Ultrasonic High-Resolution Imaging and Acoustic Tweezers Using Ultrahigh Frequency Transducer: Integrative Single-Cell Analysis.

Ultrasound imaging is a highly valuable tool in imaging human tissues due to its non-invasive and easily accessible nature. Despite advances in the field of ultrasound research, conventional transducers with frequencies lower than 20 MHz face limitations in resolution for cellular applications. To address this challenge, we employed ultrahigh frequency (UHF) transducers and demonstrated their potential applications in the field of biomedical engineering, specifically for cell imaging and acoustic tweezers. The lateral resolution achieved with a 110 MHz UHF transducer was 20 µm, and 6.5 µm with a 410 MHz transducer, which is capable of imaging single cells. The results of our experiments demonstrated the successful imaging of a single PC-3 cell and a 15 µm bead using an acoustic scanning microscope equipped with UHF transducers. Additionally, the dual-mode multifunctional UHF transducer was used to trap and manipulate single cells and beads, highlighting its potential for single-cell studies in areas such as cell deformability and mechanotransduction.

Automated cell-type classification combining dilated convolutional neural networks with label-free acoustic sensing.

This study aimed to automatically classify live cells based on their cell type by analyzing the patterns of backscattered signals of cells with minimal effect on normal cell physiology and activity. Our previous studies have demonstrated that label-free acoustic sensing using high-frequency ultrasound at a high pulse repetition frequency (PRF) can capture and analyze a single object from a heterogeneous sample. However, eliminating possible errors in the manual setting and time-consuming processes when postprocessing integrated backscattering (IB) coefficients of backscattered signals is crucial. In this study, an automated cell-type classification system that combines a label-free acoustic sensing technique with deep learning-empowered artificial intelligence models is proposed. We applied an one-dimensional (1D) convolutional autoencoder to denoise the signals and conducted data augmentation based on Gaussian noise injection to enhance the robustness of the proposed classification system to noise. Subsequently, denoised backscattered signals were classified into specific cell types using convolutional neural network (CNN) models for three types of signal data representations, including 1D CNN models for waveform and frequency spectrum analysis and two-dimensional (2D) CNN models for spectrogram analysis. We evaluated the proposed system by classifying two types of cells (e.g., RBC and PNT1A) and two types of polystyrene microspheres by analyzing their backscattered signal patterns. We attempted to discover cell physical properties reflected on backscattered signals by controlling experimental variables, such as diameter and structure material. We further evaluated the effectiveness of the neural network models and efficacy of data representations by comparing their accuracy with that of baseline methods. Therefore, the proposed system can be used to classify reliably and precisely several cell types with different intrinsic physical properties for personalized cancer medicine development.

Manipulation and Mechanical Deformation of Leukemia Cells by High-Frequency Ultrasound Single Beam.

Ultrasound single-beam acoustic tweezer system has attracted increasing attention in the field of biomechanics. Cell biomechanics play a pivotal role in leukemia cell functions. To better understand and compare the cell mechanics of the leukemia cells, herein, we fabricated an acoustic tweezer system in-house connected with a 50-MHz high-frequency cylinder ultrasound transducer. Selected leukemia cells (Jurkat, K562, and MV-411 cells) were cultured, trapped, and manipulated by high-frequency ultrasound single beam, which was transmitted from the ultrasound transducer without contacting any cells. The relative deformability of each leukemia cell was measured, characterized, and compared, and the leukemia cell (Jurkat cell) gaining the highest deformability was highlighted. Our results demonstrate that the high-frequency ultrasound single beam can be utilized to manipulate and characterize leukemia cells, which can be applied to study potential mechanisms in the immune system and cell biomechanics in other cell types.

Automated estimation of cancer cell deformability with machine learning and acoustic trapping.

