Alfred C. H. Yu

Membrane Perforation and Recovery Dynamics in Microbubble-Mediated Sonoporation

Transient sonoporation can essentially be epitomized by two fundamental processes: acoustically induced membrane perforation and its subsequent resealing. To provide insight into these processes, this article presents a new series of direct evidence on the membrane-level dynamics during and after an episode of sonoporation. Our direct observations were obtained from anchored fetal fibroblasts whose membrane topography was imaged in situ using real-time confocal microscopy. To facilitate controlled sonoporation at the single-cell level, microbubbles that can passively adhere to the cell membrane were first introduced at a 1:1 cell-to-bubble ratio. Single-pulse ultrasound exposure (1-MHz frequency, 10-cycle pulse duration, 0.85-MPa peak negative pressure in situ) was then applied to trigger microbubble pulsation/collapse, which, in turn, instigated membrane perforation. With this protocol, five membrane-level phenomena were observed: (i) localized perforation of the cell membrane was synchronized with the instant of ultrasound pulsing; (ii) perforation sites with temporal peak area <30 μm(2) were resealed successfully; (iii) during recovery, a thickened pore rim emerged, and its temporal progression corresponded with the pore closure action; (iv) membrane resealing, if successful, would generally be completed within 1 min of the onset of sonoporation, and the resealing time constant was estimated to be below 20 s; (v) membrane resealing would fail for overly large pores (>100 μm(2)) or in the absence of extracellular calcium ions. These findings serve to underscore the spatiotemporal complexity of membrane-level dynamics in sonoporation.

Eigen-based clutter filter design for ultrasound color flow imaging: a review

Proper suppression of tissue clutter is a prerequisite for visualizing flow accurately in ultrasound color flow imaging. Among various clutter suppression methods, the eigen- based filter has shown potential because it can theoretically adapt its stopband to the actual clutter characteristics even when tissue motion is present. This paper presents a formative review on how eigen-based filters should be designed to improve their practical efficacy in adaptively suppressing clutter without affecting the blood flow echoes. Our review is centered around a comparative assessment of two eigen-filter design considerations: 1) eigen-component estimation approach (single-ensemble vs. multi-ensemble formulations), and 2) filter order selection mechanism (eigenvalue-based vs. frequencybased algorithms). To evaluate the practical efficacy of existing eigen-filter designs, we analyzed their clutter suppression level in two in vivo scenarios with substantial tissue motion (intra-operative coronary imaging and thyroid imaging). Our analysis shows that, as compared with polynomial regression filters (with or without instantaneous clutter downmixing), eigen-filters that use a frequency-based algorithm for filter order selection generally give Doppler power images with better contrast between blood and tissue regions. Results also suggest that both multi-ensemble and single-ensemble eigen-estimation approaches have their own advantages and weaknesses in different imaging scenarios. It may be beneficial to develop an algorithmic way of defining the eigen-filter formulation so that its performance advantages can be better realized.

Vector Projectile Imaging: Time-Resolved Dynamic Visualization of Complex Flow Patterns

Achieving non-invasive, accurate and time-resolved imaging of vascular flow with spatiotemporal fluctuations is well acknowledged to be an ongoing challenge. In this article, we present a new ultrasound-based framework called vector projectile imaging (VPI) that can dynamically render complex flow patterns over an imaging view at millisecond time resolution. VPI is founded on three principles: (i) high-frame-rate broad-view data acquisition (based on steered plane wave firings); (ii) flow vector estimation derived from multi-angle Doppler analysis (coupled with data regularization and least-squares fitting); (iii) dynamic visualization of color-encoded vector projectiles (with flow speckles displayed as adjunct). Calibration results indicated that by using three transmit angles and three receive angles (-10°, 0°, +10° for both), VPI can consistently compute flow vectors in a multi-vessel phantom with three tubes positioned at different depths (1.5, 4, 6 cm), oriented at different angles (-10°, 0°, +10°) and of different sizes (dilated diameter: 2.2, 4.4 and 6.3 mm; steady flow rate: 2.5 mL/s). The practical merit of VPI was further illustrated through an anthropomorphic flow phantom investigation that considered both healthy and stenosed carotid bifurcation geometries. For the healthy bifurcation with 1.2-Hz carotid flow pulses, VPI was able to render multi-directional and spatiotemporally varying flow patterns (using a nominal frame rate of 416 fps or 2.4-ms time resolution). In the case of stenosed bifurcations (50% eccentric narrowing), VPI enabled dynamic visualization of flow jet and recirculation zones. These findings suggest that VPI holds promise as a new tool for complex flow analysis.

