Focused Ultrasound Hyperthermia for Targeted Drug Release from Thermosensitive Liposomes: Results from a Phase I Trial.

Purpose To demonstrate the feasibility and safety of using focused ultrasound planning models to determine the treatment parameters needed to deliver volumetric mild hyperthermia for targeted drug delivery without real-time thermometry. Materials and Methods This study was part of the Targeted Doxorubicin, or TARDOX, phase I prospective trial of focused ultrasound-mediated, hyperthermia-triggered drug delivery to solid liver tumors ( ClinicalTrials.gov identifier NCT02181075). Ten participants (age range, 49-68 years; average age, 60 years; four women) were treated from March 2015 to March 2017 by using a clinically approved focused ultrasound system to release doxorubicin from lyso-thermosensitive liposomes. Ultrasonic heating of target tumors (treated volume: 11-73 cm3 [mean ± standard deviation, 50 cm3 ± 26]) was monitored in six participants by using a minimally invasive temperature sensor; four participants were treated without real-time thermometry. For all participants, CT images were used with a patient-specific hyperthermia model to define focused ultrasound treatment plans. Feasibility was assessed by comparing model-prescribed focused ultrasound powers to those implemented for treatment. Safety was assessed by evaluating MR images and biopsy specimens for evidence of thermal ablation and monitoring adverse events. Results The mean difference between predicted and implemented treatment powers was -0.1 W ± 17.7 (n = 10). No evidence of focused ultrasound-related adverse effects, including thermal ablation, was found. Conclusion In this 10-participant study, the authors confirmed the feasibility of using focused ultrasound-mediated hyperthermia planning models to define treatment parameters that safely enabled targeted, noninvasive drug delivery to liver tumors while monitored with B-mode guidance and without real-time thermometry. Published under a CC BY 4.0 license. Online supplemental material is available for this article. See also the editorial by Dickey and Levi-Polyachenko in this issue.

Compensation of array lens effects for improved co-registration of passive acoustic mapping and B-mode images for cavitation monitoring.

Passive acoustic mapping (PAM) techniques offer a simple means of spatio-temporal cavitation monitoring during therapeutic ultrasound procedures. Implementation with a conventional diagnostic ultrasound system allows natural integration of PAM with B-mode imaging. However, the refracting properties of diagnostic array lenses may introduce PAM image registration errors that could lead to inaccuracies in treatment monitoring and guidance. To address these concerns, this paper presents lens characterization of two different array designs, analytical estimation of lens-induced source mapping errors in simple media, and experimental demonstration and correction of lens effects, reducing the depth-averaged image co-registration errors to no more than 0.52 mm.

Safety and feasibility of ultrasound-triggered targeted drug delivery of doxorubicin from thermosensitive liposomes in liver tumours (TARDOX): a single-centre, open-label, phase 1 trial.

BACKGROUND: Previous preclinical research has shown that extracorporeal devices can be used to enhance the delivery and distribution of systemically administered anticancer drugs, resulting in increased intratumoural concentrations. We aimed to assess the safety and feasibility of targeted release and enhanced delivery of doxorubicin to solid tumours from thermosensitive liposomes triggered by mild hyperthermia, induced non-invasively by focused ultrasound. METHODS: We did an open-label, single-centre, phase 1 trial in a single UK hospital. Adult patients (aged ≥18 years) with unresectable and non-ablatable primary or secondary liver tumours of any histological subtype were considered for the study. Patients received a single intravenous infusion (50 mg/m2) of lyso-thermosensitive liposomal doxorubicin (LTLD), followed by extracorporeal focused ultrasound exposure of a single target liver tumour. The trial had two parts: in part I, patients had a real-time thermometry device implanted intratumourally, whereas patients in part II proceeded without thermometry and we used a patient-specific model to predict optimal exposure parameters. We assessed tumour biopsies obtained before and after focused ultrasound exposure for doxorubicin concentration and distribution. The primary endpoint was at least a doubling of total intratumoural doxorubicin concentration in at least half of the patients treated, on an intention-to-treat basis. This study is registered with ClinicalTrials.gov, number NCT02181075, and is now closed to recruitment. FINDINGS: Between March 13, 2015, and March 27, 2017, ten patients were enrolled in the study (six patients in part I and four in part II), and received a dose of LTLD followed by focused ultrasound exposure. The treatment resulted in an average increase of 3·7 times in intratumoural biopsy doxorubicin concentrations, from an estimate of 2·34 µg/g (SD 0·93) immediately after drug infusion to 8·56 µg/g (5·69) after focused ultrasound. Increases of two to ten times were observed in seven (70%) of ten patients, satisfying the primary endpoint. Serious adverse events registered were expected grade 4 transient neutropenia in five patients and prolonged hospital stay due to unexpected grade 1 confusion in one patient. Grade 3-4 adverse events recorded were neutropenia (grade 3 in one patient and grade 4 in five patients), and grade 3 anaemia in one patient. No treatment-related deaths occurred. INTERPRETATION: The combined treatment of LTLD and non-invasive focused ultrasound hyperthermia in this study seemed to be clinically feasible, safe, and able to enhance intratumoural drug delivery, providing targeted chemo-ablative response in human liver tumours that were refractory to standard chemotherapy. FUNDING: Oxford Biomedical Research Centre, National Institute for Health Research.

