Spatial transcriptomic analysis drives PET imaging of tight junction protein expression in pancreatic cancer theranostics.

We apply spatial transcriptomics and proteomics to select pancreatic cancer surface receptor targets for molecular imaging and theranostics using an approach that can be applied to many cancers. Selected cancer surfaceome epithelial markers were spatially correlated and provided specific cancer localization, whereas the spatial correlation between cancer markers and immune- cell or fibroblast markers was low. While molecular imaging of cancer-associated fibroblasts and integrins has been proposed for pancreatic cancer, our data point to the tight junction protein claudin-4 as a theranostic target. Claudin-4 expression increased ∼16 fold in cancer as compared with normal pancreas, and the tight junction localization conferred low background for imaging in normal tissue. We developed a peptide-based molecular imaging agent targeted to claudin-4 with accumulation to ∼25% injected activity per cc (IA/cc) in metastases and ∼18% IA/cc in tumors. Our work motivates a new approach for data-driven selection of molecular targets.

Pyroptosis Induction with Nanosonosensitizer-Augmented Sonodynamic Therapy Combined with PD-L1 Blockade Boosts Efficacy against Liver Cancer.

Induction of pyroptosis can promote anti-PD-L1 therapeutic efficacy due to the release of pro-inflammatory cytokines, but current approaches can cause off target toxicity. Herein, a phthalocyanine-conjugated mesoporous silicate nanoparticle (PMSN) is designed for amplifying sonodynamic therapy (SDT) to augment oxidative stress and induce robust pyroptosis in tumors. The sub-10 nm diameter structure and c(RGDyC)-PEGylated modification enhance tumor targeting and renal clearance. The unique porous architecture of PMSN doubles ROS yield and enhances pyroptotic cell populations in tumors (25.0%) via a cavitation effect. PMSN-mediated SDT treatment efficiently reduces tumor mass and suppressed residual tumors in treated and distant sites by synergizing with PD-L1 blockade (85.93% and 77.09%, respectively). Furthermore, loading the chemotherapeutic, doxorubicin, into PMSN intensifies SDT-pyroptotic effects and increased efficacy. This is the first report of the use of SDT regimens to induce pyroptosis in liver cancer. This noninvasive and effective strategy has potential for clinical translation.

Protocol for in vitro sonoporation validation using non-targeted microbubbles for human studies of ultrasound-mediated gene delivery.

Microbubbles are currently approved for diagnostic ultrasound imaging and are under evaluation in therapeutic protocols. Here, we present a protocol for in vitro sonoporation validation using non-targeted microbubbles for gene delivery. We describe steps for computational simulation, experimental calibration, reagent preparation, ultrasound treatment, validation, and gene expression analysis. This protocol uses approved diagnostic microbubbles and parameters that are applicable for human use. For complete details on the use and execution of this protocol, please refer to Bez et al. (2017).1.

PET imaging of focused-ultrasound enhanced delivery of AAVs into the murine brain.

Rationale: Despite recent advances in the use of adeno-associated viruses (AAVs) as potential vehicles for genetic intervention of central and peripheral nervous system-associated disorders, gene therapy for the treatment of neuropathology in adults has not been approved to date. The currently FDA-approved AAV-vector based gene therapies rely on naturally occurring serotypes, such as AAV2 or AAV9, which display limited or no transport across the blood-brain barrier (BBB) if systemically administered. Recently developed engineered AAV variants have shown broad brain transduction and reduced off-target liver toxicity in non-human primates (NHPs). However, these vectors lack spatial selectivity for targeted gene delivery, a potentially critical limitation for delivering therapeutic doses in defined areas of the brain. The use of microbubbles, in conjunction with focused ultrasound (FUS), can enhance regional brain AAV transduction, but methods to assess transduction in vivo are needed. Methods: In a murine model, we combined positron emission tomography (PET) and optical imaging of reporter gene payloads to non-invasively assess the spatial distribution and transduction efficiency of systemically administered AAV9 after FUS and microbubble treatment. Capsid and reporter probe accumulation are reported as percent injected dose per cubic centimeter (%ID/cc) for in vivo PET quantification, whereas results for ex vivo assays are reported as percent injected dose per gram (%ID/g). Results: In a study spanning accumulation and transduction, mean AAV9 accumulation within the brain was 0.29 %ID/cc without FUS, whereas in the insonified region of interest of FUS-treated mice, the spatial mean and maximum reached ~2.3 %ID/cc and 4.3 %ID/cc, respectively. Transgene expression assessed in vivo by PET reporter gene imaging employing the pyruvate kinase M2 (PKM2)/[18F]DASA-10 reporter system increased up to 10-fold in the FUS-treated regions, as compared to mice receiving AAVs without FUS. Systemic injection of AAV9 packaging the EF1A-PKM2 transgene followed by FUS in one hemisphere resulted in 1) an average 102-fold increase in PKM2 mRNA concentration compared to mice treated with AAVs only and 2) a 12.5-fold increase in the insonified compared to the contralateral hemisphere of FUS-treated mice. Conclusion: Combining microbubbles with US-guided treatment facilitated a multi-hour BBB disruption and stable AAV transduction in targeted areas of the murine brain. This unique platform has the potential to provide insight and aid in the translation of AAV-based therapies for the treatment of neuropathologies.

