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.

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.

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

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

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.

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.

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.

Co-Integrated PIN-PMN-PT 2-D Array and Transceiver Electronics by Direct Assembly Using a 3-D Printed Interposer Grid Frame.

Tiled modular 2-D ultrasound arrays have the potential for realizing large apertures for novel diagnostic applications. This work presents an architecture for fabrication of tileable 2-D array modules implemented using 1-3 composites of high-bandwidth (BW) PIN-PMN-PT single-crystal piezoelectric material closely coupled with high-voltage CMOS application-specific integrated circuit (ASIC) electronics for buffering and multiplexing functions. The module, which is designed to be operated as a λ -pitch 1.75-D array, benefits from an improved electromechanical coupling coefficient and increased Curie temperature and is assembled directly on top of the ASIC silicon substrate using an interposer backing. The interposer consists of a novel 3-D printed acrylic frame that is filled with conducting and acoustically absorbing silver epoxy material. The ASIC comprises a high-voltage switching matrix with locally integrated buffering and is interfaced to a Verasonics Vantage 128, using a local field programmable gate array (FPGA) controller. Multiple prototype 5 ×6 element array modules have been fabricated by this process. The combined acoustic array and ASIC module was configured electronically by programming the switches to operate as a 1-D array with elements grouped in elevation for imaging and pulse-echo testing. The resulting array configuration had an average center frequency of 4.55 MHz, azimuthal element pitch of [Formula: see text], and exhibited average -20-dB pulsewidth of 592 ns and average -6-dB fractional BW of 77%.

Fabrication and Characterization of a Miniaturized 15-MHz Side-Looking Phased-Array Transducer Catheter.

This paper describes the development of a miniaturized 15-MHz side-looking phased-array transducer catheter. The array features a 2-2 linear composite with 64 piezoelectric elements mechanically diced into a piece of PMN-30%PT single crystal and separated by non-conductive epoxy kerfs at a 50-µm pitch, yielding a total active aperture of 3.2 mm in the azimuth direction and 1.8 mm in the elevation direction, with an elevation natural focal depth of 8.1 mm. The array includes non-conductive epoxy backing and two front matching layers. A custom flexible circuit connects the array piezoelectric elements to a bundle of 64 individual 48-AWG micro-coaxial cables enclosed within a 1.5-m long 10F catheter. Performance characterization was evaluated via finite element analysis simulations and afterwards compared against obtained measurement results, which showed an average center frequency of 17.7 MHz, an average bandwidth of 52.2% at -6 dB, and crosstalk less than -30 dB. Imaging of a tungsten fine-wire phantom resulted in axial and lateral spatial resolutions of approximately 90 µm and 420 ìm, respectively. The imaging capability was further evaluated with colorectal tissue-mimicking phantoms, demonstrating the potential suitability of the proposed phased-array transducer for the intraoperative assessment of surgical margins during minimally invasive colorectal surgery procedures.

Characterizing Deformability of Drug Resistant Patient-Derived Acute Lymphoblastic Leukemia (ALL) Cells Using Acoustic Tweezers.

The role of cell mechanics in cancer cells is a novel research area that has resulted in the identification of new mechanisms of therapy resistance. Single beam acoustic (SBA) tweezers are a promising technology for the quantification of the mechanical phenotype of cells. Our previous study showed that SBA tweezers can be used to quantify the deformability of adherent breast cancer cell lines. The physical properties of patient-derived (primary) pre-B acute lymphoblastic leukemia (ALL) cells involved in chemotherapeutic resistance have not been widely investigated. Here, we demonstrate the feasibility of analyzing primary pre-B ALL cells from four cases using SBA tweezers. ALL cells showed increased deformability with increasing acoustic pressure of the SBA tweezers. Moreover, ALL cells that are resistant to chemotherapeutic drugs were more deformable than were untreated ALL cells. We demonstrated that SBA tweezers can quantify the deformability of nonadherent leukemia cells and discriminate this mechanical phenotype in chemotherapy-resistant leukemia cells in a contact- and label-free manner.

CMOS High-Voltage Analog 1-64 Multiplexer/Demultiplexer for Integrated Ultrasound Guided Breast Needle Biopsy.