Energy Transfer Mechanisms during Molecular Delivery to Cells by Laser-Activated Carbon Nanoparticles.

Previous studies have shown that exposure of carbon black nanoparticles to nanosecond pulsed near-infrared laser causes intracellular delivery of molecules through hypothesized transient breaks in the cell membrane. The goal of this study is to determine the underlying mechanisms of sequential energy transfer from laser light to nanoparticle to fluid medium to cell. We found that laser pulses on a timescale of 10 ns rapidly heat carbon nanoparticles to temperatures on the order of 1200 K. Heat is transferred from the nanoparticles to the surrounding aqueous medium on a similar timescale, causing vaporization of the surrounding water and generation of acoustic emissions. Nearby cells can be impacted thermally by the hot bubbles and mechanically by fluid mechanical forces to transiently increase cell membrane permeability. The experimental and theoretical results indicate that transfer of momentum and/or heat from the bubbles to the cells are the dominant mechanisms of energy transfer that results in intracellular uptake of molecules. We further conclude that neither thermal expansion of the nanoparticles nor a carbon-steam chemical reaction play a significant role in the observed effects on cells, and that acoustic pressure appears to be concurrent with, but not essential to, the observed bioeffects.

In vivo ultrasonic attenuation in cetacean soft tissues.

In vivo ultrasonic attenuation was estimated for extracranial soft tissues of two Tursiops truncatus and one Delphinapterus leucas. Backscatter data was non-invasively collected as part of routine health-based ultrasound examinations using a transducer operating in the 1.6-3.7 MHz frequency range. Data collected over the proximal mandible and temporal regions was processed to yield attenuation estimates using a reference tissue phantom whose properties had been independently determined. The estimated attenuations were at the low end of the range of literature-reported values for mammalian fatty and connective tissues. A companion model-based analysis quantified errors arising from tissue composition and sound speed uncertainties.

"Large" Tank Acoustics: How Big Is Big Enough?

In this paper, we discuss the issues encountered when trying to perform hearing experiments in water-filled tanks that are several meters in lateral extent, typically large in terms of the size of the animals under study but not necessarily so with respect to the wavelengths of interest. This paper presents measurements of pressure and particle motion fields in these "large" tanks. The observed characteristics and complexities are discussed in reference to their potential impact on the planning and interpretation of hearing experiments.

Parvulescu Revisited: Small Tank Acoustics for Bioacousticians.

Researchers often perform hearing studies on fish in small tanks. The acoustic field in such a tank is considerably different from the acoustic field that occurs in the animal's natural environment. The significance of these differences is magnified by the nature of the fish's auditory system where either acoustic pressure (a scalar), acoustic particle velocity (a vector), or both may serve as the stimulus. It is essential for the underwater acoustician to understand the acoustics of small tanks to be able to carry out valid auditory research in the laboratory and to properly compare and interpret the results of others.

Hearing thresholds of swimming Pacific bluefin tuna Thunnus orientalis.

