Dual frequency cavitation event sensor with iodide dosimeter

The inertial cavitation activity depends on the sonication parameters. The purpose of this work is development of dual frequency inertial cavitation meter for therapeutic applications of ultrasound waves. In this study, the chemical effects of sonication parameters in dual frequency sonication (40 kHz and 1 MHz) were investigated in the progressive wave mode using iodide dosimetry. For this purpose, efficacy of different exposure parameters such as intensity, sonication duration, sonication mode, duty factor and net ultrasound energy on the inertial cavitation activity have been studied. To quantify cavitational effects, the KI dosimeter solution was sonicated and its absorbance at a wavelength of 350 nm was measured. The absorbance values in continuous sonication mode was significantly higher than the absorbance corresponding to the pulsed mode having duty factors of 20–80% (p < 0.05). Among different combination modes (1 MHz_100% + 40 kHz_100%, 1 MHz_100% + 40 kHz_80%, 1 MHz_80% + 40 kHz_100%, 1 MHz_80% + 40 kHz_80%), the continuous mode for dual frequency sonication is more effective than other combinations (p < 0.05). The absorbance for this combined dual frequency mode was about 1.8 times higher than that obtained from the algebraic summation of single frequency sonications. It is believed that the optimization of dual frequency sonication parameters at low-level intensity (<3 W/cm²) by optically assisted cavitation event sensor can be useful for ultrasonic treatments.     

Correlation between iodide dosimetry and terephthalic acid dosimetry to evaluate the reactive radical production due to the acoustic cavitation activity

Acoustic cavitation plays an important role in sonochemical processes and the rate of sonochemical reaction is influenced by sonication parameters. There are several methods to evaluate cavitation activity such as chemical dosimetry. In this study, to comparison between iodide dosimetry and terephthalic acid dosimetry, efficacy of sonication parameters in reactive radical production has been considered by iodide and terephthalic acid dosimetries. For this purpose, efficacy of different exposure parameters on cavitations production by 1 MHz ultrasound has been studied. The absorbance of KI dosimeter was measured by spectrophotometer and the fluorescence of terephthalic acid dosimeter was measured using spectrofluorometer after sonication. The result of experiments related to sonication time and intensity showed that with increasing time of sonication or intensity, the absorbance is increased. It has been shown that the absorbance for continuous mode is remarkably higher than for pulsing mode (p-value < 0.05). Also results show that with increasing the duty cycles of pulsed field, the inertial cavitation activity is increased. With compensation of sonication time or intensity in different duty cycles, no significant absorbance difference were observed unless 20% duty cycle. A significant correlation between the absorbance and fluorescence intensities (count) at different intensity (R = 0.971), different sonication time (R = 0.999) and different duty cycle (R = 0.967) were observed (p-value < 0.05). It is concluded that the sonication parameters having important influences on reactive radical production. These results suggest that there is a correlation between iodide dosimetry and terephthalic acid dosimetry to examine the acoustic cavitation activity in ultrasound field.     

In vitro sonodynamic cytotoxicity in regulated cavitation conditions

Sonodynamic toxicity has always been linked to the cavitation phenomenon. In this work, sonodynamic effect with Photofrin^® was evaluated with a new ultrasound device: a regulated cavitation generator. In this way, acoustic intensity was substituted with cavitation level as ultrasound parameter. Photofrin^® potentiated significantly the cavitation cytotoxicity even for low setpoints where no inertial cavitation appeared. Therefore sonodynamic mechanism was principally mechanical, facilitated by the Photofrin^® insertion in cellular cytoplasmic membranes. This assertion was also supported by the fact that sonodynamic cytotoxicity was independent from the Photofrin^® presence or absence in the extracellular medium. Reproducible sonodynamic efficiency was perfectly obtained with this new regulated cavitation generator.     

