Automatic Max-Likelihood Envelope Detection Algorithm for Quantitative High-Frame-Rate Ultrasound for Neonatal Brain Monitoring.

OBJECTIVE: Post-operative brain injury in neonates may result from disturbed cerebral perfusion, but accurate peri-operative monitoring is lacking. High-frame-rate (HFR) cerebral ultrasound could visualize and quantify flow in all detectable vessels using spectral Doppler; however, automated quantification in small vessels is challenging because of low signal amplitude. We have developed an automatic envelope detection algorithm for HFR pulsed wave spectral Doppler signals, enabling neonatal brain quantitative parameter maps during and after surgery. METHODS: HFR ultrasound data from high-risk neonatal surgeries were recorded with a custom HFR mode (frame rate = 1000 Hz) on a Zonare ZS3 system. A pulsed wave Doppler spectrogram was calculated for each pixel containing blood flow in the image, and spectral peak velocity was tracked using a max-likelihood estimation algorithm of signal and noise regions in the spectrogram, where the most likely cross-over point marks the blood flow velocity. The resulting peak systolic velocity (PSV), end-diastolic velocity (EDV) and resistivity index (RI) were compared with other detection schemes, manual tracking and RIs from regular pulsed wave Doppler measurements in 10 neonates. RESULTS: Envelope detection was successful in both high- and low-quality arterial and venous flow spectrograms. Our technique had the lowest root mean square error for EDV, PSV and RI (0.46 cm/s, 0.53 cm/s and 0.15, respectively) when compared with manual tracking. There was good agreement between the clinical pulsed wave Doppler RI and HFR measurement with a mean difference of 0.07. CONCLUSION: The max-likelihood algorithm is a promising approach to accurate, automated cerebral blood flow monitoring with HFR imaging in neonates.

An Ultrasound Matrix Transducer for High-Frame-Rate 3-D Intra-cardiac Echocardiography.

OBJECTIVE: Described here is the development of an ultrasound matrix transducer prototype for high-frame-rate 3-D intra-cardiac echocardiography. METHODS: The matrix array consists of 16 × 18 lead zirconate titanate elements with a pitch of 160 µm × 160 µm built on top of an application-specific integrated circuit that generates transmission signals and digitizes the received signals. To reduce the number of cables in the catheter to a feasible number, we implement subarray beamforming and digitization in receive and use a combination of time-division multiplexing and pulse amplitude modulation data transmission, achieving an 18-fold reduction. The proposed imaging scheme employs seven fan-shaped diverging transmit beams operating at a pulse repetition frequency of 7.7 kHz to obtain a high frame rate. The performance of the prototype is characterized, and its functionality is fully verified. RESULTS: The transducer exhibits a transmit efficiency of 28 Pa/V at 5 cm per element and a bandwidth of 60% in transmission. In receive, a dynamic range of 80 dB is measured with a minimum detectable pressure of 10 Pa per element. The element yield of the prototype is 98%, indicating the efficacy of the manufacturing process. The transducer is capable of imaging at a frame rate of up to 1000 volumes/s and is intended to cover a volume of 70° × 70° × 10 cm. CONCLUSION: These advanced imaging capabilities have the potential to support complex interventional procedures and enable full-volumetric flow, tissue, and electromechanical wave tracking in the heart.

4D Flow Patterns and Relative Pressure Distribution in a Left Ventricle Model by Shake-the-Box and Proper Orthogonal Decomposition Analysis.