SONOPORATION INDUCES APOPTOSIS AND CELL CYCLE ARREST IN HUMAN PROMYELOCYTIC LEUKEMIA CELLS

Despite being a transient biophysical phenomenon, sonoporation is known to disturb the homeostasis of living cells. This work presents new evidence on how sonoporation may lead to antiproliferation effects including cell-cycle arrest and apoptosis through disrupting various cell signaling pathways. Our findings were obtained from sonoporation experiments conducted on HL-60 human promyelocytic leukemia cells (with 1% v/v microbubbles; 1 MHz ultrasound; 0.3 or 0.5MPa peak negative pressure; 10% duty cycle; 1 kHz pulse repetition frequency; 1 min exposure period). Membrane resealing in these sonoporated cells was first verified using scanning electron microscopy. Time-lapse flow cytometry analysis of cellular deoxyribonucleic acid (DNA) contents was then performed at four post-sonoporation time points (4 h, 8 h, 12 h and 24 h). Results indicate that an increasing trend in the apoptotic cell population can be observed for at least 12 h after sonoporation, whilst viable sonoporated cells are found to temporarily accumulate in the G(2)/M (gap-2/mitosis) phase of the cell cycle. Further analysis using western blotting reveals that sonoporation-induced apoptosis involves cleavage of poly adenosine diphosphate ribose polymerase (PARP) proteins: a pro-apoptotic hallmark related to loss of DNA repair functionality. Also, mitochondrial signaling seems to have taken part in triggering this cellular event as the expression of two complementary regulators for mitochondrial release of pro-apoptotic molecules, Bcl-2 (B-cell lymphoma 2) and Bax (Bcl-2-associated X), are seen to be imbalanced in sonoporated cells. Furthermore, sonoporation is found to induce cell-cycle arrest through perturbing the expression of various cyclin and Cdk (cyclin-dependent kinase) checkpoint proteins that play an enabling role in cell-cycle progression. These bioeffects should be taken into account when using sonoporation for therapeutic purposes.

Medical Ultrasound Imaging: To GPU or Not to GPU?

Medical ultrasound imaging stands out from other modalities in providing real-time diagnostic capability at an affordable price while being physically portable. This article explores the suitability of using GPUs as the primary signal and image processors for future medical ultrasound imaging systems. A case study on synthetic aperture (SA) imaging illustrates the promise of using high-performance GPUs in such systems.

Multi-channel pre-beamformed data acquisition system for research on advanced ultrasound imaging methods

The lack of open access to the pre-beamformed data of an ultrasound scanner has limited the research of novel imaging methods to a few privileged laboratories. To address this need, we have developed a pre-beamformed data acquisition (DAQ) system that can collect data over 128 array elements in parallel from the Ultrasonix series of research-purpose ultrasound scanners. Our DAQ system comprises three system-level blocks: 1) a connector board that interfaces with the array probe and the scanner through a probe connector port; 2) a main board that triggers DAQ and controls data transfer to a computer; and 3) four receiver boards that are each responsible for acquiring 32 channels of digitized raw data and storing them to the on-board memory. This system can acquire pre-beamformed data with 12-bit resolution when using a 40-MHz sampling rate. It houses a 16 GB RAM buffer that is sufficient to store 128 channels of pre-beamformed data for 8000 to 25000 transmit firings, depending on imaging depth; corresponding to nearly a 2-s period in typical imaging setups. Following the acquisition, the data can be transferred through a USB 2.0 link to a computer for offline processing and analysis. To evaluate the feasibility of using the DAQ system for advanced imaging research, two proof-of-concept investigations have been conducted on beamforming and plane-wave B-flow imaging. Results show that adaptive beamforming algorithms such as the minimum variance approach can generate sharper images of a wire cross-section whose diameter is equal to the imaging wavelength (150 μm in our example). Also, plane wave B-flow imaging can provide more consistent visualization of blood speckle movement given the higher temporal resolution of this imaging approach (2500 fps in our example).

Flow Velocity Mapping Using Contrast Enhanced High-Frame-Rate Plane Wave Ultrasound and Image Tracking: Methods and Initial in Vitro and in Vivo Evaluation

Ultrasound imaging is the most widely used method for visualising and quantifying blood flow in medical practice, but existing techniques have various limitations in terms of imaging sensitivity, field of view, flow angle dependence, and imaging depth. In this study, we developed an ultrasound imaging velocimetry approach capable of visualising and quantifying dynamic flow, by combining high-frame-rate plane wave ultrasound imaging, microbubble contrast agents, pulse inversion contrast imaging and speckle image tracking algorithms. The system was initially evaluated in vitro on both straight and carotid-mimicking vessels with steady and pulsatile flows and in vivo in the rabbit aorta. Colour and spectral Doppler measurements were also made. Initial flow mapping results were compared with theoretical prediction and reference Doppler measurements and indicate the potential of the new system as a highly sensitive, accurate, angle-independent and full field-of-view velocity mapping tool capable of tracking and quantifying fast and dynamic flows.

