Acoustic radiation force-induced longitudinal shear wave for ultrasound-based viscoelastic evaluation.

Acoustic radiation force (ARF) is widely used to induce shear waves for evaluating the mechanical properties of biological tissues. Two shear waves can be generated when exciting with ARF: a transverse shear wave, also simply called shear wave (SW), and a longitudinal shear wave (LSW). Shear waves (SWs) have been broadly used to assess the mechanical properties. Some articles have reported that the LSW can be used to evaluate mechanical properties locally. However, existing LSW studies are mainly focused on the group velocity evaluation using optical coherence tomography (OCT). Here, we report that a LSW generated with ARF can be used to probe viscoelastic properties, including shear modulus and viscosity, using ultrasound. We took advantage of the surface boundary effect to reflect the LSW, named RLSW, to address the energy deficiency of LSW induced by ARF. We systematically evaluated the experiments with tissue-mimicking viscoelastic phantoms and validated by numerical simulations. Phase velocity and dispersion comparison between the results induced by a RLSW and a SW exhibit good agreement in both the numerical simulations and experimental results. The Kelvin-Voigt (KV) model was used to determine the shear modulus and viscosity. RLSW shows great potential to evaluate localized viscoelastic properties, which could benefit various biomedical applications such as evaluating the viscoelasticity of heterogeneous materials or microscopic lesions of tissues.

Wearable bioadhesive ultrasound shear wave elastography.

Acute liver failure (ALF) is a critical medical condition defined as the rapid development of hepatic dysfunction. Conventional ultrasound elastography cannot continuously monitor liver stiffness over the course of rapidly changing diseases for early detection due to the requirement of a handheld probe. In this study, we introduce wearable bioadhesive ultrasound elastography (BAUS-E), which can generate acoustic radiation force impulse (ARFI) to induce shear waves for the continuous monitoring of modulus changes. BAUS-E contains 128 channels with a compact design with only 24 mm in the azimuth direction for comfortable wearability. We further used BAUS-E to continuously monitor the stiffness of in vivo rat livers with ALF induced by d-galactosamine over 48 hours, and the stiffness change was observed within the first 6 hours. BAUS-E holds promise for clinical applications, particularly in patients after organ transplantation or postoperative care in the intensive care unit (ICU).

Acoustic Force Elastography Microscopy.

OBJECTIVE: Hydrogel scaffolds have attracted attention to develop cellular therapy and tissue engineering platforms for regenerative medicine applications. Among factors, local mechanical properties of scaffolds drive the functionalities of cell niche. Dynamic mechanical analysis (DMA), the standard method to characterize mechanical properties of hydrogels, restricts development in tissue engineering because the measurement provides a single elasticity value for the sample, requires direct contact, and represents a destructive evaluation preventing longitudinal studies on the same sample. We propose a novel technique, acoustic force elastography microscopy (AFEM), to evaluate elastic properties of tissue engineering scaffolds. RESULTS: AFEM can resolve localized and two-dimensional (2D) elastic properties of both transparent and opaque materials with advantages of being non-contact and non-destructive. Gelatin hydrogels, neat synthetic oligo[poly(ethylene glycol)fumarate] (OPF) scaffolds, OPF hydroxyapatite nanocomposite scaffolds and ex vivo biological tissue were examined with AFEM to evaluate the elastic modulus. These measurements of Young's modulus range from approximately 2 kPa to over 100 kPa were evaluated and are in good agreement with finite element simulations, surface wave measurements, and DMA tests. CONCLUSION: The AFEM can resolve localized and 2D elastic properties of hydrogels, scaffolds and thin biological tissues. These materials can either be transparent or non-transparent and their evaluation can be done in a non-contact and non-destructive manner, thereby facilitating longitudinal evaluation. SIGNIFICANCE: AFEM is a promising technique to quantify elastic properties of scaffolds for tissue engineering and will be applied to provide new insights for exploring elastic changes of cell-laden scaffolds for tissue engineering and material science.

Measurement of wave propagation through a tube using dual transducers for elastography in arteries.

Objective.Measuring waves induced with acoustic radiation force (ARF) in arteries has been studied over the last decade. To date, it remains a challenge to quantitatively assess the local arterial biomechanical properties. The cylindrical shape and waveguide behavior of waves propagating in the arterial wall pose complexities to determining the mechanical properties of the artery.Approach. In this paper, an artery-mimicking tube in water is examined utilizing three-dimensional measurements. The cross-section of the tube is measured while a transducer is translated over 41 different positions along the length of the tube. Motion in the radial direction is calculated using two components of motion which are measured from the two orthogonal views of the cross-section. This enables more accurate estimation of motion along the circumference of tube.Main results. The results provide more information to categorize the motion in tube wall into two types of responses: a transient response and a steady state response. The transient response is caused by ARF application and the waves travel along the length of the tube for a relatively short period of time. This corresponds to the axial and circumferential propagating waves. The two circumferential waves travel along the circumference of tube in CW (clockwise) and CCW (counter-clockwise) direction and result in a standing wave. By using a directional filter, the two waves were successfully separated, and their propagation was more clearly visualized. As a steady state response, a circumferential mode is generated showing a symmetric motion (i.e. the proximal and distal walls move in the opposite direction) following the transient response.Significance.This study provides a more comprehensive understanding of the waves produced in an artery-mimicking tube with ARF application, which will provide opportunities for improving measurement of arterial mechanical properties.