PURPOSE: Intraventricular blood flow dynamics are associated with cardiac function. Accurate, noninvasive, and easy assessments of hemodynamic quantities (such as velocity, vortex, and pressure) could be an important addition to the clinical diagnosis and treatment of heart diseases. However, the complex time-varying flow brings many challenges to the existing noninvasive image-based hemodynamic assessments. The development of reliable techniques and analysis tools is essential for the application of hemodynamic biomarkers in clinical practice. METHODS: In this study, a time-resolved particle tracking method, Shake-the-Box, was applied to reconstruct the flow in a realistic left ventricle (LV) silicone model with biological valves. Based on the obtained velocity, 4D pressure field was calculated using a Poisson equation-based pressure solver. Furthermore, flow analysis by proper orthogonal decomposition (POD) of the 4D velocity field has been performed. RESULTS: As a result of the Shake-the-Box algorithm, we have extracted: (i) particle positions, (ii) particle tracks, and finally, (iii) 4D velocity fields. From the latter, the temporal evolution of the 3D pressure field during the full cardiac cycle was obtained. The obtained maximal pressure difference extracted along the base-to-apex was about 2.7 mmHg, which is in good agreement with those reported in vivo. The POD analysis results showed a clear picture of different scale of vortices in the pulsatile LV flow, together with their time-varying information and corresponding kinetic energy content. To reconstruct 95% of the kinetic energy of the LV flow, only the first six POD modes would be required, leading to significant data reduction. CONCLUSIONS: This work demonstrated Shake-the-Box is a promising technique to accurately reconstruct the left ventricle flow field in vitro. The good spatial and temporal resolutions of the velocity measurements enabled a 4D reconstruction of the pressure field in the left ventricle. The application of POD analysis showed its potential in reducing the complexity of the high-resolution left ventricle flow measurements. For future work, image analysis, multi-modality flow assessments, and the development of new flow-derived biomarkers can benefit from fast and data-reducing POD analysis.

Continuous shear wave measurements for dynamic cardiac stiffness evaluation in pigs.

Ultrasound-based shear wave elastography is a promising technique to non-invasively assess the dynamic stiffness variations of the heart. The technique is based on tracking the propagation of acoustically induced shear waves in the myocardium of which the propagation speed is linked to tissue stiffness. This measurement is repeated multiple times across the cardiac cycle to assess the natural variations in wave propagation speed. The interpretation of these measurements remains however complex, as factors such as loading and contractility affect wave propagation. We therefore applied transthoracic shear wave elastography in 13 pigs to investigate the dependencies of wave speed on pressure-volume derived indices of loading, myocardial stiffness, and contractility, while altering loading and inducing myocardial ischemia/reperfusion injury. Our results show that diastolic wave speed correlates to a pressure-volume derived index of operational myocardial stiffness (R = 0.75, p < 0.001), suggesting that both loading and intrinsic properties can affect diastolic wave speed. Additionally, the wave speed ratio, i.e. the ratio of systolic and diastolic speed, correlates to a pressure-volume derived index of contractility, i.e. preload-recruitable stroke work (R = 0.67, p < 0.001). Measuring wave speed ratio might thus provide a non-invasive index of contractility during ischemia/reperfusion injury.

High-Frame-Rate Volumetric Porcine Renal Vasculature Imaging.

OBJECTIVE: The aim of this study was to assess the feasibility and imaging options of contrast-enhanced volumetric ultrasound kidney vasculature imaging in a porcine model using a prototype sparse spiral array. METHODS: Transcutaneous freehand in vivo imaging of two healthy porcine kidneys was performed according to three protocols with different microbubble concentrations and transmission sequences. Combining high-frame-rate transmission sequences with our previously described spatial coherence beamformer, we determined the ability to produce detailed volumetric images of the vasculature. We also determined power, color and spectral Doppler, as well as super-resolved microvasculature in a volume. The results were compared against a clinical 2-D ultrasound machine. RESULTS: Three-dimensional visualization of the kidney vasculature structure and blood flow was possible with our method. Good structural agreement was found between the visualized vasculature structure and the 2-D reference. Microvasculature patterns in the kidney cortex were visible with super-resolution processing. Blood flow velocity estimations were within a physiological range and pattern, also in agreement with the 2-D reference results. CONCLUSION: Volumetric imaging of the kidney vasculature was possible using a prototype sparse spiral array. Reliable structural and temporal information could be extracted from these imaging results.

Higher Order Singular Value Decomposition Filter for Contrast Echocardiography.

Assessing the coronary circulation with contrast-enhanced echocardiography has high clinical relevance. However, it is not being routinely performed in clinical practice because the current clinical tools generally cannot provide adequate image quality. The contrast agent's visibility in the myocardium is generally poor, impaired by motion and nonlinear propagation artifacts. The established multipulse contrast schemes (MPCSs) and the more experimental singular value decomposition (SVD) filter also fall short to solve these issues. Here, we propose a scheme to process amplitude modulation/amplitude-modulated pulse inversion (AM/AMPI) echoes with higher order SVD (HOSVD) instead of conventionally summing the complementary pulses. The echoes from the complementary pulses form a separate dimension in the HOSVD algorithm. Then, removing the ranks in that dimension with dominant coherent signals coming from tissue scattering would provide the contrast detection. We performed both in vitro and in vivo experiments to assess the performance of our proposed method in comparison with the current standard methods. A flow phantom study shows that HOSVD on AM pulsing exceeds the contrast-to-background ratio (CBR) of conventional AM and an SVD filter by 10 and 14 dB, respectively. In vivo porcine heart results also demonstrate that, compared to AM, HOSVD improves CBR in open-chest acquisition (up to 19 dB) and contrast ratio (CR) in closed-chest acquisition (3 dB).