Cell deformability is a useful feature for diagnosing various diseases (e.g., the invasiveness of cancer cells). Existing methods commonly inflict pressure on cells and observe changes in cell areas, diameters, or thickness according to the degree of pressure. Then, the Young's moduli (i.e., a measure of deformability) of cells are estimated based on the assumption that the degrees of the changes are inversely proportional to Young's moduli. However, manual measurements of the physical changes in cells are labor-intensive, and the subjectivity of the operators can intervene during this step, thereby causing considerable uncertainty. Further, because the shapes of cells are nonuniform, we cannot ensure the assumption for linear correlations of physical changes in cells with their deformability. Therefore, this study aims at measuring non-linear elastic moduli of live cells (degrees of cell deformability) automatically by employing conventional neural networks (CNN) and multilayer perceptrons (MLP) while preserving (or enhancing) the accuracy of the manual methods. First, we obtain photomicrographs of cells on multiple pressure levels using single-beam acoustic tweezers, and then, we suggest an image preprocessing method for emphasizing changes in cell areas on the photomicrographs. The CNN model is trained to measure the ratios of the cell area change at each pressure level. Then, we apply the multilayer perceptron (MLP) to learn the correlations of the cell area change ratios according to the pressure levels with cell deformability. The accuracy of the CNN was evaluated using two types of breast cancer cells: MDA-MB-231 (invasive) and MCF-7 (noninvasive). The MLP was assessed using five different beads (Young's moduli from 0.214 to 9.235 kPa), which provides standardized reference data of the non-linear elastic moduli of live cells. Finally, we validated the practicality of the proposed system by examining whether the non-linear elastic moduli estimated by the proposed system can distinguish invasive breast cancer cells from noninvasive ones.

Recent Advancements in Ultrasound Transducer: From Material Strategies to Biomedical Applications.

Ultrasound is extensively studied for biomedical engineering applications. As the core part of the ultrasonic system, the ultrasound transducer plays a significant role. For the purpose of meeting the requirement of precision medicine, the main challenge for the development of ultrasound transducer is to further enhance its performance. In this article, an overview of recent developments in ultrasound transducer technologies that use a variety of material strategies and device designs based on both the piezoelectric and photoacoustic mechanisms is provided. Practical applications are also presented, including ultrasound imaging, ultrasound therapy, particle/cell manipulation, drug delivery, and nerve stimulation. Finally, perspectives and opportunities are also highlighted.

Noninvasive Ultrasound Retinal Stimulation for Vision Restoration at High Spatiotemporal Resolution.

Objective. Retinal degeneration involving progressive deterioration and loss of function of photoreceptors is a major cause of permanent vision loss worldwide. Strategies to treat these incurable conditions incorporate retinal prostheses via electrically stimulating surviving retinal neurons with implanted devices in the eye, optogenetic therapy, and sonogenetic therapy. Existing challenges of these strategies include invasive manner, complex implantation surgeries, and risky gene therapy. Methods and Results. Here, we show that direct ultrasound stimulation on the retina can evoke neuron activities from the visual centers including the superior colliculus and the primary visual cortex (V1), in either normal-sighted or retinal degenerated blind rats in vivo. The neuron activities induced by the customized spherically focused 3.1 MHz ultrasound transducer have shown both good spatial resolution of 250 µm and temporal resolution of 5 Hz in the rat visual centers. An additional customized 4.4 MHz helical transducer was further implemented to generate a static stimulation pattern of letter forms. Conclusion. Our findings demonstrate that ultrasound stimulation of the retina in vivo is a safe and effective approach with high spatiotemporal resolution, indicating a promising future of ultrasound stimulation as a novel and noninvasive visual prosthesis for translational applications in blind patients.

Super-Resolution Ultrasound Localization Microscopy for Visualization of the Ocular Blood Flow.

