Enhancement of sonochemical production of hydroxyl radicals from pulsed cylindrically converging ultrasound waves.

Sonochemistry is the use of ultrasound to generate highly reactive radical species through the inertial collapse of a gas/vapour cavity and is a green alternative for hydrogen production, wastewater treatment, and chemical synthesis and modifications. Yet, current sonochemical reactors often are limited by their design, resulting in low efficacy and yields with slow reaction kinetics. Here, we constructed a novel sonochemical reactor design that creates cylindrically converging ultrasound waves to create an intense localised region of high acoustic pressure amplitudes (15 MPaPKPK) capable of spontaneously nucleating cavitation. Using a novel dosimetry technique, we determined the effect of acoustic parameters on the yield of hydroxyl radicals (HO), HO production rate, and ultimately the sonochemical efficiency (SE) of our reactor. Our reactor design had a significantly higher HO production rate and SE compared to other conventional reactors and across literature.

Development of a morphologically realistic mouse phantom for pre-clinical photoacoustic imaging.

BACKGROUND: Characterizations based on anatomically realistic phantoms are highly effective to perform accurate technical validation of imaging systems. Specifically for photoacoustic imaging (PAI), although a variety of phantom models with simplified geometries are reported, an unmet need still exists to establish morphologically realistic heterogeneous pre-clinical phantoms. So the development of a mouse-mimicking phantom can reduce the use of animals for the validation and standardization studies of pre-clinical PAI systems and thus eventually translate the PAI technology to clinical research. PURPOSE: Here we designed, developed, and fabricated a stable phantom that mimics the detailed morphology of a mouse, to be used as a realistic tool for PAI. METHODS: The mouse phantom, has been designed by using a combination of image modeling and 3D-printing techniques. As a tissue-mimicking material, we have used copolymer-in-oil-based material that was recently proposed by the International Photoacoustic Standardization Consortium (IPASC). In particular, the anatomically realistic phantom has been modeled by using the real atlas of a mouse as a reference. The mouse phantom includes a 3D-printed skeleton and the main abdominal organs such as the liver, spleen, and kidneys obtained by using doped copolymer-in-oil material with 3D-printed molds. In addition, the acoustic and optical properties of the tissue-mimicking material and the long-term stability have been broadly characterized. RESULTS: Furthermore, our studies showed that the phantom is durable and stable for more than 200 days, under normal storage and repeated use. Fabrication protocol is easy to reproduce. As a result, the proposed morphologically realistic mouse phantom offers durability, material compatibility, and an unprecedented realistic resemblance to the actual rodents' anatomy in PAI. CONCLUSION: This durable morphologically realistic mouse phantom would minimize the animal experiments in compliance with the 3R principle of Replacement, Reduction, and Refinement. To our knowledge, this is the first time an anatomically realistic heterogeneous mouse phantom has been proposed for PAI in pre-clinical animal imaging and tested its durability over 200 days.

Compressibility of Lithium Hexafluorophosphate Solutions in Two Carbonate Solvents.

Speed-of-sound measurements are performed to establish how the isentropic bulk modulus K s of the electrolyte system comprising lithium hexafluorophospate (LiPF6) in blends of propylene carbonate (PC) and ethyl methyl carbonate (EMC) varies with salt molality m, mass fraction of PC in the PC:EMC cosolvent f, and temperature T. Bulk moduli are calculated by combining acoustic time-of-flight data between parallel walls of a liquid-filled cuvette with densitometric data for a sequence of binary and ternary salt solutions. Correlations are presented to yield K s (m, f, T) accurately for nine compositions spanning the range m = 0-2 mol kg-1 and f = 0-1, at temperatures T ranging from 283.15 to 313.15 K. Electrolyte compressibility varies most with solvent ratio, followed by salt content and temperature, with K s ranging from 1 to 3 GPa. Composition-dependent acoustical properties elucidate the nature of speciation and solvation states in bulk electrolytes, and could be useful to identify the features of individual phases within solution-permeated porous electrodes.

Fluorescence-based chemical tools for monitoring ultrasound-induced hydroxyl radical production in aqueous solution and in cells.