Pacific bluefin tuna (Thunnus orientalis) is a highly migratory, commercially valuable species potentially vulnerable to acoustic noise generated from human activities which could impact behavior and fitness. Although significant efforts have been made to understand hearing abilities of fishes, the large size and need to continuously swim for respiration have hindered investigations with tuna and other large pelagic species. In this study, Pacific bluefin tuna were trained to respond to a pure tone sound stimulus ranging 325-800 Hz and their hearing abilities quantified using a staircase psychophysical technique. Hearing was most sensitive from 400 to 500 Hz in terms of particle motion (radial acceleration -88 dB re 1 m s(-2); vertical acceleration -86 dB re 1 m s(-2)) and sound pressure (83 dB re 1 µPa). Compared to yellowfin tuna (Thunnus albacares) and kawakawa (Euthynnus affinis), Pacific bluefin tuna has a similar bandwidth of hearing and best frequency, but greater sensitivity overall. Careful calibration of the sound stimulus and experimental tank environment, as well as the adoption of behavioral methodology, demonstrates an experimental approach highly effective for the study of large fish species in the laboratory.

Use of the swim bladder and lateral line in near-field sound source localization by fish.

We investigated the roles of the swim bladder and the lateral line system in sound localization behavior by the plainfin midshipman fish (Porichthys notatus). Reproductive female midshipman underwent either surgical deflation of the swim bladder or cryoablation of the lateral line and were then tested in a monopolar sound source localization task. Fish with nominally 'deflated' swim bladders performed similar to sham-deflated controls; however, post-experiment evaluation of swim bladder deflation revealed that a majority of 'deflated' fish (88%, seven of the eight fish) that exhibited positive phonotaxis had partially inflated swim bladders. In total, 95% (21/22) of fish that localized the source had at least partially inflated swim bladders, indicating that pressure reception is likely required for sound source localization. In lateral line experiments, no difference was observed in the proportion of females exhibiting positive phonotaxis with ablated (37%) versus sham-ablated (47%) lateral line systems. These data suggest that the lateral line system is likely not required for sound source localization, although this system may be important for fine-tuning the approach to the sound source. We found that midshipman can solve the 180 deg ambiguity of source direction in the shallow water of our test tank, which is similar to their nesting environment. We also found that the potential directional cues (phase relationship between pressure and particle motion) in shallow water differs from a theoretical free-field. Therefore, the general question of how fish use acoustic pressure cues to solve the 180 deg ambiguity of source direction from the particle motion vector remains unresolved.

Sound speed and attenuation of human pancreas and pancreatic tumors and their influence on focused ultrasound thermal and mechanical therapies.

BACKGROUND: There is increasing interest in using ultrasound for thermal ablation, histotripsy, and thermal or cavitational enhancement of drug delivery for the treatment of pancreatic cancer. Ultrasonic and thermal modelling conducted as part of the treatment planning process requires acoustic property values for all constituent tissues, but the literature contains no data for the human pancreas. PURPOSE: This study presents the first acoustic property measurements of human pancreatic samples and provides examples of how these properties impact a broad range of ultrasound therapies. METHODS: Data were collected on human pancreatic tissue samples at physiological temperature from 23 consented patients in cooperation with a hospital pathology laboratory. Propagation of ultrasound over the 2.1-4.5 MHz frequency range through samples of various thicknesses and pathologies was measured using a set of custom-built ultrasonic calipers, with the data processed to estimate sound speed and attenuation. The results were used in acoustic and thermal simulations to illustrate the impacts on extracorporeal ultrasound therapies for mild hyperthermia, thermal ablation, and histotripsy implemented with a CE-marked clinical system operating at 0.96 MHz. RESULTS: The mean sound speed and attenuation coefficient values for human samples were well below the range of values in the literature for non-human pancreata, while the human attenuation power law exponents were substantially higher. The simulated impacts on ultrasound mediated therapies for the pancreas indicated that when using the human data instead of the literature average, there was a 30% reduction in median temperature elevation in the treatment volume for mild hyperthermia and 43% smaller volume within a 60°C contour for thermal ablation, all driven by attenuation. By comparison, impacts on boiling and intrinsic threshold histotripsy were minor, with peak pressures changing by less than 15% (positive) and 1% (negative) as a consequence of the counteracting effects of attenuation and sound speed. CONCLUSION: This study provides the most complete set of speed of sound and attenuation data available for the human pancreas, and it reiterates the importance of acoustic material properties in the planning and conduct of ultrasound-mediated procedures, particularly thermal therapies.

Developing an expert panel process to refine health outcome definitions in observational data.