OBJECTIVE: The ocular vascular system plays an important role in preserving the visual function. Alterations in either anatomy or hemodynamics of the eye may have adverse effects on vision. Thus, an imaging approach that can monitor alterations of ocular blood flow of the deep eye vasculature ranging from capillary-level vessels to large supporting vessels would be advantageous for detection of early stage retinal and optic nerve diseases. METHODS: We propose a super-resolution ultrasound localization microscopy (ULM) technique that can assess both the microvessel and flow velocity of the deep eye with high resolution. Ultrafast plane wave imaging was acquired using an L22-14v linear array on a high frequency Verasonics Vantage system. A robust microbubble localization and tracking technique was applied to reconstruct ULM images. The experiment was first performed on pre-designed flow phantoms in vitro and then tested on a New Zealand white rabbit eye in vivo calibrated to various intraocular pressures (IOP) - 10 mmHg, 30 mmHg and 50 mmHg. RESULTS: We demonstrated that retinal/choroidal vessels, central retinal artery, posterior ciliary artery, and vortex vein were all visible at high resolution. In addition, reduction of vascular density and flow velocity were observed with elevated IOPs. CONCLUSION: These results indicate that super-resolution ULM is able to image the deep ocular tissue while maintaining high resolution that is comparable with optical coherence tomography angiography. SIGNIFICANCE: Capability to detect subtle changes of blood flow may be clinically important in detecting and monitoring eye diseases such as glaucoma.

Ultrasound Therapy, Chemotherapy and Their Combination for Prostate Cancer.

Prostate cancer is the second leading cause of cancer death in men. Its current treatment includes various physical and chemical approaches for the localized and advanced prostate cancer [e.g. metastatic castrate resistant prostate cancer (mCRPC)]. Although many new drugs are now available for prostate cancer, none is suitable for local treatment that can reduce adverse effects often associated with the current physical treatment. Of the drugs approved by FDA for mCRPC, the best mean improvement in overall survival is only about 4.8 months. Therefore, there is a need for improved treatment approaches for prostate cancer, especially drug-resistant cancer.Ultrasound therapy represents a useful new physical approach for the drug-resistant cancer treatment by facilitating the entry of the related chemotherapy drug into the target cancer cells. There are two versions of ultrasound: High Intensity Focused Ultrasound (HIFU) and Low Intensity Pulsed Ultrasound (LIPUS). HIFU has been a promising treatment option for prostate cancer due to its noninvasiveness and various biological effects on cancer tissue. It has been approved for the treatment of cancer and in recent years there have been numerous findings suggesting HIFU can reduce cancer cell viability and possibly reverse the spread of cancerous tumors. LIPUS is currently being studied as an alternative treatment option for prostate cancer. Preliminary studies have found LIPUS to reduce cancer cell viability without the side effects seen in HIFU. Reversible cell membrane damage caused by LIPUS could allow increased uptake of anticancer drugs, enhancing cytotoxicity and death of cancer cells. In this way, a low dose of anticancer drug is more effective toward cancer cells while there is less damage to normal cells. The combination of LIPUS with certain chemotherapeutic agents can be an exciting physical-chemical combination therapy for prostate cancer. This review will focus on this topic as well as the clinical use of HIFU to provide an understanding of their current use and future potential role for prostate cancer therapy.

Prospective assessment of breast cancer risk from multimodal multiview ultrasound images via clinically applicable deep learning.

The clinical application of breast ultrasound for the assessment of cancer risk and of deep learning for the classification of breast-ultrasound images has been hindered by inter-grader variability and high false positive rates and by deep-learning models that do not follow Breast Imaging Reporting and Data System (BI-RADS) standards, lack explainability features and have not been tested prospectively. Here, we show that an explainable deep-learning system trained on 10,815 multimodal breast-ultrasound images of 721 biopsy-confirmed lesions from 634 patients across two hospitals and prospectively tested on 912 additional images of 152 lesions from 141 patients predicts BI-RADS scores for breast cancer as accurately as experienced radiologists, with areas under the receiver operating curve of 0.922 (95% confidence interval (CI) = 0.868-0.959) for bimodal images and 0.955 (95% CI = 0.909-0.982) for multimodal images. Multimodal multiview breast-ultrasound images augmented with heatmaps for malignancy risk predicted via deep learning may facilitate the adoption of ultrasound imaging in screening mammography workflows.

2-D Ultrasonic Array-Based Optical Coherence Elastography.