We report the synthesis of hydroxyl-radical (˙OH) responsive fluorescent probes that utilise the 3,5-dihydroxybenzyl (DHB) functionality. 4-Methylumbeliferone-DHB (Umb-DHB) and resorufin-DHB (Res-DHB) in the presence of ˙OH radicals resulted in significant increases in their respective fluorescent emission intensities at 460 nm and 585 nm. The incubation of Res-DHB in HeLa cells followed by therapeutic ultrasound (1 MHz) resulted in a significant increase in fluorescence emission intensity thus permitting the ability to monitor ultrasound-induced ˙OH production in live cells.

Genetic engineering biofilms in situ using ultrasound-mediated DNA delivery.

The ability to directly modify native and established biofilms has enormous potential in understanding microbial ecology and application of biofilm in 'real-world' systems. However, efficient genetic transformation of established biofilms at any scale remains challenging. In this study, we applied an ultrasound-mediated DNA delivery (UDD) technique to introduce plasmid to established non-competent biofilms in situ. Two different plasmids containing genes coding for superfolder green fluorescent protein (sfGFP) and the flavin synthesis pathway were introduced into established bacterial biofilms in microfluidic flow (transformation efficiency of 3.9 ± 0.3 × 10-7 cells in biofilm) and microbial fuel cells (MFCs), respectively, both employing UDD. Gene expression and functional effects of genetically modified bacterial biofilms were observed, where some cells in UDD-treated Pseudomonas putida UWC1 biofilms expressed sfGFP in flow cells and UDD-treated Shewanella oneidensis MR-1 biofilms generated significantly (P < 0.05) greater (61%) bioelectricity production (21.9 ± 1.2 µA cm-2 ) in MFC than a wild-type control group (~ 13.6 ± 1.6 µA cm-2 ). The effects of UDD were amplified in subsequent growth under selection pressure due to antibiotic resistance and metabolism enhancement. UDD-induced gene transfer on biofilms grown in both microbial flow cells and MFC systems was successfully demonstrated, with working volumes of 0.16 cm3 and 300 cm3 , respectively, demonstrating a significant scale-up in operating volume. This is the first study to report on a potentially scalable direct genetic engineering method for established non-competent biofilms, which can be exploited in enhancing their capability towards environmental, industrial and medical applications.

The influence of droplet concentration on phase change and inertial cavitation thresholds associated with acoustic droplet vaporization.

Acoustic droplet vaporization (ADV) is an important process that enables the theragnostic application of acoustically activated droplets, where the nucleation of inertial cavitation (IC) activity must be precisely controlled. This Letter describes threshold pressure measurements for ADV and acoustic emissions consistent with IC activity of lipid-shelled non-superheated perfluoropentane nanodroplets over a range of physiologically relevant concentrations at 1.1-MHz. Under the frequency investigated, results show that the thresholds were relatively independent of concentration for intermediate concentrations (105, 106, and 107 droplets/ml), thus indicating an optimal range of droplet concentrations for conducting threshold studies. For the highest concentration, the difference between the threshold for IC and the threshold for ADV was greatly reduced, suggesting that it might prove difficult to induce ADV without concomitant IC in applications that employ higher concentrations.

Optimal Control of SonoVue Microbubbles to Estimate Hydrostatic Pressure.