Optical Breast Imaging: A Review of Physical Principles, Technologies, and Clinical Applications.

Optical imaging involves the propagation of light through tissue. Current optical breast imaging technologies, including diffuse optical spectroscopy, diffuse optical tomography, and photoacoustic imaging, capitalize on the selective absorption of light in the near-infrared spectrum by deoxygenated and oxygenated hemoglobin. They provide information on the morphological and functional characteristics of different tissues based on their varied interactions with light, including physiologic information on lesion vascular content and anatomic information on tissue vascularity. Fluorescent contrast agents, such as indocyanine green, are used to visualize specific tissues, molecules, or proteins depending on how and where the agent accumulates. In this review, we describe the physical principles, spectrum of technologies, and clinical applications of the most common optical systems currently being used or developed for breast imaging. Most notably, US co-registered photoacoustic imaging and US-guided diffuse optical tomography have demonstrated efficacy in differentiating benign from malignant breast masses, thereby improving the specificity of diagnostic imaging. Diffuse optical tomography and diffuse optical spectroscopy have shown promise in assessing treatment response to preoperative systemic therapy, and photoacoustic imaging and diffuse optical tomography may help predict tumor phenotype. Lastly, fluorescent imaging using indocyanine green dye performs comparably to radioisotope mapping of sentinel lymph nodes and appears to improve the outcomes of autologous tissue flap breast reconstruction.

Sonogenetic control of multiplexed genome regulation and base editing.

Manipulating gene expression in the host genome with high precision is crucial for controlling cellular function and behavior. Here, we present a precise, non-invasive, and tunable strategy for controlling the expression of multiple endogenous genes both in vitro and in vivo, utilizing ultrasound as the stimulus. By engineering a hyper-efficient dCas12a and effector under a heat shock promoter, we demonstrate a system that can be inducibly activated through thermal energy produced by ultrasound absorption. This system allows versatile thermal induction of gene activation or base editing across cell types, including primary T cells, and enables multiplexed gene activation using a single guide RNA array. In mouse models, localized temperature elevation guided by high-intensity focused ultrasound effectively triggers reporter gene expression in implanted cells. Our work underscores the potential of ultrasound as a clinically viable approach to enhance cell and gene-based therapies via precision genome and epigenome engineering.

OMIP-095: 40-Color spectral flow cytometry delineates all major leukocyte populations in murine lymphoid tissues.

High-dimensional immunoprofiling is essential for studying host response to immunotherapy, infection, and disease in murine model systems. However, the difficulty of multiparameter panel design combined with a lack of existing murine tools has prevented the comprehensive study of all major leukocyte phenotypes in a single assay. Herein, we present a 40-color flow cytometry panel for deep immunophenotyping of murine lymphoid tissues, including the spleen, blood, Peyer's patches, inguinal lymph nodes, bone marrow, and thymus. This panel uses a robust set of surface markers capable of differentiating leukocyte subsets without the use of intracellular staining, thus allowing for the use of cells in downstream functional experiments or multiomic analyses. Our panel classifies T cells, B cells, natural killer cells, innate lymphoid cells, monocytes, macrophages, dendritic cells, basophils, neutrophils, eosinophils, progenitors, and their functional subsets by using a series of co-stimulatory, checkpoint, activation, migration, and maturation markers. This tool has a multitude of systems immunology applications ranging from serial monitoring of circulating blood signatures to complex endpoint analysis, especially in pre-clinical settings where treatments can modulate leukocyte abundance and/or function. Ultimately, this 40-color panel resolves a diverse array of immune cells on the axes of time, tissue, and treatment, filling the niche for a modern tool dedicated to murine immunophenotyping.