Acoustic radiation force optical coherence elastography (ARF-OCE) has been successfully implemented to characterize the biomechanical properties of soft tissues, such as the cornea and the retina, with high resolution using single-element ultrasonic transducers for ARF excitation. Most currently proposed OCE techniques, such as air puff and ARF, have less capability to control the spatiotemporal information of the induced region of deformation, resulting in limited accuracy and low temporal resolution of the shear wave elasticity imaging. In this study, we propose a new method called 2-D ultrasonic array-based OCE imaging, which combines the advantages of 3-D dynamic electronic steering of the 2-D ultrasonic array and high-resolution optical coherence tomography (OCT). The 3-D steering capability of the 2-D array was first validated using a hydrophone. Then, the combined 2-D ultrasonic array OCE system was calibrated using a homogenous phantom, followed by an experiment on ex vivo rabbit corneal tissue. The results demonstrate that our newly developed 2-D ultrasonic array-based OCE system has the capability to map tissue biomechanical properties accurately, and therefore, has the potential to be a vital diagnostic tool in ophthalmology.

Focused Ultrasound Stimulates ER Localized Mechanosensitive PANNEXIN-1 to Mediate Intracellular Calcium Release in Invasive Cancer Cells.

Focused ultrasound (FUS) is a rapidly developing stimulus technology with the potential to uncover novel mechanosensory dependent cellular processes. Since it is non-invasive, it holds great promise for future therapeutic applications in patients used either alone or as a complement to boost existing treatments. For example, FUS stimulation causes invasive but not non-invasive cancer cell lines to exhibit marked activation of calcium signaling pathways. Here, we identify the membrane channel PANNEXIN1 (PANX1) as a mediator for activation of calcium signaling in invasive cancer cells. Knockdown of PANX1 decreases calcium signaling in invasive cells, while PANX1 overexpression enhances calcium elevations in non-invasive cancer cells. We demonstrate that FUS may directly stimulate mechanosensory PANX1 localized in endoplasmic reticulum to evoke calcium release from internal stores. This process does not depend on mechanosensory stimulus transduction through an intact cytoskeleton and does not depend on plasma membrane localized PANX1. Plasma membrane localized PANX1, however, plays a different role in mediating the spread of intercellular calcium waves via ATP release. Additionally, we show that FUS stimulation evokes cytokine/chemokine release from invasive cancer cells, suggesting that FUS could be an important new adjuvant treatment to improve cancer immunotherapy.

Characterizing the Motility of Chemotherapeutics-Treated Acute Lymphoblastic Leukemia Cells by Time-Lapse Imaging.

Drug resistance is an obstacle in the therapy of acute lymphoblastic leukemia (ALL). Whether the physical properties such as the motility of the cells contribute to the survival of ALL cells after drug treatment has recently been of increasing interest, as they could potentially allow the metastasis of solid tumor cells and the migration of leukemia cells. We hypothesized that chemotherapeutic treatment may alter these physical cellular properties. To investigate the motility of chemotherapeutics-treated B-cell ALL (B-ALL) cells, patient-derived B-ALL cells were treated with chemotherapy for 7 days and left for 12 h without chemotherapeutic treatment. Two parameters of motility were studied, velocity and migration distance, using a time-lapse imaging system. The study revealed that compared to non-chemotherapeutically treated B-ALL cells, B-ALL cells that survived chemotherapy treatment after 7 days showed reduced motility. We had previously shown that Tysabri and P5G10, antibodies against the adhesion molecules integrins α4 and α6, respectively, may overcome drug resistance mediated through leukemia cell adhesion to bone marrow stromal cells. Therefore, we tested the effect of integrin α4 or α6 blockade on the motility of chemotherapeutics-treated ALL cells. Only integrin α4 blockade decreased the motility and velocity of two chemotherapeutics-treated ALL cell lines. Interestingly, integrin α6 blockade did not affect the velocity of chemoresistant ALL cells. This study explores the physical properties of the movements of chemoresistant B-ALL cells and highlights a potential link to integrins. Further studies to investigate the underlying mechanism are warranted.