Sonodynamic inactivation of methicillin-resistant Staphylococcus aureus in planktonic condition by curcumin under ultrasound sonication

Methicillin-Resistant Staphylococcus aureus (MRSA) is an important cause of difficult-to-treat infections. The present study aims to investigate sonodynamic inactivation of MRSA in planktonic condition using curcumin under ultrasound sonication. Dark toxicity of curcumin to MRSA was investigated to choose the concentration range of curcumin used in the study. The uptake of curcumin in MRSA was observed before ultrasound sonication. After sonication colony forming units (CFUs) and bacterial viability were investigated using fluorescence assay. Additionally, chromosomal DNA fragmentation was also analyzed. Curcumin showed no dark toxicity to MRSA in the concentration range of ?500 μM. The maximum uptake of curcumin in MRSA occurred in 50 min after curcumin incubation. Counting of CFUs showed that curcumin had significantly sonodynamic killing effect on MRSA in a curcumin dose-dependent manner, and 5-log reduction in CFU was observed after curcumin treatment (40 μM) at room temperature in the dark for 50 min followed by exposure to ultrasound with intensity of 1.56 W/cm² for 5 min. The ratio of green-fluorescent intensity to red-fluorescent intensity was obviously decreased after curcumin treatment under ultrasound sonication. No significant change in chromosomal DNA was found in the cultured MRSA after the combined treatment of curcumin and ultrasound. These results demonstrated that sonodynamic action of curcumin had significant inactivation of MRSA in planktonic condition.     

The impact of bubbles on measurement of drug release from echogenic liposomes

Echogenic liposomes (ELIP) encapsulate gas bubbles and drugs within lipid vesicles, but the mechanisms of ultrasound-mediated drug release from ELIP are not well understood. The effect of cavitation activity on drug release from ELIP was investigated in flowing solutions using two fluorescent molecules: a lipophilic drug (rosiglitazone) and a hydrophilic drug substitute (calcein). ELIP samples were exposed to pulsed Doppler ultrasound from a clinical diagnostic ultrasound scanner at pressures above and below the inertial and stable cavitation thresholds. Control samples were exposed to a surfactant, Triton X-100 (positive control), or to flow alone (negative control). Fluorescence techniques were used to detect release. Encapsulated microbubbles reduced the measured fluorescence intensity and this effect should be considered when assessing drug release from ELIP. The origin of this effect is not specific to ELIP. Release of rosiglitazone or calcein compared to the negative control was only observed with detergent treatment, but not with ultrasound exposure, despite the presence of stable and inertial cavitation activity. Release of rosiglitazone or calcein from ELIP exposed to diagnostic ultrasound was not observed, even in the presence of cavitation activity. Ultrasound-mediated drug delivery strategies with ELIP will thus rely on passage of the drug-loaded liposomes to target tissues.     

Enhancement of heat transfer in forced convection by using dual low-high frequency ultrasound

Combined sonication with dual-frequency ultrasound has been investigated to enhance heat transfer in forced convection. The test section used for this study consists of a channel with, on one hand, heating blocks normal to the water flow, equipped with thermocouples, and, on the other hand, two ultrasonic emitters. One is facing the heating blocks, thus the ultrasonic field is perpendicular, and the second ultrasonic field is collinear to the water flow. Two types of ultrasonic waves were used: low-frequency ultrasound (25 kHz) to generate mainly acoustic cavitation and high-frequency ultrasound (2 MHz) well-known to induce Eckart’s acoustic streaming. A thermal approach was conducted to investigate heat transfer enhancement in the presence of ultrasound. This approach was completed with PIV measurements to assess the hydrodynamic behavior modifications under ultrasound. Sonochemiluminescence experiments were performed to account for the presence and the location of acoustic cavitation within the water flow. The results have shown a synergetic effect using combined low-and-high-frequency sonication. Enhancement of heat transfer is related to greater induced turbulence within the water flow by comparison with single-frequency sonication. However, the ultrasonically-induced turbulence is not homogeneously distributed within the water flow and the synergy effect on heat transfer enhancement depends mainly on the generation of turbulence along the heating wall. For the optimal configuration of dual-frequency sonication used in this work, a local heat transfer enhancement factor up to 366% was observed and Turbulent Kinetic Energy was enhanced by up to 84% when compared to silent regime.     