Combined near infrared photoacoustic imaging and ultrasound detects vulnerable atherosclerotic plaque.

Atherosclerosis is an inflammatory process resulting in the deposition of cholesterol and cellular debris, narrowing of the vessel lumen and clot formation. Characterization of the morphology and vulnerability of the lesion is essential for effective clinical management. Here, near-infrared auto-photoacoustic (NIRAPA) imaging is shown to detect plaque components and, when combined with ultrasound imaging, to differentiate stable and vulnerable plaque. In an ex vivo study of photoacoustic imaging of excised plaque from 25 patients, 88.2% sensitivity and 71.4% specificity were achieved using a clinically-relevant protocol. In order to determine the origin of the NIRAPA signal, immunohistochemistry, spatial transcriptomics and spatial proteomics were co-registered with imaging and applied to adjacent plaque sections. The highest NIRAPA signal was spatially correlated with bilirubin and associated blood-based residue and with the cytoplasmic contents of inflammatory macrophages bearing CD74, HLA-DR, CD14 and CD163 markers. In summary, we establish the potential to apply the NIRAPA-ultrasound imaging combination to detect vulnerable carotid plaque and a methodology for fusing molecular imaging with spatial transcriptomic and proteomic methods.

Combined near infrared photoacoustic imaging and ultrasound detects vulnerable atherosclerotic plaque.

Atherosclerosis is an inflammatory process resulting in the deposition of cholesterol and cellular debris, narrowing of the vessel lumen and clot formation. Characterization of the morphology and vulnerability of the lesion is essential for effective clinical management. Photoacoustic imaging has sufficient penetration and sensitivity to map and characterize human atherosclerotic plaque. Here, near infrared photoacoustic imaging is shown to detect plaque components and, when combined with ultrasound imaging, to differentiate stable and vulnerable plaque. In an ex vivo study of photoacoustic imaging of excised plaque from 25 patients, 88.2% sensitivity and 71.4% specificity were achieved using a clinically-relevant protocol. In order to determine the origin of the near-infrared auto-photoacoustic (NIRAPA) signal, immunohistochemistry, spatial transcriptomics and proteomics were applied to adjacent sections of the plaque. The highest NIRAPA signal was spatially correlated with bilirubin and associated blood-based residue and inflammatory macrophages bearing CD74, HLA-DR, CD14 and CD163 markers. In summary, we establish the potential to apply the NIRAPA-ultrasound imaging combination to detect vulnerable carotid plaque.

Hardwiring tissue-specific AAV transduction in mice through engineered receptor expression.

The development of transgenic mouse models that express genes of interest in specific cell types has transformed our understanding of basic biology and disease. However, generating these models is time- and resource-intensive. Here we describe a model system, SELective Expression and Controlled Transduction In Vivo (SELECTIV), that enables efficient and specific expression of transgenes by coupling adeno-associated virus (AAV) vectors with Cre-inducible overexpression of the multi-serotype AAV receptor, AAVR. We demonstrate that transgenic AAVR overexpression greatly increases the efficiency of transduction of many diverse cell types, including muscle stem cells, which are normally refractory to AAV transduction. Superior specificity is achieved by combining Cre-mediated AAVR overexpression with whole-body knockout of endogenous Aavr, which is demonstrated in heart cardiomyocytes, liver hepatocytes and cholinergic neurons. The enhanced efficacy and exquisite specificity of SELECTIV has broad utility in development of new mouse model systems and expands the use of AAV for gene delivery in vivo.

Fast volumetric ultrasound facilitates high-resolution 3D mapping of tissue compartments.