Investigation of Ultrasound-Mediated Intracellular Ca2+ Oscillations in HIT-T15 Pancreatic ß-Cell Line.

In glucose-stimulated insulin secretion (GSIS) of pancreatic ß-cells, the rise of free cytosolic Ca2+ concentration through voltage-gated calcium channels (VGCCs) triggers the exocytosis of insulin-containing granules. Recently, mechanically induced insulin secretion pathways were also reported, which utilize free cytosolic Ca2+ ions as a direct regulator of exocytosis. In this study, we aimed to investigate intracellular Ca2+ responses on the HIT-T15 pancreatic ß-cell line upon low-intensity pulsed ultrasound (LIPUS) stimulation and found that ultrasound induces two distinct types of intracellular Ca2+ oscillation, fast-irregular and slow-periodic, from otherwise resting cells. Both Ca2+ patterns depend on the purinergic signaling activated by the rise of extracellular ATP or ADP concentration upon ultrasound stimulation, which facilitates the release through mechanosensitive hemichannels on the plasma membrane. Further study demonstrated that two subtypes of purinergic receptors, P2X and P2Y, are working in a competitive manner depending on the level of glucose in the cell media. The findings can serve as an essential groundwork providing an underlying mechanism for the development of a new therapeutic approach for diabetic conditions with further validation.

Classification of Breast Cancer Cells Using the Integration of High-Frequency Single-Beam Acoustic Tweezers and Convolutional Neural Networks.

Single-beam acoustic tweezers (SBAT) is a widely used trapping technique to manipulate microscopic particles or cells. Recently, the characterization of a single cancer cell using high-frequency (>30 MHz) SBAT has been reported to determine its invasiveness and metastatic potential. Investigation of cell elasticity and invasiveness is based on the deformability of cells under SBAT's radiation forces, and in general, more physically deformed cells exhibit higher levels of invasiveness and therefore higher metastatic potential. However, previous imaging analysis to determine substantial differences in cell deformation, where the SBAT is turned ON or OFF, relies on the subjective observation that may vary and requires follow-up evaluations from experts. In this study, we propose an automatic and reliable cancer cell classification method based on SBAT and a convolutional neural network (CNN), which provides objective and accurate quantitative measurement results. We used a custom-designed 50 MHz SBAT transducer to obtain a series of images of deformed human breast cancer cells. CNN-based classification methods with data augmentation applied to collected images determined and validated the metastatic potential of cancer cells. As a result, with the selected optimizers, precision, and recall of the model were found to be greater than 0.95, which highly validates the classification performance of our integrated method. CNN-guided cancer cell deformation analysis using SBAT may be a promising alternative to current histological image analysis, and this pretrained model will significantly reduce the evaluation time for a larger population of cells.

In Vivo Visualization of Eye Vasculature Using Super-Resolution Ultrasound Microvessel Imaging.

OBJECTIVE: The choroidal vessels, which supply oxygen and nutrient to the retina, may play a pivotal role in eye disease pathogenesis such as diabetic retinopathy and glaucoma. In addition, the retrobulbar circulation that feeds the choroid shows an important pathophysiologic role in myopia and degenerative myopia. Owing to the light-absorbing retinal pigment epithelium (RPE) and optically opaque sclera, choroidal and retrobulbar vasculature were difficult to be observed using clinically accepted optical coherence tomography angiography (OCT-A) technique. Here, we have developed super-resolution ultrasound microvessel imaging technique to visualize the deep ocular vasculature. METHODS: An 18-MHz linear array transducer with compounding plane wave imaging technique and contrast agent - microbubble was implemented in this study. The centroid intensity of each microbubble was detected using image deconvolution algorithm with spatially variant point spread function, and then accumulated in successive frames in order to reconstruct microvasculature. The image deconvolution technique was first evaluated in a simulation study and experimental flow phantoms. The performance was then validated on normal rabbit eyes in vivo. RESULTS: The image deconvolution based super-resolution ultrasound microvessel imaging technique shows good performance on either simulation study or flow phantoms. In vivo rabbit eye study indicated that the micron-level choroidal and retrobulbar vessels around the optic nerve head were successfully reconstructed in multiple 2D views and 3D volume imaging. CONCLUSIONS: Our results demonstrate the capability of using super-resolution ultrasound microvessel imaging technique to image the microvasculature of the posterior pole of the eye. This efficient approach can potentially lead to a routinely performed diagnostic procedure in the field of ophthalmology.