The measurement of cardiac and aortic pressures enables diagnostic insight into cardiac contractility and stiffness. However, these pressures are currently assessed invasively using pressure catheters. It may be possible to estimate these pressures less invasively by applying microbubble ultrasound contrast agents as pressure sensors. The aim of this study was to investigate the subharmonic response of the microbubble ultrasound contrast agent SonoVue (Bracco Spa, Milan, Italy) at physiological pressures using a static pressure phantom. A commercially available cell culture cassette with Luer connections was used as a static pressure chamber. SonoVue was added to the phantom, and radio frequency data were recorded on the ULtrasound Advanced Open Platform (ULA-OP). The mean subharmonic amplitude over a 40% bandwidth was extracted at 0-200-mmHg hydrostatic pressures, across 1.7-7.0-MHz transmit frequencies and 3.5%-100% maximum scanner acoustic output. The Rayleigh-Plesset equation for single-bubble oscillations and additional hysteresis experiments were used to provide insight into the mechanisms underlying the subharmonic pressure response of SonoVue. The subharmonic amplitude of SonoVue increased with hydrostatic pressure up to 50 mmHg across all transmit frequencies and decreased thereafter. A decreasing microbubble surface tension may drive the initial increase in the subharmonic amplitude of SonoVue with hydrostatic pressure, while shell buckling and microbubble destruction may contribute to the subsequent decrease above 125-mmHg pressure. In conclusion, a practical operating regime that may be applied to estimate cardiac and aortic blood pressures from the subharmonic signal of SonoVue has been identified.

Photo- and Sono-Dynamic Therapy: A Review of Mechanisms and Considerations for Pharmacological Agents Used in Therapy Incorporating Light and Sound.

As irreplaceable energy sources of minimally invasive treatment, light and sound have, separately, laid solid foundations in their clinic applications. Constrained by the relatively shallow penetration depth of light, photodynamic therapy (PDT) typically involves involves superficial targets such as shallow seated skin conditions, head and neck cancers, eye disorders, early-stage cancer of esophagus, etc. For ultrasound-driven sonodynamic therapy (SDT), however, to various organs is facilitated by the superior... transmission and focusing ability of ultrasound in biological tissues, enabling multiple therapeutic applications including treating glioma, breast cancer, hematologic tumor and opening blood-brain-barrier (BBB). Considering the emergence of theranostics and precision therapy, these two classic energy sources and corresponding sensitizers are worth reevaluating. In this review, three typical therapies using light and sound as a trigger, PDT, SDT, and combined PDT and SDT are introduced. The therapeutic dynamics and current designs of pharmacological sensitizers involved in these therapies are presented. By introducing both the history of the field and the most up-to-date design strategies, this review provides a systemic summary on the development of PDT and SDT and fosters inspiration for researchers working on 'multi-modal' therapies involving light and sound.

HIFU-induced changes in optical scattering and absorption of tissue over nine orders of thermal dose.

The optical properties of tissue change during thermal ablation. Multi-modal methods such as acousto-optic (AO) and photo-acoustic (PA) imaging may provide a real-time, direct measure of lesion formation. Baseline changes in optical properties have been previously measured over limited ranges of thermal dose for tissues exposed to a temperature-controlled water bath, however, there is scant data for optical properties of lesions created by HIFU. In this work, the optical scattering and absorption coefficients from 400-1300 nm of excised chicken breast exposed to HIFU were measured using an integrating sphere spectrophotometric technique. HIFU-induced spatiotemporal temperature elevations were measured using an infrared camera and used to calculate the thermal dose delivered to a localized region of tissue. Results obtained over a range of thermal dose spanning 9 orders of magnitude show that the reduced scattering coefficient increases for HIFU exposures exceeding a threshold thermal dose of CEM43 = 600 ± 81 cumulative equivalent minutes. HIFU-induced thermal damage results in changes in scattering over all optical wavelengths, with a 2.5-fold increase for thermal lesions exceeding 70 °C. The tissue absorption coefficient was also found to increase for thermally lesioned tissue, however, the magnitude was strongly dependent on the optical wavelength and there was substantial sample-to-sample variability, such that the existence of a threshold thermal dose could not be determined. Therapeutic windows, where the optical penetration depth is expected to be greatest, were identified in the near infrared regime centered near 900 nm and 1100 nm. These data motivate further research to improve the real-time AO and PA sensing of lesion formation during HIFU therapy as an alternative to thermometry.

Effect of Temperature on the Size Distribution, Shell Properties, and Stability of Definity®.