High-frame-rate ultrasound color-encoded speckle imaging of complex flow dynamics.

Realization of flow imaging at high frame rates is essential to the visualization of complex flow patterns with fast-changing spatiotemporal dynamics. In this study, we present an experimental demonstration of a novel ultrasound-based high-frame-rate flow visualization technique called color-encoded speckle imaging (CESI), which depicts flow information in a hybrid form comprising flow speckle pattern and color-encoded velocity mapping. This technique works by integrating two key principles: (i) using broad-view data acquisition schemes like plane wave compounding to obtain image data at frame rates well beyond the video display range and (ii) deriving and displaying both flow speckles and velocity estimates from the acquired broad-view image data. CESI was realized on a channel-domain ultrasound imaging research platform, and its performance was evaluated in the context of monitoring complex flow dynamics inside a carotid bifurcation flow phantom with 25% eccentric stenosis at the inlet of the internal carotid artery. Results show that, using an imaging frame rate of 2000 frames per second (based on plane wave compounding with five steering angles), CESI can effectively render flow acceleration and deceleration with visual continuity. It is also effective in depicting how stenosis-related flow disturbance events, such as flow jet formation and post-stenotic flow recirculation, evolve spatiotemporally over a pulse cycle. We anticipate that CESI can represent a rational approach to rendering flow information in ultrasound-based vascular diagnoses.

Single-ensemble-based eigen-processing methods for color flow imaging - Part I. The Hankel-SVD filter

Because of their adaptability to the slow-time signal contents, eigen-based filters have shown potential in improving the flow detection performance of color flow images. This paper proposes a new eigen-based filter called the Hankel-SVD filter that is intended to process each slow- time ensemble individually. The new filter is derived using the notion of principal Hankel component analysis, and it achieves clutter suppression by retaining only the principal components whose order is greater than the clutter eigen- space dimension estimated from a frequency-based analysis algorithm. To assess its efficacy, the Hankel-SVD filter was first applied to synthetic slow-time data (ensemble size: 10) simulated from two different sets of flow parameters that model: (1) arterial imaging (blood velocity: 0 to 38.5 cm/s, tissue motion: up to 2 mm/s, transmit frequency: 5 MHz, pulse repetition period: 0.4 ms) and 2) deep vessel imaging (blood velocity: 0 to 19.2 cm/s, tissue motion: up to 2 cm/s, transmit frequency: 2 MHz, pulse repetition period: 2.0 ms). In the simulation analysis, the post-filter clutter- to-blood signal ratio (CBR) was computed as a function of blood velocity. Results show that for the same effective stopband size (50 Hz), the Hankel-SVD filter has a narrower transition region in the post-filter CBR curve than that of another type of adaptive filter called the clutter- downmixing filter. The practical efficacy of the proposed filter was tested by application to in vivo color flow data obtained from the human carotid arteries (transmit frequency: 4 MHz, pulse repetition period: 0.333 ms, ensemble size: 10). The resulting power images show that the Hankel-SVD filter can better distinguish between blood and moving- tissue regions (about 9 dB separation in power) than the clutter-downmixing filter and a fixed-rank multi-ensemble- based eigen-filter (which showed a 2 to 3 dB separation).

Hardness Anisotropy of Acetaminophen Crystals

The anisotropy of acetaminophen hardness was demonstrated using both Vickers and Knoop indentation hardness measurements. Based on a model of Knoop hardness anisotropy proposed by Brookes et al. (1), it was concluded that plastic flow in acetaminophen crystals occurs primarily as a result of slip in the (010)(001) system. This conclusion was corroborated with the results of the Vickers indentation tests. The apparent brittleness of acetaminophen was rationalized because only one slip system appeared to be operative. Under these conditions generalized plastic flow cannot occur, since this requires the operation of at least five independent slip systems (2). The high stress concentrations that result from flow lead to fracture. Therefore acetaminophen is more precisely classified as being semiductile. When a material deforms plastically as a result of slip in only one slip system, considerable crystal realignment can occur during compaction. This in turn can facilitate capping during decompression and ejection, since the cleavage plane, (010), would become aligned with the direction of highest tensile stress.