OBJECTIVES: Drug safety surveillance using observational data requires valid adverse event, or health outcome of interest (HOI) measurement. The objectives of this study were to develop a method to review HOI definitions in claims databases using (1) web-based digital tools to present de-identified patient data, (2) a systematic expert panel review process, and (3) a data collection process enabling analysis of concepts-of-interest that influence panelists' determination of HOI. METHODS: De-identified patient data were presented via an interactive web-based dashboard to enable case review and determine if specific HOIs were present or absent. Criteria for determining HOIs and their severity were provided to each panelist. Using a modified Delphi method, six panelist pairs independently reviewed approximately 200 cases across each of three HOIs (acute liver injury, acute kidney injury, and acute myocardial infarction) such that panelist pairs independently reviewed the same cases. Panelists completed an assessment within the dashboard for each case that included their assessment of the presence or absence of the HOI, HOI severity (if present), and data contributing to their decision. Discrepancies within panelist pairs were resolved during a consensus process. RESULTS: Dashboard development was iterative, focusing on data presentation and recording panelists' assessments. Panelists reported quickly learning how to use the dashboard. The assessment module was used consistently. The dashboard was reliable, enabling an efficient review process for panelists. Modifications were made to the dashboard and review process when necessary to facilitate case review. Our methods should be applied to other health outcomes of interest to further refine the dashboard and case review process. CONCLUSION: The expert review process was effective and was supported by the web-based dashboard. Our methods for case review and classification can be applied to future methods for case identification in observational data sources.

Protein-Decorated Microbubbles for Ultrasound-Mediated Cell Surface Manipulation.

Delivering cargo to the cell membranes of specific cell types in the body is a major challenge for a range of treatments, including immunotherapy. This study investigates employing protein-decorated microbubbles (MBs) and ultrasound (US) to "tag" cellular membranes of interest with a specific protein. Phospholipid-coated MBs were produced and functionalized with a model protein using a metallochelating complex through an NTA(Ni) and histidine residue interaction. Successful "tagging" of the cellular membrane was observed using microscopy in adherent cells and was promoted by US exposure. Further modification of the MB surface to enable selective binding to target cells was then achieved by functionalizing the MBs with a targeting protein (transferrin) that specifically binds to a receptor on the target cell membrane. Attachment and subsequent transfer of material from MBs functionalized with transferrin to the target cells significantly increased, even in the absence of US. This work demonstrates the potential of these MBs as a platform for the noninvasive delivery of proteins to the surface of specific cell types.

Local acoustic particle motion guides sound-source localization behavior in the plainfin midshipman fish, Porichthys notatus.

Sound-source localization behavior was studied in the plainfin midshipman fish (Porichthys notatus) by making use of the naturally occurring phonotaxis response of gravid females to playback of the male's advertisement call. The observations took place outdoors in a circular concrete tank. A dipole sound projector was placed at the center of the tank and an 80-90 Hz tone (the approximate fundamental frequency to the male's advertisement call) was broadcast to gravid females that were released from alternative sites approximately 100 cm from the source. The phonotaxic responses of females to the source were recorded, analyzed and compared with the sound field. One release site was approximately along the vibratory axis of the dipole source, and the other was approximately orthogonal to the vibratory axis. The sound field in the tank was fully characterized through measurements of the sound pressure field using hydrophones and acoustic particle motion using an accelerometer. These measurements confirmed that the sound field was a nearly ideal dipole. When released along the dipole vibratory axis, the responding female fish took essentially straight paths to the source. However, when released approximately 90 deg to the source's vibratory axis, the responding females took highly curved paths to the source that were approximately in line with the local particle motion axes. These results indicate that the acoustic cues used by fish during sound-source localization include the axes of particle motion of the local sound field.

PAX (Passive-Active Crossing) Method for Sub-Millimeter Coregistration of Passive Acoustic Mapping and B-Mode Images.

Nonlinear ultrasonic emissions produced during a therapeutic ultrasound procedure can be detected, localized, and quantified through a class of methods that can be referred to as passive acoustic mapping (PAM). While a variety of PAM beamforming algorithms may be employed, they share a common limitation that a single sound speed is specified for both phase steering of array elements and for calculation of source power or energy. The specified value may be inadequate whether derived from B-mode-based metrics or literature values for constituent materials. This study employed experiments and simulations with linear and curvilinear array geometries to investigate the impact of in situ sound speed uncertainties on source localization in layered media. The data were also used to evaluate a new method for optimizing coregistration of PAM and B-mode images. Coregistration errors as large as 10 mm were observed with the curvilinear array, which also showed much greater sound speed sensitivity than the linear array. Errors with both array geometries were typically reduced to the order of 0.1 mm using the proposed optimization method regardless of beamformer choice or whether the array was calibrated. In a further step toward reliable implementation of PAM, the current work provides an approach that can help ensure that therapeutic ultrasound procedures are accurately guided by cavitation emissions.