Physical characterization of an ultrasound contrast agent (UCA) aids in its safe and effective use in diagnostic and therapeutic applications. The goal of this study was to investigate the impact of temperature on the size distribution, shell properties, and stability of Definity®, a U.S. Food and Drug Administration-approved UCA used for left ventricular opacification. A Coulter counter was modified to enable particle size measurements at physiologic temperatures. The broadband acoustic attenuation spectrum and size distribution of Definity® were measured at room temperature (25 °C) and physiologic temperature (37 °C) and were used to estimate the viscoelastic shell properties of the agent at both temperatures. Attenuation and size distribution was measured over time to assess the effect of temperature on the temporal stability of Definity®. The attenuation coefficient of Definity® at 37 °C was as much as 5 dB higher than the attenuation coefficient measured at 25 °C. However, the size distributions of Definity® at 25 °C and 37 °C were similar. The estimated shell stiffness and viscosity decreased from 1.76 ± 0.18 N/m and 0.21 × 10-6 ± 0.07 × 10-6 kg/s at 25 °C to 1.01 ± 0.07 N/m and 0.04 × 10-6 ± 0.04 × 10-6 kg/s at 37 °C, respectively. Size-dependent differences in dissolution rates were observed within the UCA population at both 25 °C and 37 °C. Additionally, cooling the diluted UCA suspension from 37 °C to 25 °C accelerated the dissolution rate. These results indicate that although temperature affects the shell properties of Definity® and can influence the stability of Definity®, the size distribution of this agent is not affected by a temperature increase from 25 °C to 37 °C.

Loss of gas from echogenic liposomes exposed to pulsed ultrasound.

The destruction of echogenic liposomes (ELIP) in response to pulsed ultrasound excitations has been studied acoustically previously. However, the mechanism underlying the loss of echogenicity due to cavitation nucleated by ELIP has not been fully clarified. In this study, an ultra-high speed imaging approach was employed to observe the destruction phenomena of single ELIP exposed to ultrasound bursts at a center frequency of 6 MHz. We observed a rapid size reduction during the ultrasound excitation in 139 out of 397 (35%) ultra- high-speed recordings. The shell dilation rate, which is defined as the microbubble wall velocity divided by the instantaneous radius, [Formula: see text] /R, was extracted from the radius versus time response of each ELIP, and was found to be correlated with the deflation. Fragmentation and surface mode vibrations were also observed and are shown to depend on the applied acoustic pressure and initial radius. Results from this study can be utilized to optimize the theranostic application of ELIP, e.g. by tuning the size distribution or the excitation frequency.

Effect of Frequency-Dependent Attenuation on Predicted Histotripsy Waveforms in Tissue-Mimicking Phantoms.

Tissue-mimicking phantoms are employed for the assessment of shocked histotripsy pulses in vitro. These broadband shock waves are critical for tissue ablation and are influenced by the frequency-dependent attenuation of the medium. The density, sound speed and attenuation spectra (2-25 MHz) were measured for phantoms that mimic key histotripsy targets. The influence of non-linear propagation relative to the attenuation was described in terms of Gol'dberg number. An expression was derived to estimate the bandwidth of shocked histotripsy pulses for power law-dependent attenuation. The expression is independent of the fundamental frequency of the histotripsy pulse for linear frequency-dependent attenuation.

Trans-Stent B-Mode Ultrasound and Passive Cavitation Imaging.

Angioplasty and stenting of a stenosed artery enable acute restoration of blood flow. However, restenosis or a lack of re-endothelization can subsequently occur depending on the stent type. Cavitation-mediated drug delivery is a potential therapy for these conditions, but requires that particular types of cavitation be induced by ultrasound insonation. Because of the heterogeneity of tissue and stochastic nature of cavitation, feedback mechanisms are needed to determine whether the sustained bubble activity is induced. The objective of this study was to determine the feasibility of passive cavitation imaging through a metal stent in a flow phantom and an animal model. In this study, an endovascular stent was deployed in a flow phantom and in porcine femoral arteries. Fluorophore-labeled echogenic liposomes, a theragnostic ultrasound contrast agent, were injected proximal to the stent. Cavitation images were obtained by passively recording and beamforming the acoustic emissions from echogenic liposomes insonified with a low-frequency (500 kHz) transducer. In vitro experiments revealed that the signal-to-noise ratio for detecting stable cavitation activity through the stent was greater than 8 dB. The stent did not significantly reduce the signal-to-noise ratio. Trans-stent cavitation activity was also detected in vivo via passive cavitation imaging when echogenic liposomes were insonified by the 500-kHz transducer. When stable cavitation was detected, delivery of the fluorophore into the arterial wall was observed. Increased echogenicity within the stent was also observed when echogenic liposomes were administered. Thus, both B-mode ultrasound imaging and cavitation imaging are feasible in the presence of an endovascular stent in vivo. Demonstration of this capability supports future studies to monitor restenosis with contrast-enhanced ultrasound and pursue image-guided ultrasound-mediated drug delivery to inhibit restenosis.