Bioadhesive ultrasound for long-term continuous imaging of diverse organs.

Continuous imaging of internal organs over days could provide crucial information about health and diseases and enable insights into developmental biology. We report a bioadhesive ultrasound (BAUS) device that consists of a thin and rigid ultrasound probe robustly adhered to the skin via a couplant made of a soft, tough, antidehydrating, and bioadhesive hydrogel-elastomer hybrid. The BAUS device provides 48 hours of continuous imaging of diverse internal organs, including blood vessels, muscle, heart, gastrointestinal tract, diaphragm, and lung. The BAUS device could enable diagnostic and monitoring tools for various diseases.

In vitro and in vivo effects of AVA4746, a novel competitive antagonist of the ligand binding of VLA-4, in B-cell acute lymphoblastic leukemia.

Treatment of resistant or recurrent acute lymphoblastic leukemia (ALL) remains a challenge. It was previously demonstrated that the adhesion molecule integrin α4, referred to hereafter as α4, mediates the cell adhesion-mediated drug resistance (CAM-DR) of B-cell ALL by binding to vascular cell adhesion molecule-1 (VCAM-1) on bone marrow stroma. In addition, it was previously observed that the blockade of α4 with natalizumab or inhibition using the small molecule antagonist TBC3486 sensitized relapsed ALL cells to chemotherapy. However, α4-targeted therapy is not clinically available for the treatment of leukemia to date. In the present study, the use of a novel non-peptidic small molecule integrin α4 antagonist, AVA4746, as a potential new approach to combat drug-resistant B-ALL was explored. An in vitro co-culture = model of primary B-ALL cells and an in vivo xenograft model of patient-derived B-ALL cells were utilized for evaluation of AVA4746. VLA-4 conformation activation, cell adhesion/de-adhesion, endothelial tube formation, in vivo leukemia cell mobilization and survival assays were performed. AVA4746 exhibited high affinity for binding to B-ALL cells, where it also efficiently blocked ligand-binding to VCAM-1. In addition, AVA4746 caused the functional de-adhesion of primary B-ALL cells from VCAM-1. Inhibition of α4 using AVA4746 also prevented angiogenesis in vitro and when applied in combination with chemotherapy consisting of Vincristine, Dexamethasone and L-asparaginase, it prolonged the survival of ~33% of the mice in an in vivo xenograft model of B-ALL. These data implicate the potential of targeting the α4-VCAM-1 interaction using AVA4746 for the treatment of drug-resistant B-lineage ALL.

Evaluation of Robustness of Local Phase Velocity Imaging in Homogenous Tissue-Mimicking Phantoms.

Shear wave elastography (SWE) is a method of evaluating mechanical properties of soft tissues. Most current implementations of SWE report the group velocity for shear wave velocity, which assumes an elastic, isotropic, homogenous and incompressible tissue. Local phase velocity imaging (LPVI) is a novel method of phase velocity reconstruction that allows for accurate evaluation of shear wave velocity at specified frequencies. This method's robustness was evaluated in 11 elastic and 8 viscoelastic phantoms using linear and curvilinear arrays. We acquired data with acoustic radiation force push beams with different focal depths and F-numbers and reconstructed phase velocity images over a wide range of frequencies. Regardless of phantom, push beam focal depth and reconstruction frequency, an F-number around 3.0 was found to produce the largest usable area in the phase velocity reconstructions. For elastic phantoms scanned with a linear array, the optimal focal depth, frequency range and maximum region of interest (ROI) were 20-30 mm, 100-400 Hz and 2.70 cm2, respectively. For viscoelastic phantoms scanned with a linear array, the optimal focal depth, frequency and maximum ROI were 20-30 mm, 100-300 Hz and 1.54 cm2, respectively. For the curvilinear array in the same phantoms, optimal focal depth, frequency range and maximum ROIs were 45-60 mm, 100-400 and 100-300 Hz and 1.54 cm2, respectively. In further work, LPVI reconstructions from inclusion phantoms will be evaluated to simulate non-homogeneous tissues. Additionally, LPVI will be evaluated in larger-volume phantoms to account for wave reflection from the containers when using the curvilinear array.

Two-dimensional (2D) dynamic vibration optical coherence elastography (DV-OCE) for evaluating mechanical properties: a potential application in tissue engineering.

