with a single short-burst FUS exposure (currently, the reconstruction can be obtained with 0.01 ms in a 1.5 MHz FUS exposure, which equates to a 10-cycle excitation). It is highly possible that the burst length can be further reduced and the SNR can been further improved. We showed that higher MI level leads to higher SNR, and introducing frame-averaging may further increase SNR (it is estimated that SNR increase was approximately∝ (N − 20) 2 , where N = frame number; in dB). Yet, there are still other considerations in addition to improve SNR; for example, the acoustic pressure should not be high to induce brain tissue damage (therefore limits the exposure level increase). Second, a lower number of averaged frames should be used to guarantee sufficient real-time capability for beam pattern visualization and monitoring (therefore limits the frame-averaging number).
For in vivo animal FUS-BBB opening, the energy level must be sufficiently high to achieve the therapeutic effect. The level of ultrasonic energy deposition dominates the success of the therapeutic effect induction. For example, for ultrasound-induced blood-brain barrier opening, the ultrasound exposure threshold is 0.46, whereas a MI exceeding 0.8 creates safety concerns because RBC extravasations could be occurring during intervention. Thus, exposure calibration including correct position and exact energy are critical because the energy required for therapy comes with a high risk and may induce damage to the brain tissue. We showed that the reconstruction emission SI level well reflect the treatment outcomes (see Fig. 9). Increased exposure level can be well reflected in the SI level increase (a 150% exposure MI increase reflected a 160% SI increase of acoustic wave reconstruction map), and can well reflect the focal beam distortion during exposure (SI level can well reflect true energy deposition in the brain even when identical exposure level was delivered). It implies the potential of this approach in serving as valid imaging feedback for monitoring the FUS-BBB opening procedure on small animals.
In transcranial FUS-BBB opening, the skull causes ultrasound propagation energy attenuation, wave diffraction, and focal beam distortion 33 . Since the skull structure is inhomogeneous, it can be challenging to predict the FUS wave penetration. Skull-insertion effects have also been shown to degrade the reliability of transcranial passive mapping 13,28 , and create detection limits associated with skull thickness 34 and density 35 . All of these effects depend on the FUS exposure frequency 34,35 , and should be taken into account along with nonlinearity effects 36 and incident angle effects 37 . We also previously determined that the ultrasound exposure when transmitting through rat, swine, and human skull can have 10%, 30%, and 60% decay, respectively 38 . It might be a challenge to successfully reconstruct the backscattered acoustic wave when penetrating the human skull, particularly for the frequency range currently applied for clinical diagnostic ultrasound. A possible solution is to reduce the transmission/reception ultrasound frequency to be close or identical to the frequency applied in therapeutic ultrasound 28 , but may with the price of spatial resolution reduction.
Due to data-accessing limitation of the employed diagnostic ultrasound system, we only implemented semi real-time beam pattern visualization and monitoring with the rate of 5s/frame, and is insufficient to realize the real-time ultrasound exposure level regulation at current stage. Yet, the proposed method provide possibility to be implemented to serve as a feedback for ultrasound exposure level control during FUS intervention 39 . Future Scientific RepoRts | 6:27939 | DOI: 10.1038/srep27939 directions toward real-time implementation may include improvement of data-accessing capability of channel radio-frequency data of the imaging engine, parallel-processing and algorithm implementation in graphic processing unit (GPU), or future simplification of the implemented algorithm.

Conclusions
In summary, we describe a synchronized backscattered ultrasound acoustic-wave reconstruction approach to visualize short-burst low-pressure FUS beam patterns in this study. We demonstrate the feasibility of using this approach to guide FUS-BBB opening via small animal experiments, and we have shown that the reconstruction of short-burst low-exposure-level ultrasonic beam is possible without the involvement of acoustic cavitation or microbubbles. Our results suggest that the proposed approach provides potential in guiding FUS brain drug delivery.

Materials and Methods
System implementation and experimental setup. The experiments were performed in an acrylic tank (30 cm L × 20 cm W × 20 cm H) filled with degassed water. In order to determine the general applicability of the proposed approach, we tested four different FUS frequencies and configurations: (1, 2) a dual-confocal 0.55/1. A commercialized diagnostic ultrasound imaging platform (Z.one, ZONARE Medical Systems, Inc., Mountain View, CA) was employed in this study. In order to synchronously perform the experiments between FUS exposure and diagnostic ultrasound imaging frame, the ultrasound system was set up to generate the trigger output for system synchronization, with the use of a delay generator (MODEL DG535, Digital Delay/Pulse Generator, Stanford Research Systems). The delay generator was used to externally trigger the signal generator (Agilent 33220A, USA) and then feed into a power amplifier (Model 100A250A, Amplifier Research, USA) for FUS energy exposure output. A power meter (Model 4421, BIRD, USA) was connected to monitor the FUS exposure power. During ultrasound sonication, a IQscan research package with a L10-5 linear array transducer (Bandwidth: 10-5 MHz) was used to receive IQ data signals for backscattered acoustic wave reconstruction, while the PC port was employed to send commands to the imaging system through RS-232 communications and to collect and store data for offline processing.
To acquire passive signals, diagnostic ultrasound platform was operated in receive-only mode (with the transmitter turned off), and synchronization between the receiver and the ultrasonic firing was achieved using a frame-trigger signal tapped from the ultrasound probe connector. A command script was created on a PC to automatically set up the desired system settings, and to acquire a number of frames of passive channel IQ data using a 64-element aperture. The data capture cycle was first conducted using the left-half aperture, and then repeated using the right-half aperture. The left and right half-aperture signal I/Q frames were combined to determine the effective 128-element aperture in the off-line process. Due to data-accessing limitation of the employed diagnostic ultrasound system, the time required time to reconstruct a single frame (including the data transmission and processing) was about 5 s (see the Supplementary movies). Figure S2A describes the system setup for the in vitro experiments, which can be divided into three parts: synchronization, FUS exposure and diagnostic ultrasound imaging. The homogeneous graphite phantoms used in the study were made of degassed water, agarose and graphite powder in certain proportion, with a size of 50 × 50 × 70 mm 3 , a sound speed of 1541 m/s at 22 °C, a density of 1040 kg/m 3 and an attenuation coefficient of 0.45 dB/cm/MHz 40,41 . The phantoms were immersed in a tank filled with degassed water and carefully fixed to prevent any motion during US imaging and FUS sonication. Under the tissue-mimicking phantom, a rubber-type sound absorber was placed to reduce ultrasound wave reflection from the acrylic tank bottom, as well as the transducers with their orientation been fine-tuned to eliminate the standing waves.