Ultrasound guided needle biopsy is an important method for collection of breast cancer tissue. In this paper, we report on the design and testing of a high-voltage 1 to 64 Multiplexer/Demultiplexer (MUX/De-MUX) integrated circuit (IC) for ultrasound-guided breast biopsy applications implemented in a high-voltage CMOS process. The IC is intended to be incorporated inside the breast biopsy needle and is designed to fit inside the needle inner diameter of 2.38 mm. The MUX/De-MUX electronics are made up of three parts, including a low-voltage 6 to 64 decoder, a level shifter to convert from low voltage to high voltage, and analog high-voltage switches. Experimental results show a -3-dB bandwidth of over 70 MHz, Rds (on) of , -2.279-dB insertion loss, and -17.5-dB off isolation at 70 MHz with low-voltage input. Finally, we present results obtained via synthetic aperture imaging using the fabricated MUX/De-Mux device and a high-frequency ultrasound array. This device and technique hold promise for high-frequency imaging probes where a limited number of elements are used and the depth of penetration is short such as in breast biopsy and intravascular applications.

Low-Intensity Ultrasound Modulates Ca2+ Dynamics in Human Mesenchymal Stem Cells via Connexin 43 Hemichannel.

In recent years, ultrasound has gained attention in new biological applications due to its ability to induce specific biological responses at the cellular level. Although the biophysical mechanisms underlying the interaction between ultrasound and cells are not fully understood, many agree on a pivotal role of Ca2+ signaling through mechanotransduction pathways. Because Ca2+ regulates a vast range of downstream cellular processes, a better understanding of how ultrasound influences Ca2+ signaling could lead to new applications for ultrasound. In this study, we investigated the mechanism of ultrasound-induced Ca2+ mobilization in human mesenchymal stem cells using 47 MHz focused ultrasound to stimulate single cells at low intensities (~ 110 mW/cm2). We found that ultrasound exposure triggers opening of connexin 43 hemichannels on the plasma membrane, causing release of ATP into the extracellular space. That ATP then binds to G-protein-coupled P2Y1 purinergic receptors on the membrane, in turn activating phospholipase C, which evokes production of inositol trisphosphate and release of Ca2+ from intracellular stores.

Bias-Voltage Stabilizer for HVHF Amplifiers in VHF Pulse-Echo Measurement Systems.

The impact of high-voltage-high-frequency (HVHF) amplifiers on echo-signal quality is greater with very-high-frequency (VHF, ≥100 MHz) ultrasound transducers than with low-frequency (LF, ≤15 MHz) ultrasound transducers. Hence, the bias voltage of an HVHF amplifier must be stabilized to ensure stable echo-signal amplitudes. We propose a bias-voltage stabilizer circuit to maintain stable DC voltages over a wide input range, thus reducing the harmonic-distortion components of the echo signals in VHF pulse-echo measurement systems. To confirm the feasibility of the bias-voltage stabilizer, we measured and compared the deviations in the gain of the HVHF amplifier with and without a bias-voltage stabilizer. Between -13 and 26 dBm, the measured gain deviations of a HVHF amplifier with a bias-voltage stabilizer are less than that of an amplifier without a bias-voltage stabilizer. In order to confirm the feasibility of the bias-voltage stabilizer, we compared the pulse-echo responses of the amplifiers, which are typically used for the evaluation of transducers or electronic components used in pulse-echo measurement systems. From the responses, we observed that the amplitudes of the echo signals of a VHF transducer triggered by the HVHF amplifier with a bias-voltage stabilizer were higher than those of the transducer triggered by the HVHF amplifier alone. The second, third, and fourth harmonic-distortion components of the HVHF amplifier with the bias-voltage stabilizer were also lower than those of the HVHF amplifier alone. Hence, the proposed scheme is a promising method for stabilizing the bias voltage of an HVHF amplifier, and improving the echo-signal quality of VHF transducers.

Functional Assay of Cancer Cell Invasion Potential Based on Mechanotransduction of Focused Ultrasound.

Cancer cells undergo a number of biophysical changes as they transform from an indolent to an aggressive state. These changes, which include altered mechanical and electrical properties, can reveal important diagnostic information about disease status. Here, we introduce a high-throughput, functional technique for assessing cancer cell invasion potential, which works by probing for the mechanically excitable phenotype exhibited by invasive cancer cells. Cells are labeled with fluorescent calcium dye and imaged during stimulation with low-intensity focused ultrasound, a non-contact mechanical stimulus. We show that cells located at the focus of the stimulus exhibit calcium elevation for invasive prostate (PC-3 and DU-145) and bladder (T24/83) cancer cell lines, but not for non-invasive cell lines (BPH-1, PNT1A, and RT112/84). In invasive cells, ultrasound stimulation initiates a calcium wave that propagates from the cells at the transducer focus to other cells, over distances greater than 1 mm. We demonstrate that this wave is mediated by extracellular signaling molecules and can be abolished through inhibition of transient receptor potential channels and inositol trisphosphate receptors, implicating these proteins in the mechanotransduction process. If validated clinically, our technology could provide a means to assess tumor invasion potential in cytology specimens, which is not currently possible. It may therefore have applications in diseases such as bladder cancer, where cytologic diagnosis of tumor invasion could improve clinical decision-making.