Ultrasonic Characterization of Ibidi µ-Slide I Luer Channel Slides for Studies With Ultrasound Contrast Agents.

Understanding and controlling the ultrasound contrast agent (UCA)'s response to an applied ultrasound pressure field are crucial when investigating ultrasound imaging sequences and therapeutic applications. The magnitude and frequency of the applied ultrasonic pressure waves affect the oscillatory response of the UCA. Therefore, it is important to have an ultrasound compatible and optically transparent chamber in which the acoustic response of the UCA can be studied. The aim of our study was to determine the in situ ultrasound pressure amplitude in the ibidi µ -slide I Luer channel, an optically transparent chamber suitable for cell culture, including culture under flow, for all microchannel heights (200, 400, 600, and [Formula: see text]). First, the in situ pressure field in the 800- [Formula: see text] high channel was experimentally characterized using Brandaris 128 ultrahigh-speed camera recordings of microbubbles (MBs) and a subsequent iterative processing method, upon insonification at 2 MHz, 45° incident angle, and 50-kPa peak negative pressure (PNP). Control studies in another cell culture chamber, the CLINIcell, were compared with the obtained results. The pressure amplitude was -3.7 dB with respect to the pressure field without the ibidi µ -slide. Second, using finite-element analysis, we determined the in situ pressure amplitude in the ibidi with the 800- [Formula: see text] channel (33.1 kPa), which was comparable to the experimental value (34 kPa). The simulations were extended to the other ibidi channel heights (200, 400, and [Formula: see text]) with either 35° or 45° incident angle, and at 1 and 2 MHz. The predicted in situ ultrasound pressure fields were between -8.7 and -1.1 dB of the incident pressure field depending on the listed configurations of ibidi slides with different channel heights, applied ultrasound frequencies, and incident angles. In conclusion, the determined ultrasound in situ pressures demonstrate the acoustic compatibility of the ibidi µ -slide I Luer for different channel heights, thereby showing its potential for studying the acoustic behavior of UCAs for imaging and therapy.

Analytic prediction of droplet vaporization events to estimate the precision of ultrasound-based proton range verification.

BACKGROUND: The safety and efficacy of proton therapy is currently hampered by range uncertainties. The combination of ultrasound imaging with injectable radiation-sensitive superheated nanodroplets was recently proposed for in vivo range verification. The proton range can be estimated from the distribution of nanodroplet vaporization events, which is stochastically related to the stopping distribution of protons, as nanodroplets are vaporized by protons reaching their maximal LET at the end of their range. PURPOSE: Here, we aim to estimate the range estimation precision of this technique. As for any stochastic measurement, the precision will increase with the sample size, that is, the number of detected vaporizations. Thus, we first develop and validate a model to predict the number of vaporizations, which is then applied to estimate the range verification precision for a set of conditions (droplet size, droplet concentration, and proton beam parameters). METHODS: Starting from the thermal spike theory, we derived a model that predicts the expected number of droplet vaporizations in an irradiated sample as a function of the droplet size, concentration, and number of protons. The model was validated by irradiating phantoms consisting of size-sorted perfluorobutane droplets dispersed in an aqueous matrix. The number of protons was counted with an ionization chamber, and the droplet vaporizations were recorded and counted individually using high frame rate ultrasound imaging. After validation, the range estimate precision was determined for different conditions using a Monte Carlo algorithm. RESULTS: A good agreement between theory and experiments was observed for the number of vaporizations, especially for large (5.8 ± 2.2 µm) and medium (3.5 ± 1.1 µm) sized droplets. The number of events was lower than expected in phantoms with small droplets (2.0 ± 0.7 µm), but still within the same order of magnitude. The inter-phantom variability was considerably larger (up to 30x) than predicted by the model. The validated model was then combined with Monte Carlo simulations, which predicted a theoretical range retrieval precision improving with the square-root of the number of vaporizations, and degrading at high beam energies due to range straggling. For single pencil beams with energies between 70 and 240 MeV, a range verification precision below 1% of the range required perfluorocarbon concentrations in the order of 0.3-2.4 µM. CONCLUSION: We proposed and experimentally validated a model to provide a quick estimate of the number of vaporizations for a given set of conditions (droplet size, droplet concentration, and proton beam parameters). From this model, promising range verification performances were predicted for realistic perfluorocarbon concentrations. These findings are an incentive to move towards preclinical studies, which are critical to assess the achievable droplet distribution in and around the tumor, and hence the in vivo range verification precision.