Impulse response method for characterization of echogenic liposomes.

An optical characterization method is presented based on the use of the impulse response to characterize the damping imparted by the shell of an air-filled ultrasound contrast agent (UCA). The interfacial shell viscosity was estimated based on the unforced decaying response of individual echogenic liposomes (ELIP) exposed to a broadband acoustic impulse excitation. Radius versus time response was measured optically based on recordings acquired using an ultra-high-speed camera. The method provided an efficient approach that enabled statistical measurements on 106 individual ELIP. A decrease in shell viscosity, from 2.1 × 10(-8) to 2.5 × 10(-9) kg/s, was observed with increasing dilatation rate, from 0.5 × 10(6) to 1 × 10(7) s(-1). This nonlinear behavior has been reported in other studies of lipid-shelled UCAs and is consistent with rheological shear-thinning. The measured shell viscosity for the ELIP formulation used in this study [κs = (2.1 ± 1.0) × 10(-8) kg/s] was in quantitative agreement with previously reported values on a population of ELIP and is consistent with other lipid-shelled UCAs. The acoustic response of ELIP therefore is similar to other lipid-shelled UCAs despite loading with air instead of perfluorocarbon gas. The methods described here can provide an accurate estimate of the shell viscosity and damping for individual UCA microbubbles.

Broadband attenuation measurements of phospholipid-shelled ultrasound contrast agents.

The aim of this study was to characterize the frequency-dependent acoustic attenuation of three phospholipid-shelled ultrasound contrast agents (UCAs): Definity, MicroMarker and echogenic liposomes. A broadband through-transmission technique allowed for measurement over 2 to 25 MHz with a single pair of transducers. Viscoelastic shell parameters of the UCAs were estimated using a linearized model developed by N. de Jong, L. Hoff, T. Skotland and N. Bom (Ultrasonics 1992; 30:95-103). The effect of diluent on the attenuation of these UCA suspensions was evaluated by performing attenuation measurements in 0.5% (w/v) bovine serum albumin and whole blood. Changes in attenuation and shell parameters of the UCAs were investigated at room temperature (25°C) and physiologic temperature (37°C). The attenuation of the UCAs diluted in 0.5% (w/v) bovine serum albumin was found to be identical to the attenuation of UCAs in whole blood. For each UCA, attenuation was higher at 37°C than at 25°C, underscoring the importance of conducting characterization studies at physiologic temperature. Echogenic liposomes exhibited a larger increase in attenuation at 37°C versus 25°C than either Definity or MicroMarker.

Relationship between cavitation and loss of echogenicity from ultrasound contrast agents.