Numerical investigation of the inertial cavitation threshold by dual-frequency excitation in the fluid and tissue

Inertial cavitation thresholds, which are defined as bubble growth by 2-fold from the equilibrium radius, by two types of ultrasonic excitation (at the classical single-frequency mode and dual-frequency mode) were calculated. The effect of the dual-frequency excitation on the inertial cavitation threshold in the different surrounding media (fluid and tissue) was studied, and the paramount parameters (driving frequency, amplitude ratio, phase difference, and frequency ratio) were also optimized to maximize the inertial cavitation. The numerical prediction confirms the previous experimental results that the dual-frequency excitation is capable of reducing the inertial cavitation threshold in comparison to the single-frequency one at the same output power. The dual-frequency excitation at the high frequency (i.e., 3.1 + 3.5 MHz vs. 1.1 + 1.3 MHz) is preferred in this study. The simulation results suggest that the same amplitudes of individual components, zero phase difference, and large frequency difference are beneficial for enhancing the bubble cavitation. Overall, this work may provide a theoretical model for further investigation of dual-frequency excitation and guidance of its applications for a better outcome.     

Sonochemiluminescence observation and acoustic detection of cavitation induced by pulsed HIFU at a tissue–fluid interface

The aim of this study is to investigate the mechanism of the erosion process induced by 1.2 MHz pulsed high-intensity focused ultrasound (pulsed HIFU). By using Sonochemiluminescence (SCL) photograph, the initiation and maintenance of active cavitation were observed. In order to understand the role of both inertial cavitation and stable cavitation, a passive cavitation detection (PCD) transducer was used. Since the exposure variables of HIFU are important in the controlled ultrasound tissue erosion, the influence of pulse length (PL) and duty cycle (DC, T_on:T_off) has been examined. The results of tissue hole, SCL observation and acoustic detection revealed that the erosion was highly efficient for shorter PL. For higher DCs, the area of SCL increased with increasing PL. For lower DCs, the area of SCL increased with increasing PL from 10 to 20 μs and then kept constant. For all PLs, the intensity of SCL decreased with lower DC. For all DCs, the intensity of SCL per unit area (the ratio of SCL intensity to SCL area) also decreased with increasing PL from 10 to 80 μs, which suggested that the higher the intensity of SCL is, the higher the efficiency of tissue erosion is. At DC of 1:10, the position of the maximum pixel in SCL pictures was distant from the tissue–fluid interface with the increasing PL because of shielding effect. By the comparison of inertial cavitation dose (ICD) and the stable cavitation dose (SCD), the mechanisms associated with inertial cavitation are very likely to be the key factor of the erosion process.     

Evaluation of correlation between chemical dosimetry and subharmonic spectrum analysis to examine the acoustic cavitation

Currently several therapeutic applications of ultrasound in cancer treatment are under progress which uses cavitation phenomena to deliver their effects. There are several methods to evaluate cavitation activity such as chemical dosimetry and measurement of subharmonic signals. In this study, the cavitation activity induced by the ultrasound irradiation on exposure parameters has been measured by terephthalic acid chemical dosimetry and subharmonic analysis. Experiments were performed in the near 1 MHz fields in the progressive wave mode and effect of duty cycles changes with 2 W/cm² intensity (I_SATA) and acoustic intensity changes in continuous mode on both fluorescence intensity and subharmonic intensity were measured. The dependence between fluorescence intensity of terephthalic acid chemical dosimetry and subharmonic intensity analysis were analyzed by Pearson correlation (p-value < 0.05). It has been shown that the subharmonic intensity and the fluorescence intensity for continuous mode is higher than for pulsing mode (p-value < 0.05). Also results show that there is a significant difference between the subharmonic intensity and the fluorescence intensity with sonication intensity (p-value < 0.05). A significant correlation between the fluorescence intensity and subharmonic intensity at different duty cycles (R = 0.997, p-value < 0.05) and different intensities (R = 0.985, p-value < 0.05) were shown. The subharmonic intensity (μW/cm²) significantly correlated with the fluorescence intensity (count) (R = 0.901; p < 0.05) and the fluorescence intensity due to chemical dosimetry could be estimated with subharmonic intensity due to subharmonic spectrum analysis. It is concluded that there is dependence between terephthalic acid chemical dosimetry and subharmonic spectrum analysis to examine the acoustic cavitation activity.     