Intra-renal microcirculatory alterations on non-traumatic hemorrhagic shock induced acute kidney injury in pigs.

Acute kidney injury (AKI) is frequently seen in patients with hemorrhagic shock due to hypotension, tissue hypoxia, and inflammation despite adequate resuscitation. There is a lack of information concerning the alteration of renal microcirculation and perfusion during shock and resuscitation. The aim of this study was to investigate the possible role of renal microcirculatory alterations on development of renal dysfunction in a pig model of non-traumatic hemorrhagic shock (HS) induced AKI.Fully instrumented female pigs were divided into the two groups as Control (n = 6) and HS (n = 11). HS was achieved by withdrawing blood until mean arterial pressure (MAP) reached around 50 mmHg. After an hour cessation period, fluid resuscitation with balanced crystalloid was started for the duration of 1 h. The systemic and renal hemodynamics, renal microcirculatory perfusion (contrast-enhanced ultrasound (CEUS)) and the sublingual microcirculation were measured.CEUS peak enhancement was significantly increased in HS during shock, early-, and late resuscitation indicating perfusion defects in the renal cortex (p < 0.05 vs. baseline, BL) despite a stable renal blood flow (RBF) and urine output. Following normalization of systemic hemodynamics, we observed persistent hypoxia (high lactate) and high red blood cell (RBC) velocity just after initiation of resuscitation resulting in further endothelial and renal damage as shown by increased plasma sialic acid (p < 0.05 vs. BL) and NGAL levels. We also showed that total vessel density (TVD) and functional capillary density (FCD) were depleted during resuscitation (p < 0.05).In this study, we showed that the correction of systemic hemodynamic variables may not be accompanied with the improvement of renal cortical perfusion, intra-renal blood volume and renal damage following fluid resuscitation. We suggest that the measurement of renal injury biomarkers, systemic and renal microcirculation can be used for guiding to the optimization of fluid therapies.

Coupling Two Ultra-high-Speed Cameras to Elucidate Ultrasound Contrast-Mediated Imaging and Therapy.

Ultrasound contrast-mediated medical imaging and therapy both rely on the dynamics of micron- and nanometer-sized ultrasound cavitation nuclei, such as phospholipid-coated microbubbles and phase-change droplets. Ultrasound cavitation nuclei respond non-linearly to ultrasound on a nanosecond time scale that necessitates the use of ultra-high-speed imaging to fully visualize these dynamics in detail. In this study, we developed an ultra-high-speed optical imaging system that can record up to 20 million frames per second (Mfps) by coupling two small-sized, commercially available, 10-Mfps cameras. The timing and reliability of the interleaved cameras needed to achieve 20 Mfps was validated using two synchronized light-emitting diode strobe lights. Once verified, ultrasound-activated microbubble responses were recorded and analyzed. A unique characteristic of this coupled system is its ability to be reconfigured to provide orthogonal observations at 10 Mfps. Acoustic droplet vaporization was imaged from two orthogonal views, by which the 3-D dynamics of the phase transition could be visualized. This optical imaging system provides the temporal resolution and experimental flexibility needed to further elucidate the dynamics of ultrasound cavitation nuclei to potentiate the clinical translation of ultrasound-mediated imaging and therapy developments.

Improving Lateral Resolution in 3-D Imaging With Micro-beamforming Through Adaptive Beamforming by Deep Learning.