Mechanical properties in tissues are an important indicator because they are associated with disease states. One of the well-known excitation sources in optical coherence elastography (OCE) to determine mechanical properties is acoustic radiation force (ARF); however, a complicated focusing alignment cannot be avoided. Another excitation source is a piezoelectric (PZT) stack to obtain strain images via compression, which can affect the intrinsic mechanical properties of tissues in tissue engineering. In this study, we report a new technique called two-dimensional (2D) dynamic vibration OCE (DV-OCE) to evaluate 2D wave velocities without tedious focusing alignment procedures and is a non-contact method with respect to the samples. The three-dimensional (3D) Fourier transform was utilized to transfer the traveling waves (x, y, t) into 3D k-space (kx, ky, f). A spatial 2D wavenumber filter and multi-angle directional filter were employed to decompose the waves with omni-directional components into four individual traveling directions. The 2D local wave velocity algorithm was used to calculate a 2D wave velocity map. Six materials, two homogeneous phantoms with 10 mm thickness, two homogeneous phantoms with 2 mm thickness, one heterogeneous phantom with 2 mm diameter inclusion and an ex vivo porcine kidney, were examined in this study. In addition, the ARF-OCE was used to evaluate wave velocities for comparison. Numerical simulations were performed to validate the proposed 2D dynamic vibration OCE technique. We demonstrate that the experimental results were in a good agreement with the results from ARF-OCE (transient OCE) and numerical simulations. Our proposed 2D dynamic vibration OCE could potentially pave the way for mechanical evaluation in tissue engineering and for laboratory translation with easy-to-setup and contactless advantages.

Optical coherence viscometry.

We report a technique, named optical coherence viscometry (OCV), to measure the viscosity of Newtonian fluids in a noncontact manner. According to linear wave theory with small amplitudes, capillary waves are associated with fluid mechanical properties. To perform this measurement and avoid the overdamped effects of capillary waves in viscous fluids, transient acoustic radiation force was applied to generate capillary waves. Within a very limited field-of-view using optical coherence tomography, wave motion acquired in the time domain was analyzed using Fourier methods to study the wave velocity dispersion and attenuation relationships for capillary waves, which can reduce the fluid quantity drastically into tissue culture scale. We measure the viscosities of water, water-glycerol solutions with three concentrations, and biological plasma using the proposed OCV and compare the experimental results to theoretical calculations. OCV is sensitive to wave perturbations and can be a promising technique for measuring the viscosity of biological fluids and could be applied in future applications for measurements for lipid membranes in cell biology and tissue engineering investigation.

Characterizing thrombus with multiple red blood cell compositions by optical coherence tomography attenuation coefficient.

Embolectomy is one of the emergency procedures performed to remove emboli. Assessing the composition of human blood clots is an important diagnostic factor and could provide guidance for an appropriate treatment strategy for interventional physicians. Immunostaining has been used to identity compositions of clots as a gold-standard procedure, but it is time-consuming and cannot be performed in situ. Here, we proposed that the optical attenuation coefficient of optical coherence tomography (OCT) can be a reliable indicator as a new imaging modality to differentiate clot compositions. Fifteen human blood clots with multiple red blood cell (RBC) compositions from 21% to 95% were prepared using healthy human whole blood. A homogeneous gelatin phantom experiment and numerical simulation based on the Lambert-Beer's law were examined to verify the validity of the attenuation coefficient estimation. The results displayed that optical attenuation coefficients were strongly correlated with RBC compositions. We reported that attenuation coefficients could be a promising biomarker to guide the choice of an appropriate interventional device in a clinical setting and assist in characterizing blood clots.

Characterizing blood clots using acoustic radiation force optical coherence elastography and ultrasound shear wave elastography.

Thromboembolism in a cerebral blood vessel is associated with high morbidity and mortality. Mechanical thrombectomy (MT) is one of the emergenc proceduresperformed to remove emboli. However, the interventional approaches such as aspiration catheters or stent retriever are empirically selected. An inappropriate selection of surgical devices can influence the success rate during embolectomy, which can lead to an increase in brain damage. There has been growing interest in the study of clot composition and using a priori knowledge of clot composition to provide guidance for an appropriate treatment strategy for interventional physicians. Developing imaging tools which can allow interventionalists to understand clot composition could affect management and device strategy. In this study, we investigated how clots of different compositions can be characterized by using acoustic radiation force optical coherence elastography (ARF-OCE) and compared with ultrasound shear wave elastography (SWE). Five different clots compositions using human blood were fabricated into cylindrical forms from fibrin-rich (21% red blood cells, RBCs) to RBC-rich (95% RBCs). Using the ARF-OCE and SWE, we characterized the wave velocities measured in the time-domain. In addition, the semi-analytical finite element model was used to explore the relationship between the phase velocities with various frequency ranges and diameters of the clots. The study demonstrated that the wave group velocities generally decrease as RBC content increases in ARF-OCE and SWE. The correlation of the group velocities from the OCE and SWE methods represented a good agreement as RBC composition is larger than 39%. Using the phase velocity dispersion analysis applied to ARF-OCE data, we estimated the shear wave velocities decoupling the effects of the geometry and material properties of the clots. The study demonstrated that the composition of the clots can be characterized by elastographic methods using ARF-OCE and SWE, and OCE demonstrated better ability to discriminate between clots of different RBC compositions, compared to the ultrasound-based approach, especially in clots with low RBC compositions.