Study of a peak in cavitation activity from HIFU exposures using TA fluorescence

Methods of measuring the sound field and focal region of a 1.05 MHz high intensity focused ultrasound (HIFU) are described in this paper. 1.05 MHz pulsed HIFU with intensity 2400 W/cm(2) with a 1:1 duty cycle ("on" phase equaled "off" phase) was used to irradiate terephthalic acid (TA). Pulse periods of 0.5 ms, 1 ms, 3.3 ms, 10 ms, 15 ms, 33 ms, 0.1s and 1s were used. The irradiation time was 2 min. To indicate the intensity of inertial cavitation activity, the fluorescence intensity of hydroxyterephthalic acid (HTA) was measured. The result shows that the cavitation activity of pulsed HIFU peaks at a pulse period of 10 ms, cavitation activity is significantly greater for pulse periods from 2 to 20 ms than for others.     

Stabilizing in vitro ultrasound-mediated gene transfection by regulating cavitation

It is well known that acoustic cavitation can facilitate the inward transport of genetic materials across cell membranes (sonoporation). However, partially due to the unstationary behavior of the initiation and leveling of cavitation, the sonoporation effect is usually unstable, especially in low intensity conditions. A system which is able to regulate the cavitation level during sonication by modulating the applied acoustic intensity with a feedback loop is implemented and its effect on in vitro gene transfection is tested. The regulated system provided better time stability and reproducibility of the cavitation levels than the unregulated conditions. Cultured hepatoma cells (BNL) mixed with 10 μg luciferase plasmids are exposed to 1-MHz pulsed ultrasound with or without cavitation regulation, and the gene transfection efficiency and cell viability are subsequently assessed. Experimental results show that for all exposure intensities (low, medium, and high), stable and intensity dependent, although not higher, gene expression could be achieved in the regulated cavitation system than the unregulated conditions. The cavitation regulation system provides a better control of cavitation and its bioeffect which are crucial important for clinical applications of ultrasound-mediated gene transfection.     

On-demand regulation and enhancement of the nucleation in acoustic droplet vaporization using dual-frequency focused ultrasound

Acoustic droplet vaporization (ADV) plays an important role in focused ultrasound theranostics. Better understanding of the relationship between the ultrasound parameters and the ADV nucleation could provide an on-demand regulation and enhancement of ADV for improved treatment outcome. In this work, ADV nucleation was performed in a dual-frequency focused ultrasound configuration that consisted of a continuous low-frequency ultrasound and a short high-frequency pulse. The combination was modelled to investigate the effects of the driving frequency and acoustic power on the nucleation rate, efficiency, onset time, and dimensions of the nucleation region. The results showed that the inclusion of short pulsed high-frequency ultrasound significantly increased the nucleation rate with less energy, reduced the nucleation onset time, and changed the length–width ratio of the nucleation region, indicating the dual-frequency ultrasound mode yields an efficient enhancement of the ADV nucleation, compared to a single-frequency ultrasound mode. Furthermore, the acoustic and temperature fields varied independently with the dual-frequency ultrasound parameters. This facilitated the spatial and temporal control over the ADV nucleation, and opens the door to the possibility to realize on-demand regulation of the ADV occurrence in ultrasound theranostics. In addition, the improved energy efficacy that is obtained with the dual-frequency configuration lowered the requirements on hardware system, increasing its flexibility and could facilitate its implementation in practical applications.     