Dual-Array Passive Acoustic Mapping for Cavitation Imaging With Enhanced 2-D Resolution.

Passive acoustic mapping (PAM) techniques have been developed for the purposes of detecting, localizing, and quantifying cavitation activity during therapeutic ultrasound procedures. Implementation with conventional diagnostic ultrasound arrays has allowed planar mapping of bubble acoustic emissions to be overlaid with B-mode anatomical images, with a variety of beamforming approaches providing enhanced resolution at the cost of extended computation times. However, no passive signal processing techniques implemented to date have overcome the fundamental physical limitation of the conventional diagnostic array aperture that results in point spread functions with axial/lateral beamwidth ratios of nearly an order of magnitude. To mitigate this problem, the use of a pair of orthogonally oriented diagnostic arrays was recently proposed, with potential benefits arising from the substantially expanded range of observation angles. This article presents experiments and simulations intended to demonstrate the performance and limitations of the dual-array system concept. The key finding of this study is that source pair resolution of better than 1 mm is now possible in both dimensions of the imaging plane using a pair of 7.5-MHz center frequency conventional arrays at a distance of 7.6cm. With an eye toward accelerating computations for real-time applications, channel count reductions of up to a factor of eight induce negligible performance losses. Modest sensitivities to sound speed and relative array position uncertainties were identified, but if these can be kept on the order of 1% and 1 mm, respectively, then the proposed methods offer the potential for a step improvement in cavitation monitoring capability.

Evaluation of Loading Strategies to Improve Tumor Uptake of Gemcitabine in a Murine Orthotopic Bladder Cancer Model Using Ultrasound and Microbubbles.

In this study we compared three different microbubble-based approaches to the delivery of a widely used chemotherapy drug, gemcitabine: (i) co-administration of gemcitabine and microbubbles (Gem+MB); (ii) conjugates of microbubbles and gemcitabine-loaded liposomes (GemlipoMB); and (iii) microbubbles with gemcitabine directly bound to their surfaces (GembioMB). Both in vitro and in vivo investigations were carried out, respectively, in the RT112 bladder cancer cell line and in a murine orthotopic muscle-invasive bladder cancer model. The in vitro (in vivo) ultrasound exposure conditions were a 1 (1.1) MHz centre frequency, 0.07 (1.0) MPa peak negative pressure, 3000 (20,000) cycles and 100 (0.5) Hz pulse repetition frequency. Ultrasound exposure produced no significant increase in drug uptake either in vitro or in vivo compared with the drug-only control for co-administered gemcitabine and microbubbles. In vivo, GemlipoMB prolonged the plasma circulation time of gemcitabine, but only GembioMB produced a statistically significant increase in cleaved caspase 3 expression in the tumor, indicative of gemcitabine-induced apoptosis.

Magnetic microbubble mediated chemo-sonodynamic therapy using a combined magnetic-acoustic device.

Recent pre-clinical studies have demonstrated the potential of combining chemotherapy and sonodynamic therapy for the treatment of pancreatic cancer. Oxygen-loaded magnetic microbubbles have been explored as a targeted delivery vehicle for this application. Despite preliminary positive results, a previous study identified a significant practical challenge regarding the co-alignment of the magnetic and ultrasound fields. The aim of this study was to determine whether this challenge could be addressed through the use of a magnetic-acoustic device (MAD) combining a magnetic array and ultrasound transducer in a single unit, to simultaneously concentrate and activate the microbubbles at the target site. in vitro experiments were performed in tissue phantoms and followed by in vivo treatment of xenograft pancreatic cancer (BxPC-3) tumours in a murine model. In vitro, a 1.4-fold (p < .01) increase in the deposition of a model therapeutic payload within the phantom was achieved using the MAD compared to separate magnetic and ultrasound devices. In vivo, tumours treated with the MAD had a 9% smaller mean volume 8 days after treatment, while tumours treated with separate devices or microbubbles alone were respectively 45% and 112% larger. This substantial and sustained decrease in tumour volume suggests that the proposed drug delivery approach has the potential to be an effective neoadjuvant therapy for pancreatic cancer patients.

Broadband Ultrasonic Attenuation Estimation and Compensation With Passive Acoustic Mapping.