In vitro experiments.
The pressure levels of the FUS transducers were measured with a calibrated hydrophone (polyvinylidene-difluoride-type, HNP-0400, ONDA, Sunnyvale, CA, USA; calibrated range: 1-20 MHz). In order to verify the detection limit of the proposed reconstruction approach, a FUS sonication experiment with various exposure levels and burst lengths was performed. We used four exposure frequencies (0.55, 1.1, 1.5, and 2 MHz) with the exposure peak negative pressure ranging from 0.391-1.194 MPa (equivalent to acoustic power varying from 3 to 30 W, respectively) and the burst length ranging from 0.01 to 10-ms (tested in 1.5-MHz FUS transducer exposure).

Magnetic Resonance Imaging (MRI).
Besides of ultrasound imaging and the reconstructed backscattered acoustic wave reconstruction mapping, MRI scans of the rat brain were also performed one day prior to in vivo animal experiment to serve as a high-resolution anatomical reference for backscattered ultrasound reconstructed maps. In order to align MR and diagnostic ultrasound imaging, the shaved rats' head were attached two fiducial markers on shaved scalp throughout experimental process, which were both detectable in MRI and diagnostic imaging. It is thought that while in-prior obtained MRI provides a high-resolution anatomical reference, ultrasound imaging provides a high temporal resolution anatomical position orientation to allow coordination of the reconstructed focal beam pattern distribution. A 7-Tesla magnetic resonance scanner (Bruker ClinScan, Germany) and a 4-channel surface coil were employed. During imaging acquisition, the animals were anesthetized through inhalation of 2% isoflurane throughout the MRI process, placed in an acrylic holder and positioned at the center of the magnet. A gradient echo FLASH sequence was set to acquire animal T1-weighted images (pulse repetition time (TR)/echo time (TE) = 300/3.81 ms; FOV = 21 × 25 mm 2 ; in-plane resolution = 0.25 × 0.2 mm 2 ; slice thickness = 0.5 mm; flip angle = 70°).

In vivo experiments. All animal procedures were approved by the Institutional Animal Care and Use
Committee of Chang-Gung University and adhered to the experimental animal care guidelines. A total of 12 adult male rats (250-300 g) were exposed to high-intensity FUS-BBB sonication in the study. The experimental setup for the in vivo experiments was shown in Fig. S2B. A center-opened 1.5-MHz FUS transducer was used to generate concentrated ultrasound energy, with the diagnostic ultrasound probe fit-in and was confocally aligned with the focused transducer for passive backscattered emissions receiving. One arbitrary-function generator was used to produce the driving signal, which was fed to a power amplifier operating in short-period mode. Animals were anesthetized by intraperitoneal injection of chlorohydrate (30 mg/kg). The scalp of animal was shaved with clippers. PE-50 catheter was inserted into the tail vein. The animal was placed directly on an acrylic water tank (with a window of 4 × 4 cm 2 bottom sealed with a thin film to allow ultrasound penetration), and its head attached tightly to the thin-film window with gel filling in the air gap. Diagnostic ultrasound probe was installed in the center-opened confocally aligned with the FUS transducer. For backscattered emission map reconstruction, a number of FUS exposure bursts were delivered (0.11-0.705 MPa; burst length = 10 ms) prior to microbubble administration to allow backscattered emissions signal collection. SonoVue ® SF6-coated ultrasound microbubbles (2-5 μ m, 30 μ g/kg; Bracco Diagnostics Inc., Milan, Italy) were administered intravenously before treatment. The animal hemisphere brain site was then exposed to burst-tone mode ultrasound to locally open the BBB (peak negative pressure = 0.11-0.705 MPa measured after penetrating through the skull; burst length = 10 ms; pulse repetition frequency = 1 Hz; exposure time = 60 s). Four animal groups were performed. Group A defines to be the low-exposure (0.11 MPa) control group (n = 1). Group B and C both conducted 0.42 to 0.44 MPa exposures, but in group B (n = 3) we introduced an imperfect layer matching by not perfectly shaving the fur to purposely leave gas gap in the interface to compare the perfect layer matching of group C (n = 5). Group D introduce a high exposure level to 0.7 MPa (n = 3).
Image signal-to-noise ratios (SNR) and statistical analyses. We analyzed the SNR in each region of interest (ROI) of the reconstructed maps to evaluate the beam detection capability, with the SNR defined as: where P s and P n denote the signal power and noise power in the region of interest, respectively. The above quantitative data analyses were based on data acquired from the regions of interest (ROI) located in the background and the beam pattern (with the ROI dimension selected to be 1.5 mm × 1.5 mm). The unpaired Student's t-test was used for statistically analyzing differences between groups of the SNR and peak signal intensity for both in vitro and in vivo study. Differences were recognized as statistically significant at p < 0.05.