Ultrasound contrast agents (UCAs) have the potential to nucleate cavitation and promote both beneficial and deleterious bioeffects in vivo. Previous studies have elucidated the pulse-duration-dependent pressure amplitude threshold for rapid loss of echogenicity due to UCA fragmentation. Previous studies have demonstrated that UCA fragmentation was concomitant with inertial cavitation. The purpose of this study was to evaluate the relationship between stable and inertial cavitation thresholds and loss of echogenicity of UCAs as a function of pulse duration. Determining the relationship between cavitation thresholds and loss of echogenicity of UCAs would enable monitoring of cavitation based upon the onscreen echogenicity in clinical applications. Two lipid-shelled UCAs, echogenic liposomes (ELIP) and Definity®, were insonified by a clinical ultrasound scanner in duplex spectral Doppler mode at four pulse durations ('sample volumes') in both a static system and a flow system. Cavitation emissions from the UCAs insonified by Doppler pulses were recorded using a passive cavitation detection system and stable and inertial cavitation thresholds ascertained. Loss of echogenicity from ELIP and Definity® was assessed within regions of interest on B-mode images. A numerical model based on UCA rupture predicted the functional form of the loss of echogenicity from ELIP and Definity®. Stable and inertial cavitation thresholds were found to have a weak dependence on pulse duration. Stable cavitation thresholds were lower than inertial cavitation thresholds. The power of cavitation emissions was an exponential function of the loss of echogenicity over the investigated range of acoustic pressures. Both ELIP and Definity® lost more than 80% echogenicity before the onset of stable or inertial cavitation. Once this level of echogenicity loss occurred, both stable and inertial cavitation were detected in the physiologic flow phantom. These results imply that stable and inertial cavitation are necessary in order to trigger complete loss of echogenicity acoustically from UCAs and this finding can be used when planning diagnostic and therapeutic applications.

Comparison of electrical conductivities of various brain phantom gels: Developing a 'Brain Gel Model'

The use of conducting gels to mimic brain and other tissues is of increasing interest in the development of new medical devices. Currently, there are few such models that can be utilized at physiologic temperatures. In this work, the conductivities of agar, agarose and gelatin gels were manipulated by varying NaCl concentration from 0-1 mg/ml. The AC conductivity was measured at room and physiological temperatures (37°C) in the 100-500 Hz frequency range. Conductivity (σ) was nearly independent of frequency but increased linearly with NaCl concentration and was higher at physiological temperatures in these gels. A formula for predicting conductivity as a function of NaCl concentration was derived for each gel type. The overall goal is to develop a 'brain gel model', for studying low frequency electrical properties of the brain and other tissues at physiological temperatures.

Experimental validation of a finite-difference model for the prediction of transcranial ultrasound fields based on CT images.

The prevalence of stroke worldwide and the paucity of effective therapies have triggered interest in the use of transcranial ultrasound as an adjuvant to thrombolytic therapy. Previous studies have shown that 120 kHz ultrasound enhanced thrombolysis and allowed efficient penetration through the temporal bone. The objective of our study was to develop an accurate finite-difference model of acoustic propagation through the skull based on computed tomography (CT) images. The computational approach, which neglected shear waves, was compared with a simple analytical model including shear waves. Acoustic pressure fields from a two-element annular array (120 and 60 kHz) were acquired in vitro in four human skulls. Simulations were performed using registered CT scans and a source term determined by acoustic holography. Mean errors below 14% were found between simulated pressure fields and corresponding measurements. Intracranial peak pressures were systematically underestimated and reflections from the contralateral bone were overestimated. Determination of the acoustic impedance of the bone from the CT images was the likely source of error. High correlation between predictions and measurements (R(2) = 0.93 and R(2) = 0.88 for transmitted and reflected waves amplitude, respectively) demonstrated that this model is suitable for a quantitative estimation of acoustic fields generated during 40-200 kHz ultrasound-enhanced ischemic stroke treatment.

The effect of static pressure on the inertial cavitation threshold.

The amplitude of the acoustic pressure required to nucleate a gas or vapor bubble in a fluid, and to have that bubble undergo an inertial collapse, is termed the inertial cavitation threshold. The magnitude of the inertial cavitation threshold is typically limited by mechanisms other than homogeneous nucleation such that the theoretical maximum is never achieved. However, the onset of inertial cavitation can be suppressed by increasing the static pressure of the fluid. The inertial cavitation threshold was measured in ultrapure water at static pressures up to 30 MPa (300 bars) by exciting a radially symmetric standing wave field in a spherical resonator driven at a resonant frequency of 25.5 kHz. The threshold was found to increase linearly with the static pressure; an exponentially decaying temperature dependence was also found. The nature and properties of the nucleating mechanisms were investigated by comparing the measured thresholds to an independent analysis of the particulate content and available models for nucleation.