There is an increased desire for miniature ultrasound probes with small apertures to provide volumetric images at high frame rates for in-body applications. Satisfying these increased requirements makes simultaneous achievement of a good lateral resolution a challenge. As micro-beamforming is often employed to reduce data rate and cable count to acceptable levels, receive processing methods that try to improve spatial resolution will have to compensate the introduced reduction in focusing. Existing beamformers do not realize sufficient improvement and/or have a computational cost that prohibits their use. Here we propose the use of adaptive beamforming by deep learning (ABLE) in combination with training targets generated by a large aperture array, which inherently has better lateral resolution. In addition, we modify ABLE to extend its receptive field across multiple voxels. We illustrate that this method improves lateral resolution both quantitatively and qualitatively, such that image quality is improved compared with that achieved by existing delay-and-sum, coherence factor, filtered-delay-multiplication-and-sum and Eigen-based minimum variance beamformers. We found that only in silica data are required to train the network, making the method easily implementable in practice.

A Tiled Ultrasound Matrix Transducer for Volumetric Imaging of the Carotid Artery.

High frame rate three-dimensional (3D) ultrasound imaging would offer excellent possibilities for the accurate assessment of carotid artery diseases. This calls for a matrix transducer with a large aperture and a vast number of elements. Such a matrix transducer should be interfaced with an application-specific integrated circuit (ASIC) for channel reduction. However, the fabrication of such a transducer integrated with one very large ASIC is very challenging and expensive. In this study, we develop a prototype matrix transducer mounted on top of multiple identical ASICs in a tiled configuration. The matrix was designed to have 7680 piezoelectric elements with a pitch of 300 µm × 150 µm integrated with an array of 8 × 1 tiled ASICs. The performance of the prototype is characterized by a series of measurements. The transducer exhibits a uniform behavior with the majority of the elements working within the -6 dB sensitivity range. In transmit, the individual elements show a center frequency of 7.5 MHz, a -6 dB bandwidth of 45%, and a transmit efficiency of 30 Pa/V at 200 mm. In receive, the dynamic range is 81 dB, and the minimum detectable pressure is 60 Pa per element. To demonstrate the imaging capabilities, we acquired 3D images using a commercial wire phantom.

Sparse 2-D PZT-on-PCB Arrays With Density Tapering.

Two-dimensional (2-D) arrays offer volumetric imaging capabilities without the need for probe translation or rotation. A sparse array with elements seeded in a tapering spiral pattern enables one-to-one connection to an ultrasound machine, thus allowing flexible transmission and reception strategies. To test the concept of sparse spiral array imaging, we have designed, realized, and characterized two prototype probes designed at 2.5-MHz low-frequency (LF) and 5-MHz high-frequency (HF) center frequencies. Both probes share the same electronic design, based on piezoelectric ceramics and rapid prototyping with printed circuit board substrates to wire the elements to external connectors. Different center frequencies were achieved by adjusting the piezoelectric layer thickness. The LF and HF prototype probes had 88% and 95% of working elements, producing peak pressures of 21 and 96 kPa/V when focused at 5 and 3 cm, respectively. The one-way -3-dB bandwidths were 26% and 32%. These results, together with experimental tests on tissue-mimicking phantoms, show that the probes are viable for volumetric imaging.

Accelerated 2-D Real-Time Refraction-Corrected Transcranial Ultrasound Imaging.

In a recent study, we proposed a technique to correct aberration caused by the skull and reconstruct a transcranial B-mode image with a refraction-corrected synthetic aperture imaging (SAI) scheme. Given a sound speed map, the arrival times were calculated using a fast marching technique (FMT), which solves the Eikonal equation and, therefore, is computationally expensive for real-time imaging. In this article, we introduce a two-point ray tracing method, based on Fermat's principle, for fast calculation of the travel times in the presence of a layered aberrator in front of the ultrasound probe. The ray tracing method along with the reconstruction technique is implemented on a graphical processing unite (GPU). The point spread function (PSF) in a wire phantom image reconstructed with the FMT and the GPU implementation was studied with numerical synthetic data and experiments with a bone-mimicking plate and a sagittally cut human skull. The numerical analysis showed that the error on travel times is less than 10% of the ultrasound temporal period at 2.5 MHz. As a result, the lateral resolution was not significantly degraded compared with images reconstructed with FMT-calculated travel times. The results using the synthetic, bone-mimicking plate, and skull dataset showed that the GPU implementation causes a lateral/axial localization error of 0.10/0.20, 0.15/0.13, and 0.26/0.32 mm compared with a reference measurement (no aberrator in front of the ultrasound probe), respectively. For an imaging depth of 70 mm, the proposed GPU implementation allows reconstructing 19 frames/s with full synthetic aperture (96 transmission events) and 32 frames/s with multiangle plane wave imaging schemes (with 11 steering angles) for a pixel size of [Formula: see text]. Finally, refraction-corrected power Doppler imaging is demonstrated with a string phantom and a bone-mimicking plate placed between the probe and the moving string. The proposed approach achieves a suitable frame rate for clinical scanning while maintaining the image quality.