Volumetric ultrasound imaging has the potential for operator-independent acquisition and enhanced field of view. Panoramic acquisition has many applications across ultrasound; spanning musculoskeletal, liver, breast, and pediatric imaging; and image-guided therapy. Challenges in high-resolution human imaging, such as subtle motion and the presence of bone or gas, have limited such acquisition. These issues can be addressed with a large transducer aperture and fast acquisition and processing. Programmable, ultrafast ultrasound scanners with a high channel count provide an unprecedented opportunity to optimize volumetric acquisition. In this work, we implement nonlinear processing and develop distributed beamformation to achieve fast acquisition over a 47-centimeter aperture. As a result, we achieve a 50-micrometer -6-decibel point spread function at 5 megahertz and resolve in-plane targets. A large volume scan of a human limb is completed in a few seconds, and in a 2-millimeter dorsal vein, the image intensity difference between the vessel center and surrounding tissue was ~50 decibels, facilitating three-dimensional reconstruction of the vasculature.

Multiomic analysis for optimization of combined focal and immunotherapy protocols in murine pancreatic cancer.

Background: Although combination immunotherapies incorporating local and systemic components have shown promising results in treating solid tumors, varied tumor microenvironments (TMEs) can impact immunotherapeutic efficacy. Method: We designed and evaluated treatment strategies for breast and pancreatic cancer combining magnetic resonance-guided focused ultrasound (MRgFUS) ablation and antibody therapies. With a combination of single-cell sequencing, spectral flow cytometry, and histological analyses, we profiled an immune-suppressed KPC (Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx1-Cre) pancreatic adenocarcinoma (MT4) model and a dense epithelial neu deletion (NDL) HER2+ mammary adenocarcinoma model with a greater fraction of lymphocytes, natural killer cells and activated dendritic cells. We then performed gene ontology analysis, spectral and digital cytometry to assess the immune response to combination immunotherapies and correlation with survival studies. Result: Based on gene ontology analysis, adding ablation to immunotherapy enriched immune cell migration pathways in the pancreatic cancer model and extensively enriched wound healing pathways in the breast cancer model. With CIBERSORTx digital cytometry, aCD40 + aPD-1 immunotherapy combinations enhanced dendritic cell activation in both models. In the MT4 TME, adding the combination of aCD40 antibody and checkpoint inhibitors (aPD-1 and aCTLA-4) with ablation was synergistic, increasing activated natural killer cells and T cells in distant tumors. Furthermore, ablation with immunotherapy upregulated critical Ly6c myeloid remodeling phenotypes that enhance T-cell effector function and increased granzyme and protease encoding genes by as much as 100-fold. Ablation combined with immunotherapy then extended survival in the MT4 model to a greater extent than immunotherapy alone. Conclusion: In summary, TME profiling informed a successful multicomponent treatment protocol incorporating ablation and facilitated differentiation of TMEs in which ablation is most effective.

Multimodal imaging of capsid and cargo reveals differential brain targeting and liver detargeting of systemically-administered AAVs.

The development of gene delivery vehicles with high organ specificity when administered systemically is a critical goal for gene therapy. We combine optical and positron emission tomography (PET) imaging of 1) reporter genes and 2) capsid tags to assess the temporal and spatial distribution and transduction of adeno-associated viruses (AAVs). AAV9 and two engineered AAV vectors (PHP.eB and CAP-B10) that are noteworthy for maximizing blood-brain barrier transport were compared. CAP-B10 shares a modification in the 588 loop with PHP.eB, but also has a modification in the 455 loop, added with the goal of reducing off-target transduction. PET and optical imaging revealed that the additional modifications retained brain receptor affinity. In the liver, the accumulation of AAV9 and the engineered AAV capsids was similar (∼15% of the injected dose per cc and not significantly different between capsids at 21 h). However, the engineered capsids were primarily internalized by Kupffer cells rather than hepatocytes, and liver transduction was greatly reduced. PET reporter gene imaging after engineered AAV systemic injection provided a non-invasive method to monitor AAV-mediated protein expression over time. Through comparison with capsid tagging, differences between brain localization and transduction were revealed. In summary, AAV capsids bearing imaging tags and reporter gene payloads create a unique and powerful platform to assay the pharmacokinetics, cellular specificity and protein expression kinetics of AAV vectors in vivo, a key enabler for the field of gene therapy.

Improving plane wave ultrasound imaging through real-time beamformation across multiple arrays.