Four-dimensional (4D) phase velocity optical coherence elastography in heterogeneous materials and biological tissue.

The variations of mechanical properties in soft tissues are biomarkers used for clinical diagnosis and disease monitoring. Optical coherence elastography (OCE) has been extensively developed to investigate mechanical properties of various biological tissues. These methods are generally based on time-domain data and measure the time-of-flight of the localized shear wave propagations to estimate the group velocity. However, there is considerable information that can be obtained from examining the mechanical properties such as wave propagation velocities at different frequencies. Here we propose a method to evaluate phase velocity, wave velocity at various frequencies, in four-dimensional space (x, y, z, f), called 4D-OCE phase velocity. The method enables local estimates of the phase velocity of propagating mechanical waves in a medium. We acquired and analyzed data with this method from a homogeneous reference phantom, a heterogeneous phantom material with four different excitation cases, and ex vivo porcine kidney tissue. The 3D-OCE group velocity was also estimated to compare with 4D-OCE phase velocity. Moreover, we performed numerical simulation of wave propagations to illustrate the boundary behavior of the propagating waves. The proposed 4D-OCE phase velocity is capable of providing further information in OCE to better understand the spatial variation of mechanical properties of various biological tissues with respect to frequency.

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.

Fluid surface tension evaluation using capillary wave measurement with optical coherence tomography.

The surface tension of biological fluids is an important parameter because the mechanical properties of fluids are closely linked with hematological diseases and other pathophysiologies. Capillary waves are associated with fluid mechanical properties. Here, we propose a method that utilizes the acoustic radiation force (ARF) to generate propagating waves and optical coherence tomography (OCT) to measure the wave motion. This ARF-OCT method is capable of evaluating the surface tension of fluids, water and porcine whole blood in this study, based on the dispersion relation of capillary waves. Two-dimensional Fourier transforms were used to decompose frequency components of wave motion images to obtain a k-space representation and estimate the wave phase velocity. The phase velocity of capillary waves was obtained from the experimental results and compared to theoretical calculations. The surface tensions of water and porcine whole blood were determined from the experimental results. We first report that capillary waves measured with OCT can be a new promising modality for measuring the surface tension of fluids. The proposed method could be used to differentiate actual pathologic fluids or blood from those taken from healthy subjects and as a biomarker in future biomedical applications.

Optical coherence tomography for evaluating capillary waves in blood and plasma.

Capillary waves are associated with fluid mechanical properties. Optical coherence tomography (OCT) has previously been used to determine the viscoelasticity of soft tissues or cornea. Here we report that OCT was able to evaluate phase velocities of capillary waves in fluids. The capillary waves of water, porcine whole blood and plasma on the interfacial surface, air-fluid in this case, are discussed in theory, and phase velocities of capillary waves were estimated by both our OCT experiments and theoretical calculations. Our experiments revealed highly comparable results with theoretical calculations. We concluded that OCT would be a promising tool to evaluate phase velocities of capillary waves in fluids. The methods described in this study could be applied to determine surface tensions and viscosities of fluids for differentiating hematological diseases in the future potential biological applications.

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.

Acoustic radiation force optical coherence elastography for evaluating mechanical properties of soft condensed matters and its biological applications.

Evaluating mechanical properties of biological soft tissues and viscous mucus is challenging because of complicated dynamic behaviors. Soft condensed matter models have been successfully used to explain a number of dynamical behaviors. Here, we reported that optical coherence elastography (OCE) is capable of quantifying mechanical properties of soft condensed matters, micellar fluids. A 7.5 MHz focused transducer was utilized to generate acoustic radiation force exerted on the surface of soft condensed matters in order to produce Rayleigh waves. The waves were recorded by optical coherence tomography (OCT). The Kelvin-Voigt model was adopted to evaluate shear modulus and loss modulus of soft condensed matters. The results reported that various concentrations of micellar fluids can provide reasonable ranges of elasticity from 65.71 to 428.78 Pa and viscosity from 0.035 to 0.283 Pa·s, which are close to ranges for actual biological samples, like mucus. OCE might be a promising tool to differentiate pathologic mucus samples from healthy cases as advanced applications in the future.

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.