Time-resolved absolute radius estimation of vibrating contrast microbubbles using an acoustical camera.

Ultrasound (US) contrast agents consist of microbubbles ranging from 1 to 10 µm in size. The acoustical response of individual microbubbles can be studied with high-frame-rate optics or an "acoustical camera" (AC). The AC measures the relative microbubble oscillation while the optical camera measures the absolute oscillation. In this article, the capabilities of the AC are extended to measure the absolute oscillations. In the AC setup, microbubbles are insonified with a high- (25 MHz) and low-frequency US wave (1-2.5 MHz). Other than the amplitude modulation (AM) from the relative size change of the microbubble (employed in Renaud, Bosch, van der Steen, and de Jong (2012a). "An 'acoustical camera' for in vitro characterization of contrast agent microbubble vibrations," Appl. Phys. Lett. 100(10), 101911, the high-frequency response from individual vibrating microbubbles contains a phase modulation (PM) from the microbubble wall displacement, which is the extension described here. The ratio of PM and AM is used to determine the absolute radius, R0. To test this sizing, the size distributions of two monodisperse microbubble populations ( R = 2.1 and 3.5 µm) acquired with the AC were matched to the distribution acquired with a Coulter counter. As a result of measuring the absolute size of the microbubbles, this "extended AC" can capture the full radial dynamics of single freely floating microbubbles with a throughput of hundreds of microbubbles per hour.

An iterative method to evaluate one-dimensional pulsed nonlinear elastic wavefields and mixing of elastic waves in solids.

Over the last 15 years, literature on nondestructive testing has shown that the generation of higher harmonics and nonlinear mixing of waves could be used to obtain the nonlinearity parameters of an elastic medium and thereby gather information about its state, e.g., aging and fatigue. To design ultrasound measurement setups based on these phenomena, efficient numerical modeling tools are needed. In this paper, the iterative nonlinear contrast source method for numerical modeling of nonlinear acoustic waves is extended to the one-dimensional elastic case. In particular, nonlinear mixing of two collinear bulk waves (one compressional, one shear) in a homogeneous, isotropic medium is considered, taking into account its third-order elastic constants ( A, B, and C). The obtained results for nonlinear propagation are in good agreement with a benchmark solution based on the modified Burgers equation. The results for the resonant waves that are caused by the one-way and two-way mixing of primary waves are in quantitative agreement with the results in the literature [Chen, Tang, Zhao, Jacobs, and Qu, J. Acoust. Soc. Am. 136(5), 2389-2404 (2014)]. The contrast source approach allows the identification of the propagating and evanescent components of the scattered wavefield in the wavenumber-frequency domain, which provides physical insight into the mixing process and explains the propagation direction of the resonant wave.

Multi-angle data acquisition to compensate transducer finite size in photoacoustic tomography.

In photoacoustic tomography (PAT) systems, the tangential resolution decreases due to the finite size of the transducer as the off-center distance increases. To address this problem, we propose a multi-angle detection approach in which the transducer used for data acquisition rotates around its center (with specific angles) as well as around the scanning center. The angles are calculated based on the central frequency and diameter of the transducer and the radius of the region-of-interest (ROI). Simulations with point-like absorbers (for point-spread-function evaluation) and a vasculature phantom (for quality assessment), and experiments with ten 0.5 mm-diameter pencil leads and a leaf skeleton phantom are used for evaluation of the proposed approach. The results show that a location-independent tangential resolution is achieved with 150 spatial sampling and central rotations with angles of ±8°/±16°. With further developments, the proposed detection strategy can replace the conventional detection (rotating a transducer around ROI) in PAT.

Dispersing and Sonoporating Biofilm-Associated Bacteria with Sonobactericide.