Several active and passive techniques have been developed to detect, localize, and quantify cavitation activity during therapeutic ultrasound procedures. Much of the prior cavitation monitoring research has been conducted using lossless in vitro systems or small animal models in which path attenuation effects were minimal. However, the performance of these techniques may be substantially degraded by attenuation between the internal therapeutic target and the external monitoring system. As a further step toward clinical application of passive acoustic mapping (PAM), this paper presents methods for attenuation estimation and compensation based on broadband cavitation data measured with a linear ultrasound array. Soft tissue phantom experiment results are used to illustrate: 1) the impact of realistic attenuation on PAM; 2) the possibility of estimating attenuation from cavitation data; 3) cavitation source energy estimation following attenuation compensation; and 4) the impact of sound speed uncertainty on PAM-related processing. Cavitation-based estimates of attenuation were within 1.5%-6.2% of the values found from conventional through-transmission measurements. Tissue phantom attenuation reduced the PAM energy estimate by an order of magnitude, but array data compensation using the cavitation-based attenuation spectrum enabled recovery of the PAM energy estimate to within 2.9%-5.9% of the values computed in the absence of the phantom. Sound speed uncertainties were found to modestly impact attenuation-compensated PAM energies, inducing errors no larger than 28% for a 40-m/s path-averaged speed error. Together, the results indicate the potential to significantly enhance the quantitative capabilities of PAM for ensuring therapeutic safety and efficacy.

Diffraction Effects and Compensation in Passive Acoustic Mapping.

Over the last decade, a variety of noninvasive techniques have been developed to monitor therapeutic ultrasound procedures in support of safety or efficacy assessments. One class of methods employs diagnostic ultrasound arrays to sense acoustic emissions, thereby providing a means to passively detect, localize, and quantify the strength of nonlinear sources, including cavitation. Real array element diffraction patterns may differ substantially from those presumed in existing beamforming algorithms. However, diffraction compensation has received limited treatment in passive and active imaging, and measured diffraction data have yet to be used for array response correction. The objectives of this paper were to identify differences between ideal and real element diffraction patterns, and to quantify the impact of diffraction correction on cavitation mapping beamformer performance. These objectives were addressed by performing calibration measurements on a diagnostic linear array, using the results to calculate diffraction correction terms, and applying the corrections to cavitation emission data collected from soft tissue phantom experiments. Measured diffraction patterns were found to differ significantly from those of ideal element forms, particularly at higher frequencies and shorter distances from the array. Diffraction compensation of array data resulted in cavitation energy estimates elevated by as much as a factor of 5, accompanied by the elimination of a substantial bias between two established beamforming algorithms. These results illustrate the importance of using measured array responses to validate analytical field models and to minimize observation biases in imaging applications where quantitative analyses are critical for assessment of therapeutic safety and efficacy.

Passive Acoustic Mapping Using Data-Adaptive Beamforming Based on Higher Order Statistics.

Sources of nonlinear acoustic emissions, particularly those associated with cavitation activity, play a key role in the safety and efficacy of current and emerging therapeutic ultrasound applications, such as oncological drug delivery, blood-brain barrier opening, and histotripsy. Passive acoustic mapping (PAM) is the first technique to enable real-time and non-invasive imaging of cavitation activity during therapeutic ultrasound exposure, through the recording and passive beamforming of broadband acoustic emissions using an array of ultrasound detectors. Initial limitations in PAM spatial resolution led to the adoption of optimal data-adaptive beamforming algorithms, such as the robust capon beamformer (RCB), that provide improved interference suppression and calibration error mitigation compared to non-adaptive beamformers. However, such approaches are restricted by the assumption that the recorded signals have a Gaussian distribution. To overcome this limitation and further improve the source resolvability of PAM, we propose a new beamforming approach termed robust beamforming by linear programming (RLPB). Along with the variance, this optimization-based method uses higher-order-statistics of the recorded signals, making no prior assumption on the statistical distribution of the acoustic signals. The RLPB is found via numerical simulations to improve resolvability over time exposure acoustics and RCB. In vitro experimentation yielded improved resolvability with respect to the source-to-array distance on the order of 22% axially and 13% transversely relative to RCB, whilst successfully accounting for array calibration errors. The improved resolution and decreased dependence on accurate calibration of RLPB is expected to facilitate the clinical translation of PAM for diagnostic, including super-resolution, and therapeutic ultrasound applications.