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.

Mechanogenetics for the remote and noninvasive control of cancer immunotherapy.

While cell-based immunotherapy, especially chimeric antigen receptor (CAR)-expressing T cells, is becoming a paradigm-shifting therapeutic approach for cancer treatment, there is a lack of general methods to remotely and noninvasively regulate genetics in live mammalian cells and animals for cancer immunotherapy within confined local tissue space. To address this limitation, we have identified a mechanically sensitive Piezo1 ion channel (mechanosensor) that is activatable by ultrasound stimulation and integrated it with engineered genetic circuits (genetic transducer) in live HEK293T cells to convert the ultrasound-activated Piezo1 into transcriptional activities. We have further engineered the Jurkat T-cell line and primary T cells (peripheral blood mononuclear cells) to remotely sense the ultrasound wave and transduce it into transcriptional activation for the CAR expression to recognize and eradicate target tumor cells. This approach is modular and can be extended for remote-controlled activation of different cell types with high spatiotemporal precision for therapeutic applications.

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.

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.

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.

High-Resolution Shear Wave Imaging of the Human Cornea Using a Dual-Element Transducer.

Estimating the corneal elasticity can provide valuable information for corneal pathologies and treatments. Ophthalmologic pathologies will invariably cause changes to the elasticity of the cornea. For example, keratoconus and the phototoxic effects of ultraviolet radiation usually increase the corneal elasticity. This makes a quantitative estimation of the elasticity of the human cornea important for ophthalmic diagnoses. The present study investigated the use of a proposed high-resolution shear wave imaging (HR-SWI) method based on a dual-element transducer (comprising an 8-MHz element for pushing and a 32-MHz element for imaging) for measuring the group shear wave velocity (GSWV) of the human cornea. An empirical Young's modulus formula was used to accurately convert the GSWV to Young's modulus. Four quantitative parameters, bias, resolution, contrast, and contrast-to-noise ratio (CNR), were measured in gelatin phantoms with two different concentrations (3% and 7%) to evaluate the performance of HR-SWI. The biases of gelatin phantoms (3% and 7%) were 5.88% and 0.78%, respectively. The contrast and CNR were 0.76, 1.31 and 3.22, 2.43 for the two-side and two-layer phantoms, respectively. The measured image resolutions of HR-SWI in the lateral and axial directions were 72 and 140 µm, respectively. The calculated phase SWV (PSWV) and their corresponding Young's modulus from six human donors were 2.45 ± 0.48 m/s (1600 Hz) and 11.52 ± 7.81 kPa, respectively. All the experimental results validated the concept of HR-SWI and its ability for measuring the human corneal elasticity.

An adjustable multi-scale single beam acoustic tweezers based on ultrahigh frequency ultrasonic transducer.

This paper reports the fabrication, characterization, and microparticle manipulation capability of an adjustable multi-scale single beam acoustic tweezers (SBAT) that is capable of flexibly changing the size of "tweezers" like ordinary metal tweezers with a single-element ultrahigh frequency (UHF) ultrasonic transducer. The measured resonant frequency of the developed transducer at 526 MHz is the highest frequency of piezoelectric single crystal based ultrasonic transducers ever reported. This focused UHF ultrasonic transducer exhibits a wide bandwidth (95.5% at -10 dB) due to high attenuation of high-frequency ultrasound wave, which allows the SBAT effectively excite with a wide range of excitation frequency from 150 to 400 MHz by using the "piezoelectric actuator" model. Through controlling the excitation frequency, the wavelength of ultrasound emitted from the SBAT can be changed to selectively manipulate a single microparticle of different sizes (3-100 µm) by using only one transducer. This concept of flexibly changing "tweezers" size is firstly introduced into the study of SBAT. At the same time, it was found that this incident ultrasound wavelength play an important role in lateral trapping and manipulation for microparticle of different sizes. Biotechnol. Bioeng. 2017;114: 2637-2647. © 2017 Wiley Periodicals, Inc.

Acoustic Radiation Force Optical Coherence Elastography of Corneal Tissue.

We report on a real-time acoustic radiation force optical coherence elastography (ARF-OCE) system to map the relative elasticity of corneal tissue. A modulated ARF is used as excitation to vibrate the cornea while OCE serves as detection of tissue response. To show feasibility of detecting mechanical contrast using this method, we performed tissue-equivalent agarose phantom studies with inclusions of a different stiffness. We obtained 3-D elastograms of a healthy cornea and a highly cross-linked cornea. Finally we induced a stiffness change on a small portion of a cornea and observed the differences in displacement.

A Review of Intravascular Ultrasound-based Multimodal Intravascular Imaging: The Synergistic Approach to Characterizing Vulnerable Plaques.