Bacteria encased in a biofilm poses significant challenges to successful treatment, since both the immune system and antibiotics are ineffective. Sonobactericide, which uses ultrasound and microbubbles, is a potential new strategy for increasing antimicrobial effectiveness or directly killing bacteria. Several studies suggest that sonobactericide can lead to bacterial dispersion or sonoporation (i.e., cell membrane permeabilization); however, real-time observations distinguishing individual bacteria during and directly after insonification are missing. Therefore, in this study, we investigated, in real-time and at high-resolution, the effects of ultrasound-induced microbubble oscillation on Staphylococcus aureus biofilms, without or with an antibiotic (oxacillin, 1 µg/mL). Biofilms were exposed to ultrasound (2 MHz, 100-400 kPa, 100-1000 cycles, every second for 30 s) during time-lapse confocal microscopy recordings of 10 min. Bacterial responses were quantified using post hoc image analysis with particle counting. Bacterial dispersion was observed as the dominant effect over sonoporation, resulting from oscillating microbubbles. Increasing pressure and cycles both led to significantly more dispersion, with the highest pressure leading to the most biofilm removal (up to 83.7%). Antibiotic presence led to more variable treatment responses, yet did not significantly impact the therapeutic efficacy of sonobactericide, suggesting synergism is not an immediate effect. These findings elucidate the direct effects induced by sonobactericide to best utilize its potential as a biofilm treatment strategy.

Imaging Scheme for 3-D High-Frame-Rate Intracardiac Echography: A Simulation Study.

Atrial fibrillation (AF) is the most common cardiac arrhythmia and is normally treated by RF ablation. Intracardiac echography (ICE) is widely employed during RF ablation procedures to guide the electrophysiologist in navigating the ablation catheter, although only 2-D probes are currently clinically used. A 3-D ICE catheter would not only improve visualization of the atrium and ablation catheter, but it might also provide the 3-D mapping of the electromechanical wave (EW) propagation pattern, which represents the mechanical response of cardiac tissue to electrical activity. The detection of this EW needs 3-D high-frame-rate imaging, which is generally only realizable in tradeoff with channel count and image quality. In this simulation-based study, we propose a high volume rate imaging scheme for a 3-D ICE probe design that employs 1-D micro-beamforming in the elevation direction. Such a probe can achieve a high frame rate while reducing the channel count sufficiently for realization in a 10-Fr catheter. To suppress the grating-lobe (GL) artifacts associated with micro-beamforming in the elevation direction, a limited number of fan-shaped beams with a wide azimuthal and narrow elevational opening angle are sequentially steered to insonify slices of the region of interest. An angular weighted averaging of reconstructed subvolumes further reduces the GL artifacts. We optimize the transmit beam divergence and central frequency based on the required image quality for EW imaging (EWI). Numerical simulation results show that a set of seven fan-shaped transmission beams can provide a frame rate of 1000 Hz and a sufficient spatial resolution to visualize the EW propagation on a large 3-D surface.

Internalization of targeted microbubbles by endothelial cells and drug delivery by pores and tunnels.

Ultrasound insonification of microbubbles can locally enhance drug delivery by increasing the cell membrane permeability. To aid development of a safe and effective therapeutic microbubble, more insight into the microbubble-cell interaction is needed. In this in vitro study we aimed to investigate the initial 3D morphology of the endothelial cell membrane adjacent to individual microbubbles (n = 301), determine whether this morphology was affected upon binding and by the type of ligand on the microbubble, and study its influence on microbubble oscillation and the drug delivery outcome. High-resolution 3D confocal microscopy revealed that targeted microbubbles were internalized by endothelial cells, while this was not the case for non-targeted or IgG1-κ control microbubbles. The extent of internalization was ligand-dependent, since αvß3-targeted microbubbles were significantly more internalized than CD31-targeted microbubbles. Ultra-high-speed imaging (~17 Mfps) in combination with high-resolution confocal microscopy (n = 246) showed that microbubble internalization resulted in a damped microbubble oscillation upon ultrasound insonification (2 MHz, 200 kPa peak negative pressure, 10 cycles). Despite damped oscillation, the cell's susceptibility to sonoporation (as indicated by PI uptake) was increased for internalized microbubbles. Monitoring cell membrane integrity (n = 230) showed the formation of either a pore, for intracellular delivery, or a tunnel (i.e. transcellular perforation), for transcellular delivery. Internalized microbubbles caused fewer transcellular perforations and smaller pore areas than non-internalized microbubbles. In conclusion, studying microbubble-mediated drug delivery using a state-of-the-art imaging system revealed receptor-mediated microbubble internalization and its effect on microbubble oscillation and resulting membrane perforation by pores and tunnels.