Ultrasound imaging is a widely used diagnostic tool but has limitations in the imaging of deep lesions or obese patients where the large depth to aperture size ratio (f-number) reduces image quality. Reducing the f-number can improve image quality, and in this work, we combined three commercial arrays to create a large imaging aperture of 100 mm and 384 elements. To maintain the frame rate given the large number of elements, plane wave imaging was implemented with all three arrays transmitting a coherent wavefront. On wire targets at a depth of 100 mm, the lateral resolution is significantly improved; the lateral resolution was 1.27 mm with one array (1/3 of the aperture) and 0.37 mm with the full aperture. After creating virtual receiving elements to fill the inter-array gaps, an autoregressive filter reduced the grating lobes originating from the inter-array gaps by - 5.2 dB. On a calibrated commercial phantom, the extended field-of-view and improved spatial resolution were verified. The large aperture facilitates aberration correction using a singular value decomposition-based beamformer. Finally, after approval of the Stanford Institutional Review Board, the three-array configuration was applied in imaging the liver of a volunteer, validating the potential for enhanced resolution.

Highly Integrated Multiplexing and Buffering Electronics for Large Aperture Ultrasonic Arrays.

Large aperture ultrasonic arrays can be implemented by tiling together multiple pretested modules of high-density acoustic arrays with closely integrated multiplexing and buffering electronics to form a larger aperture with high yield. These modular arrays can be used to implement large 1.75D array apertures capable of focusing in elevation for uniform slice thickness along the axial direction which can improve image contrast. An important goal for large array tiling is obtaining high yield and sensitivity while reducing extraneous image artifacts. We have been developing tileable acoustic-electric modules for the implementation of large array apertures utilizing Application Specific Integrated Circuits (ASICs) implemented using 0.35 µ m high voltage (50 V) CMOS. Multiple generations of ASICs have been designed and tested. The ASICs were integrated with high-density transducer arrays for acoustic testing and imaging. The modules were further interfaced to a Verasonics Vantage imaging system and were used to image industry standard ultrasound phantoms. The first-generation modules comprise ASICs with both multiplexing and buffering electronics on-chip and have demonstrated a switching artifact which was visible in the images. A second-generation ASIC design incorporates low switching injection circuits which effectively mitigate the artifacts observed with the first-generation devices. Here, we present the architecture of the two ASIC designs and module types as well imaging results that demonstrate reduction in switching artifacts for the second-generation devices.

A Review of Imaging Methods to Assess Ultrasound-Mediated Ablation.

Ultrasound ablation techniques are minimally invasive alternatives to surgical resection and have rapidly increased in use. The response of tissue to HIFU ablation differs based on the relative contributions of thermal and mechanical effects, which can be varied to achieve optimal ablation parameters for a given tissue type and location. In tumor ablation, similar to surgical resection, it is desirable to include a safety margin of ablated tissue around the entirety of the tumor. A factor in optimizing ablative techniques is minimizing the recurrence rate, which can be due to incomplete ablation of the target tissue. Further, combining focal ablation with immunotherapy is likely to be key for effective treatment of metastatic cancer, and therefore characterizing the impact of ablation on the tumor microenvironment will be important. Thus, visualization and quantification of the extent of ablation is an integral component of ablative procedures. The aim of this review article is to describe the radiological findings after ultrasound ablation across multiple imaging modalities. This review presents readers with a general overview of the current and emerging imaging methods to assess the efficacy of ultrasound ablative treatments.

A theranostic 3D ultrasound imaging system for high resolution image-guided therapy.

Microbubble contrast agents are a diagnostic tool with broad clinical impact and an increasing number of indications. Many therapeutic applications have also been identified. Yet, technologies for ultrasound guidance of microbubble-mediated therapy are limited. In particular, arrays that are capable of implementing and imaging microbubble-based therapy in three dimensions in real-time are lacking. We propose a system to perform and monitor microbubble-based therapy, capable of volumetric imaging over a large field-of-view. To propel the promise of the theranostic treatment strategies forward, we have designed and tested a unique array and system for 3D ultrasound guidance of microbubble-based therapeutic protocols based on the frequency, temporal and spatial requirements. Methods: Four 256-channel plane wave scanners (Verasonics, Inc, WA, USA) were combined to control a 1024-element planar array with 1.3 and 2.5 MHz therapeutic and imaging transmissions, respectively. A transducer aperture of ~40×15 mm was selected and Field II was applied to evaluate the point spread function. In vitro experiments were performed on commercial and custom phantoms to assess the spatial resolution, image contrast and microbubble-enhanced imaging capabilities. Results: We found that a 2D array configuration with 64 elements separated by λ-pitch in azimuth and 16 elements separated by 1.5λ-pitch in elevation ensured the required flexibility. This design, of 41.6 mm × 16 mm, thus provided both an extended field-of-view, up to 11 cm x 6 cm at 10 cm depth and steering of ±18° in azimuth and ±12° in elevation. At a depth of 16 cm, we achieved a volume imaging rate of 60 Hz, with a contrast ratio and resolution, respectively, of 19 dB, 0.8 mm at 3 cm and 20 dB and 2.1 mm at 12.5 cm. Conclusion: A single 2D array for both imaging and therapeutics, integrated with a 1024 channel scanner can guide microbubble-based therapy in volumetric regions of interest.