A combined ultrasonic B-mode and color Doppler system for the classification of breast masses using neural network.

OBJECTIVES: To develop a dual-modal neural network model to characterize ultrasound (US) images of breast masses. MATERIALS AND METHODS: A combined US B-mode and color Doppler neural network model was developed to classify US images of the breast. Three datasets with breast masses were originally detected and interpreted by 20 experienced radiologists according to Breast Imaging-Reporting and Data System (BI-RADS) lexicon ((1) training set, 103212 masses from 45,433 + 12,519 patients. (2) held-out validation set, 2748 masses from 1197 + 395 patients. (3) test set, 605 masses from 337 + 78 patients). The neural network was first trained on training set. Then, the trained model was tested on a held-out validation set to evaluate agreement on BI-RADS category between the model and the radiologists. In addition, the model and a reader study of 10 radiologists were applied to the test set with biopsy-proven results. To evaluate the performance of the model in benign or malignant classifications, the receiver operating characteristic curve, sensitivities, and specificities were compared. RESULTS: The trained dual-modal model showed favorable agreement with the assessment performed by the radiologists (κ = 0.73; 95% confidence interval, 0.71-0.75) in classifying breast masses into four BI-RADS categories in the validation set. For the binary categorization of benign or malignant breast masses in the test set, the dual-modal model achieved the area under the ROC curve (AUC) of 0.982, while the readers scored an AUC of 0.948 in terms of the ROC convex hull. CONCLUSION: The dual-modal model can be used to assess breast masses at a level comparable to that of an experienced radiologist. KEY POINTS: • A neural network model based on ultrasonic imaging can classify breast masses into different Breast Imaging-Reporting and Data System categories according to the probability of malignancy. • A combined ultrasonic B-mode and color Doppler neural network model achieved a high level of agreement with the readings of an experienced radiologist and has the potential to automate the routine characterization of breast masses.

Investigation of cell mechanics using single-beam acoustic tweezers as a versatile tool for the diagnosis and treatment of highly invasive breast cancer cell lines: an in vitro study.

Advancements in diagnostic systems for metastatic cancer over the last few decades have played a significant role in providing patients with effective treatment by evaluating the characteristics of cancer cells. Despite the progress made in cancer prognosis, we still rely on the visual analysis of tissues or cells from histopathologists, where the subjectivity of traditional manual interpretation persists. This paper presents the development of a dual diagnosis and treatment tool using an in vitro acoustic tweezers platform with a 50 MHz ultrasonic transducer for label-free trapping and bursting of human breast cancer cells. For cancer cell detection and classification, the mechanical properties of a single cancer cell were quantified by single-beam acoustic tweezers (SBAT), a noncontact assessment tool using a focused acoustic beam. Cell-mimicking phantoms and agarose hydrogel spheres (AHSs) served to standardize the biomechanical characteristics of the cells. Based on the analytical comparison of deformability levels between the cells and the AHSs, the mechanical properties of the cells could be indirectly measured by interpolating the Young's moduli of the AHSs. As a result, the calculated Young's moduli, i.e., 1.527 kPa for MDA-MB-231 (highly invasive breast cancer cells), 2.650 kPa for MCF-7 (weakly invasive breast cancer cells), and 2.772 kPa for SKBR-3 (weakly invasive breast cancer cells), indicate that highly invasive cancer cells exhibited a lower Young's moduli than weakly invasive cells, which indicates a higher deformability of highly invasive cancer cells, leading to a higher metastasis rate. Single-cell treatment may also be carried out by bursting a highly invasive cell with high-intensity, focused ultrasound.