Single-Beam Acoustic Trapping of Red Blood Cells and Polystyrene Microspheres in Flowing Red Blood Cell Saline and Plasma Suspensions.

Single-beam acoustic tweezers (SBATs) represent a new technology for particle and cell trapping. The advantages of SBATs are their deep penetration into tissues, reduction of tissue damage and ease of application to in vivo studies. The use of these tools for applications in drug delivery in vivo must meet the following conditions: large penetration depth, strong trapping force and tissue safety. A reasonable penetration depth for SBATs in the development of in vivo applications was established in a previous study conducted in water with zero velocity. However, capturing objects in flowing fluid can provide more meaningful results. In this study, we investigated the capability of SBATs to trap red blood cells (RBCs) and polystyrene microspheres in flowing RBC suspensions. Two different types of RBC suspension were prepared in this work: an RBC phosphate-buffered saline (PBS) suspension and an RBC plasma suspension. The results indicated that SBATs successfully trapped RBCs and polystyrene microspheres in a flowing RBC PBS suspension with an average steady velocity of 1.6 cm/s in a 2-mm-diameter polyimide. Furthermore, SBATs were found able to trap RBCs in a flowing RBC PBS suspension at speeds as high as 7.9 cm/s in a polyimide tube, which is higher than the velocity in capillaries (0.03 cm/s) and approaches the velocity in arterioles and venules. Moreover, the results also indicated that polystyrene microspheres can be trapped in an RBC plasma suspension, where aggregation is observed. This work represents a step forward in using this tool in actual in vivo experimentation.

Cell Deformation by Single-beam Acoustic Trapping: A Promising Tool for Measurements of Cell Mechanics.

We demonstrate a noncontact single-beam acoustic trapping method for the quantification of the mechanical properties of a single suspended cell with label-free. Experimentally results show that the single-beam acoustic trapping force results in morphological deformation of a trapped cell. While a cancer cell was trapped in an acoustic beam focus, the morphological changes of the immobilized cell were monitored using bright-field imaging. The cell deformability was then compared with that of a trapped polystyrene microbead as a function of the applied acoustic pressure for a better understanding of the relationship between the pressure and degree of cell deformation. Cell deformation was found to become more pronounced as higher pressure levels were applied. Furthermore, to determine if this acoustic trapping method can be exploited in quantifying the cell mechanics in a suspension and in a non-contact manner, the deformability levels of breast cancer cells with different degrees of invasiveness due to acoustic trapping were compared. It was found that highly-invasive breast cancer cells exhibited greater deformability than weakly-invasive breast cancer cells. These results clearly demonstrate that the single-beam acoustic trapping technique is a promising tool for non-contact quantitative assessments of the mechanical properties of single cells in suspensions with label-free.

Power Amplifier Linearizer for High Frequency Medical Ultrasound Applications.

Power amplifiers (PAs) are used to produce high-voltage excitation signals to drive ultrasonic transducers. A larger dynamic range of linear PAs allows higher contrast resolution, a highly desirable characteristic in ultrasonic imaging. The linearity of PAs can be improved by reducing the nonlinear harmonic distortion components of high-voltage output signals. In this paper, a linearizer circuit is proposed to reduce output signal harmonics when working in conjunction with a PA. The PA performance with and without the linearizer was measured by comparing the output power 1-dB compression point (OP1dB), and the second- and third-order harmonic distortions (HD2 and HD3, respectively). The results show that the PA with the linearizer circuit had higher OP1dB (31.7 dB) and lower HD2 (-61.0 dB) and HD3 (-42.7 dB) compared to those of the PA alone (OP1dB (27.1 dB), HD2 (-38.2 dB), and HD3 (-36.8 dB)) at 140 MHz. A pulse-echo measurement was also performed to further evaluate the capability of the linearizer circuit. The HD2 of the echo signal received by the transducer using a PA with the linearizer (-24.8 dB) was lower than that obtained for the PA alone (-16.6 dB). The linearizer circuit is capable of improving the linearity performance of PA by lowering harmonic distortions.

Ultrasonic scattering measurements of a live single cell at 86 MHz.