Catheter-based intravascular imaging modalities are being developed to visualize pathologies in coronary arteries, such as high-risk vulnerable atherosclerotic plaques known as thin-cap fibroatheroma, to guide therapeutic strategy at preventing heart attacks. Mounting evidences have shown three distinctive histopathological features-the presence of a thin fibrous cap, a lipid-rich necrotic core, and numerous infiltrating macrophages-are key markers of increased vulnerability in atherosclerotic plaques. To visualize these changes, the majority of catheter-based imaging modalities used intravascular ultrasound (IVUS) as the technical foundation and integrated emerging intravascular imaging techniques to enhance the characterization of vulnerable plaques. However, no current imaging technology is the unequivocal "gold standard" for the diagnosis of vulnerable atherosclerotic plaques. Each intravascular imaging technology possesses its own unique features that yield valuable information although encumbered by inherent limitations not seen in other modalities. In this context, the aim of this review is to discuss current scientific innovations, technical challenges, and prospective strategies in the development of IVUS-based multi-modality intravascular imaging systems aimed at assessing atherosclerotic plaque vulnerability.

Transparent Lead lanthanum zirconate titanate (PLZT) ceramic fibers for High-frequency Ultrasonic Transducer Applications.

This paper presents fabrication of transparent lanthanum zirconate titanate (PLZT) fibers using extrusion technique. The diameter of the sintered PLZT fiber is about 400-µm, and the fibers exhibit very good transparency. Measured dielectric constant, remnant polarization and coercive field of PLZT fiber were found to be 2340, 22.5-µC/cm2, and 9.8-kV/cm, respectively. The transparent piezoelectric materials may exhibit great potential for Photoacoustic (PA) imaging and hybrid intravascular imaging combining OCT and ultrasound imaging by using the transparent fiber as the path of light propagation and ultrasonic transducer material. In our study, these transparent PLZT fibers were designed to fabricate two types of high-frequency ultrasonic transducers: small aperture single PLZT fiber/epoxy composite and large aperture 1-3 PLZT fiber/epoxy composite ultrasonic transducers. Besides, a 20-µm tungsten wire phantom and the cornea of the porcine eye were also imaged with the 1-3 PLZT fiber/epoxy composite ultrasonic transducer to demonstrate its imaging capability.

Jitter reduction technique for acoustic radiation force impulse microscopy via photoacoustic detection.

We demonstrate a jitter noise reduction technique for acoustic radiation force impulse microscopy via photoacoustic detection (PA-ARFI), which promises to be capable of measuring cell mechanics. To reduce the jitter noise induced by Q-switched pulsed laser operated at high repetition frequency, photoacoustic signals from the surface of an ultrasound transducer are aligned by cross-correlation and peak-to-peak detection, respectively. Each method is then employed to measure the displacements of a target sample in an agar phantom and a breast cancer cell due to ARFI application, followed by the quantitative comparison between their performances. The suggested methods for PA-ARFI significantly reduce jitter noises, thus allowing us to measure displacements of a target cell due to ARFI application by less than 3 µm.

Imaging and characterizing shear wave and shear modulus under orthogonal acoustic radiation force excitation using OCT Doppler variance method.

We report on a novel acoustic radiation force orthogonal excitation optical coherence elastography (ARFOE-OCE) technique for imaging shear wave and quantifying shear modulus under orthogonal acoustic radiation force (ARF) excitation using the optical coherence tomography (OCT) Doppler variance method. The ARF perpendicular to the OCT beam is produced by a remote ultrasonic transducer. A shear wave induced by ARF excitation propagates parallel to the OCT beam. The OCT Doppler variance method, which is sensitive to the transverse vibration, is used to measure the ARF-induced vibration. For analysis of the shear modulus, the Doppler variance method is utilized to visualize shear wave propagation instead of Doppler OCT method, and the propagation velocity of the shear wave is measured at different depths of one location with the M scan. In order to quantify shear modulus beyond the OCT imaging depth, we move ARF to a deeper layer at a known step and measure the time delay of the shear wave propagating to the same OCT imaging depth. We also quantitatively map the shear modulus of a cross-section in a tissue-equivalent phantom after employing the B scan.

Angled-focused 45 MHz PMN-PT single element transducer for intravascular ultrasound imaging.

A transducer with an angled and focused aperture for intravascular ultrasound imaging has been developed. The acoustic stack for the angled-focused transducer was made of PMN-PT single crystal with one matching layer, one protective coating layer, and a highly damped backing layer. It was then press-focused to a desired focal length and inserted into a thin needle housing with an angled tip. A transducer with an angled and unfocused aperture was also made, following the same fabrication procedure, to compare the performance of the two transducers. The focused and unfocused transducers were tested to measure their center frequencies, bandwidths, and spatial resolutions. Lateral resolution of the angled-focused transducer (AFT) improved more than two times compared to that of the angled-unfocused transducer (AUT). A tissue-mimicking phantom in water and a rabbit aorta tissue sample in rabbit blood were scanned using AFT and AUT. Imaging with AFT offered improved contrast, over imaging with AUT, of the tissue-mimicking phantom and the rabbit aorta tissue sample by 23 dB and 8 dB, respectively. The results show that AFT has strong potential to provide morphological and pathological information of coronary arteries with high resolution and high contrast.