Integrin Antibody Decreases Deformability of Patient-Derived Pre-B Acute Lymphocytic Leukemia Cells as Measured by High-Frequency Acoustic Tweezers.

OBJECTIVES: This article reports a study of cell mechanics in patient-derived (primary) B-cell acute lymphocytic leukemia (ALL) cells treated with antibodies against integrins. Leukemia cell adhesion to stromal cells mediates chemotherapeutic drug resistance, also known as cell adhesion-mediated chemotherapeutic drug resistance. We have previously shown that antibodies against integrin α4 and α6 adhesion molecules can de-adhere ALL cells from stromal cells or counter-receptors. Because drug-resistant cells are more deformable, as evaluated by single-beam acoustic tweezers, we hypothesized that changes in cell mechanics might contribute to the de-adhesive effect of integrin-targeting antibodies. METHODS: In this study, the deformability of primary pre-B ALL cells was evaluated by single-beam acoustic tweezers after treatments with the de-adhering antibody Tysabri or P5G10 against integrin α4 and α6 adhesion molecules. RESULTS: We demonstrated that primary ALL cells treated with P5G10 expressed decreased deformability compared with immunoglobulin G1 -treated control cells (P < .05). Tysabri did not show an effect on deformability (P > .05). CONCLUSIONS: These results suggest that decreased deformability is associated with an integrin α6 blockade. Further assessments of the functional roles of deformability and integrin blockades in B-ALL cell drug resistance and deformability, respectively, are necessary.

A One-Sided Acoustic Trap for Cell Immobilization Using 30-MHz Array Transducer.

Biological studies often involve the investigation of immobilized (or trapped) particles and cells. Various trapping methods without touching, such as optical, magnetic, and acoustic tweezers, have been developed to trap small particles. Here, we present the manipulation of a single cell or multiple cells using ultrasound-array-based single-beam acoustic tweezers (UA-SBATs). In SBATs, only a one-sided tightly focused acoustic beam produces a high acoustic gradient force-a mechanism that mirrors that of optical tweezers. As a result, targeted cells can be attracted to the beam center and immobilized within its trapping zone. Since an array transducer allows acoustic beam steering and scanning electronically instead of mechanical translation, it can manipulate cells more simply and quickly compared with single-element transducers, especially in biocompatible setup. In this experiment, a customized 30-MHz array transducer with an interdigitally bonded (IB) 2-2 piezocomposite was employed to immobilize MCF-12F cells. Cells were attracted to the center of the beam and laterally displaced with the array transducer without any damages to the cells. These findings suggest that UA-SBAT can be a promising tool for cell manipulation and may pave the way for exploring new biological applications.

Monitoring of Adult Zebrafish Heart Regeneration Using High-Frequency Ultrasound Spectral Doppler and Nakagami Imaging.

This paper reports the feasibility of Nakagami imaging in monitoring the regeneration process of zebrafish hearts in a noninvasive manner. In addition, spectral Doppler waveforms that are typically used to access the diastolic function were measured to validate the performance of Nakagami imaging. A 30-MHz high-frequency ultrasound array transducer was used to acquire backscattered echo signal for spectral Doppler and Nakagami imaging. The performances of both methods were validated with flow and tissue-mimicking phantom experiments. For in vivo experiments, both spectral Doppler and Nakagami imaging were simultaneously obtained from adult zebrafish with amputated hearts. Longitudinal measurements were performed for five zebrafish. From the experiments, the E/A ratio measured using spectral Doppler imaging increased at 3 days post-amputation (3 dpa) and then decreased to the value before amputation, which were consistent with previous studies. Similar results were obtained from the Nakagami imaging where the Nakagami parameter value increased at 3 dpa and decreased to its original value. These results suggested that the Nakagami and spectral Doppler imaging would be useful techniques in monitoring the regeneration of heart or tissues.