Cell separation and sorting techniques have been employed biomedical applications such as cancer diagnosis and cell gene expression analysis. The capability to accurately measure ultrasonic scattering properties from cells is crucial in making an ultrasonic cell sorter a reality if ultrasound scattering is to be used as the sensing mechanism as well. To assess the performance of sensing and identifying live single cells with high-frequency ultrasound, an 86-MHz lithium niobate press-focused single-element acoustic transducer was used in a high-frequency ultrasound scattering measurement system that was custom designed and developed for minimizing noise and allowing better mobility. Peak-to-peak echo amplitude, integrated backscatter (IB) coefficient, spectral parameters including spectral slope and intercept, and midband fit from spectral analysis of the backscattered echoes were measured and calculated from a live single cell of two different types on an agar surface: leukemia cells (K562 cells) and red blood cells (RBCs). The amplitudes of echo signals from K562 cells and RBCs were 48.25 ± 11.98 mV(pp) and 56.97 ± 7.53 mV(pp), respectively. The IB coefficient was -89.39 ± 2.44 dB for K562 cells and -89.00 ± 1.19 dB for RBCs. The spectral slope and intercept were 0.30 ± 0.19 dB/MHz and -56.07 ± 17.17 dB, respectively, for K562 cells and 0.78 ± 0.092 dB/MHz and -98.18 ± 8.80 dB, respectively, for RBCs. Midband fits of K562 cells and RBCs were -31.02 ± 3.04 dB and -33.51 ± 1.55 dB, respectively. Acoustic cellular discrimination via these parameters was tested by Student's t-test. Their values, except for the IB value, showed statistically significant difference (p < 0.001). This paper reports for the first time that ultrasonic scattering measurements can be made on a live single cell with a highly focused high-frequency ultrasound microbeam at 86 MHz. These results also suggest the feasibility of ultrasonic scattering as a sensing mechanism in the development of ultrasonic cell sorters.

High-resolution harmonic motion imaging (HR-HMI) for tissue biomechanical property characterization.

BACKGROUND: Elastography, capable of mapping the biomechanical properties of biological tissues, serves as a useful technique for clinicians to perform disease diagnosis and determine stages of many diseases. Many acoustic radiation force (ARF) based elastography, including acoustic radiation force impulse (ARFI) imaging and harmonic motion imaging (HMI), have been developed to remotely assess the elastic properties of tissues. However, due to the lower operating frequencies of these approaches, their spatial resolutions are insufficient for revealing stiffness distribution on small scale applications, such as cancerous tumor margin detection, atherosclerotic plaque composition analysis and ophthalmologic tissue characterization. Though recently developed ARF-based optical coherence elastography (OCE) methods open a new window for the high resolution elastography, shallow imaging depths significantly limit their usefulness in clinics. METHODS: The aim of this study is to develop a high-resolution HMI method to assess the tissue biomechanical properties with acceptable field of view (FOV) using a 4 MHz ring transducer for efficient excitation and a 40 MHz needle transducer for accurate detection. Under precise alignment of two confocal transducers, the high-resolution HMI system has a lateral resolution of 314 µm and an axial resolution of 147 µm with an effective FOV of 2 mm in depth. RESULTS: The performance of this high resolution imaging system was validated on the agar-based tissue mimicking phantoms with different stiffness distributions. These data demonstrated the imaging system's improved resolution and sensitivity on differentiating materials with varying stiffness. In addition, ex vivo imaging of a human atherosclerosis coronary artery demonstrated the capability of high resolution HMI in identifying layer-specific structures and characterizing atherosclerotic plaques based on their stiffness differences. CONCLUSIONS: All together high resolution HMI appears to be a promising ultrasound-only technology for characterizing tissue biomechanical properties at the microstructural level to improve the image-based diseases diagnosis in multiple clinical applications.

Pulse electrodeposition of Ni-W alloy for trench filling in microelectromechanical systems.

The effect of pulse current (PC) on Ni-W alloy electrodeposition was investigated to fill the trenches with aspect ratio 1:2 for MEMS applications. Rapid deposition of top side in trench produced a void in electrodeposits with direct current (DC). Morphology of Ni-W electrodeposition inside trench was strongly influenced by the applied current density and the current off-time (t(off)). Enhanced filling phenomena were observed with increasing current off-time because of decreasing concentration gradient between top and bottom in trench. The complete Ni-W filling of trench was achieved with a current density of 200 mA/cm2 and a current on- and off-time of 100 ms and 300 ms, respectively. Amorphous structure was revealed in both DC and PC electrodeposits and the concentration of W in Ni-W electrodeposits for complete filling of trench was about 24% in atomic ratio.