A sidelobe suppressing near-field beamforming approach for ultrasound array imaging.

A method is proposed to suppress sidelobe level for near-field beamforming in ultrasound array imaging. An optimization problem is established, and the second-order cone algorithm is used to solve the problem to obtain the weight vector based on the near-field response vector of a transducer array. The weight vector calculation results show that the proposed method can be used to suppress the sidelobe level of the near-field beam pattern of a transducer array. Ultrasound images following the application of weight vector to the array of a wire phantom are obtained by simulation with the Field II program, and the images of a wire phantom and anechoic sphere phantom are obtained experimentally with a 64-element 26 MHz linear phased array. The experimental and simulation results agree well and show that the proposed method can achieve a much lower sidelobe level than the conventional delay and sum beamforming method. The wire phantom image is demonstrated to focus much better and the contrast of the anechoic sphere phantom image improved by applying the proposed beamforming method.

Modification of microstructure on PZT films for ultrahigh frequency transducer.

Porous Lead zirconate titanate (PZT) films may have promising applications in high frequency ultrasonic transducers for their capability to modify electrical properties for better electrical and acoustic matching. In this work, porous PZT films in range of several micrometers were fabricated using a chemical solution deposition (CSD) method modified with polyvinylpyrrolidone (PVP) as a pore-foaming agent. The crystalline phase, microstructure and electrical properties of the porous films were investigated as a function of PVP contents, molecular weights and annealing temperatures. It was found that the electrical properties were closely associated with the porosity.

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.

Piezoelectric single crystals for ultrasonic transducers in biomedical applications.

Piezoelectric single crystals, which have excellent piezoelectric properties, have extensively been employed for various sensors and actuators applications. In this paper, the state-of-art in piezoelectric single crystals for ultrasonic transducer applications is reviewed. Firstly, the basic principles and design considerations of piezoelectric ultrasonic transducers will be addressed. Then, the popular piezoelectric single crystals used for ultrasonic transducer applications, including LiNbO3 (LN), PMN-PT and PIN-PMN-PT, will be introduced. After describing the preparation and performance of the single crystals, the recent development of both the single-element and array transducers fabricated using the single crystals will be presented. Finally, various biomedical applications including eye imaging, intravascular imaging, blood flow measurement, photoacoustic imaging, and microbeam applications of the single crystal transducers will be discussed.

Fully motorized optical-resolution photoacoustic microscopy.

We have developed fully motorized optical-resolution photoacoustic microscopy (OR-PAM), which integrates five complementary scanning modes and simultaneously provides a high imaging speed and a wide field of view (FOV) with 2.6 µm lateral resolution. With one-dimensional (1D) motion-mode mechanical scanning, we measured the blood flow through a cross section of a blood vessel in vivo. With two-dimensional (2D) optical scanning at a laser repetition rate of 40 kHz, we achieved a 2 kHz B-scan rate over a range of 50 µm with 20 A-lines and 50 Hz volumetric-scan rate over a FOV of 50 µm × 50 µm with 400 A-lines, which enabled real-time tracking of cellular dynamics in vivo. With synchronized 1D optical and 2D mechanical hybrid scanning, we imaged a 10 mm × 8 mm FOV within three minutes, which is 20 times faster than the conventional mechanical scan in our second-generation OR-PAM. With three-dimensional mechanical contour scanning, we maintained the optimal signal-to-noise ratio and spatial resolution of OR-PAM while imaging objects with uneven surfaces, which is essential for quantitative studies.

Trimodality imaging system and intravascular endoscopic probe: combined optical coherence tomography, fluorescence imaging and ultrasound imaging.

In this Letter, we present a trimodality imaging system and an intravascular endoscopic probe for the detection of early-stage atherosclerotic plaques. The integrated system is able to acquire optical coherence tomography (OCT), fluorescence, and ultrasound images and simultaneously display them in real time. A trimodality intravascular endoscopic probe of 1.2 mm in diameter and 7 mm in length was fabricated based on a dual-modality optical probe that integrates OCT and fluorescence imaging functions and a miniature ultrasound transducer. The probe is capable of rotating at up to 600 rpm. Ex vivo images from rabbit aorta and human coronary arteries showed that this combined system is capable of providing high resolution, deep penetration depth and specific molecular fluorescence contrast simultaneously.

Urogenital photoacoustic endoscope.

Photoacoustic (PA) endoscopy for human urogenital imaging has the potential to diagnose many important diseases, such as endometrial and prostate cancers. We have specifically developed a 12.7 mm diameter, rigid, side-scanning PA endoscopic probe for such applications. The key features of this endoscope are the streamlined structure for smooth cavity introduction and the proximal actuation mechanism for fast scanning. Here we describe the probe's composition and scanning mechanism and present in vivo experimental results suggesting its potential for comprehensive clinical applications.