Alternative medicine: therapeutic effects on gastric original signet ring carcinoma via ascorbate and combination with sodium alpha lipoate.

BACKGROUND: Gastric signet ring cell carcinoma (SRCC) is an aggressive gastric adenocarcinoma with a poor prognosis when diagnosed at an advanced stage. As alternative medicine, two natural supplements (ascorbate (AA) and sodium alpha lipoate (LA)) have been shown to inhibit various cancers with mild side effects. METHODS: These two natural supplements and a series of combinations (AA&LA, AA+LA and LA + AA) were incubated with non-SRCC cells (GPM-1), patient-derived gastric origin SRCC (GPM-2), gastric-origin SRCCs (HSC-39 and KATO-3), human pancreatic (MIA PaCa-2) and ovarian (SKOV-3) cells for evaluating their therapeutic effects. Moreover, these treatments were applied in 3D-cultured organoids to reveal the feasibility of these approaches for in vivo study. RESULTS: Analyzing their antioxidant capabilities and dose-response curves, we observed that all four gastric cell lines, including three patient-derived cell lines were sensitive to ascorbate (~ 10 mM). The influence of ascorbate incubation time was studied, with a 16-h incubation found to be optimal for in vitro studies. Moreover, a simultaneous combination of AA and LA (AA&LA) did not significantly inhibit cell proliferation, while prior LA treatment increased the growth inhibition of AA therapy (LA + AA). Anti-cancer efficacy of AA was further confirmed in 3D-cultured SRCC (KATO-3) organoids. CONCLUSIONS: This study highlights the potential of AA and LA + AA in treating gastric origin SRCC, and demonstrates the influence of order in which the drugs are administered.

In situ T-cell transfection by anti-CD3-conjugated lipid nanoparticles leads to T-cell activation, migration, and phenotypic shift.

Ex vivo programming of T cells can be efficacious but is complex and expensive; therefore, the development of methods to transfect T cells in situ is important. We developed and optimized anti-CD3-targeted lipid nanoparticles (aCD3-LNPs) to deliver tightly packed, reporter gene mRNA specifically to T cells. In vitro, targeted LNPs efficiently delivered mCherry mRNA to Jurkat T cells, and T-cell activation and depletion were associated with aCD3 antibody coating on the surface of LNPs. aCD3-LNPs, but not non-targeted LNPs, accumulated within the spleen following systemic injection, with mCherry and Fluc signals visible within 30 min after injection. At 24 h after aCD3-LNP injection, 2-4% of all splenic T cells and 2-7% of all circulating T cells expressed mCherry, and this was dependent on aCD3 coating density. Targeting and transfection were accompanied by systemic CD25+, OX40+, and CD69+ T-cell activation with temporary CD3e ligand loss and depletion of splenic and circulating subsets. Migration of splenic CD8a+ T cells from the white-pulp to red-pulp, and differentiation from naïve to memory and effector phenotypes, followed upon aCD3-LNP delivery. Additionally, aCD3-LNP injection stimulated the secretion of myeloid-derived chemokines and T-helper cytokines into plasma. Lastly, we administered aCD3-LNPs to tumor bearing mice and found that transfected T cells localized within tumors and tumor-draining lymph nodes following immunotherapy treatment. In summary, we show that CD3-targeted transfection is feasible, yet associated with complex immunological consequences that must be further studied for potential therapeutic applications.

Erratum to "Highly Integrated Multiplexing and Buffering Electronics for Large Aperture Ultrasonic Arrays".

[This corrects the article DOI: 10.34133/2022/9870386.].