Simulations of lesion detection using a combined phased array LHMI-technique

Ultrasound based elasticity imaging techniques have been developed during the past decades. Some of these techniques are based on an internal radiation force stimulation in which a transient or dynamic radiation force is produced by using a single or dual-frequency sonication. In addition, sonication and data acquisition can be implemented using combined or separate transducers. In this simulation study of lesion detection using localized harmonic motion imaging (LHMI), we used a combined phased array designed for simultaneous thermal ablation and lesion detection. In the sonication mode, a focused single-frequency amplitude-modulated sonication is used to induce harmonic motion and in the tracking mode, some of the array elements are used for pulse-echo tracking of the induced displacements. The results showed that the size of the lesion affected the induced displacement around the sonication point. The displacement tracking simulations demonstrated that these changes in the displacement distributions can be detected using only a few of the array elements in the tracking mode but the exact size of the lesion can not be detected accurately. The simulations also showed that two lesions having the radius of 2.5mm can be distinguished if distance between these lesions is at least 2.5mm.     

Exploring the effects of pulsed ultrasound at 205 and 616 kHz on the sonochemical degradation of octylbenzene sulfonate

Compared to continuous wave (CW) ultrasound, pulsed wave (PW) ultrasound has been shown to result in enhanced sonochemical degradation of octylbenzene sulfonate (OBS). However, pulsed ultrasound was investigated under limited pulsing conditions. In this study, pulse-enhanced degradation of OBS was investigated over a broad range of pulsing conditions and at two ultrasonic frequencies (616 and 205 kHz). The rate of OBS degradation was compared to the rate of formation of 2-hydroxyterephthalic acid (HTA) following sonolysis of aqueous terephthalic acid (TA) solutions. This study shows that sonication mode and ultrasound frequency affect both OBS degradation and HTA formation rates, but not necessarily in the same way. Unlike TA, OBS, being a surface active solute, alters the cavitation bubble field by adsorbing to the gas/solution interface of cavitation bubbles. Enhanced OBS degradation rates during pulsing are attributed to this adsorption process. However, negative or smaller pulse enhancements compared to enhanced HTA formation rates are attributed to a decrease in the high-energy stable bubble population and a corresponding increase in the transient bubble population. Therefore, sonochemical activity as determined from TA sonolysis cannot be used as a measure of the effect of pulsing on the rate of degradation of surfactants in water. Over relatively long sonolysis times, a decrease in the rate of OBS degradation was observed under CW, but not under PW conditions. We propose that the generation and accumulation of surface active and volatile byproducts on the surface and inside of cavitation bubbles, respectively, during CW sonolysis is a contributing factor to this effect. This result suggests that there are practical applications to the use of pulsed ultrasound as a method to degrade surface active contaminants in water.     

Effect of polymer surface activity on cavitation nuclei stability against dissolution

The persistence of acoustic cavitation in a pulsed wave ultrasound regime depends upon the ability of cavitation nuclei, i.e., bubbles, to survive the off time between pulses. Due to the dependence of bubble dissolution on surface tension, surface-active agents may affect the stability of bubbles against dissolution. In this study, measurements of bubble dissolution rates in solutions of the surface-active polymer poly(propyl acrylic acid) (PPAA) were conducted to test this premise. The surface activity of PPAA varies with solution pH and concentration of dissolved polymer molecules. The surface tension of PPAA solutions (55-72 dynes/cm) that associated with the polymer surface activity was measured using the Wilhelmy plate technique. Samples of these polymer solutions then were exposed to 1.1 MHz high intensity focused ultrasound, and the dissolution of bubbles created by inertial cavitation was monitored using an active cavitation detection scheme. Analysis of the pulse echo data demonstrated that bubble dissolution time was inversely proportional to the surface tension of the solution. Finally, comparison of the experimental results with dissolution times computed from the Epstein-Plesset equation suggests that the radii of residual bubbles from inertial cavitation increase as the surface tension decreases.     

Stability of alteplase in presence of cavitation

Several experimental studies have demonstrated that ultrasound (US) can accelerate enzymatic fibrinolysis and this effect is further enhanced in the presence of ultrasound contrast agents (UCA). Although UCA have been shown to be safe when administered to ischemic stroke patients, safety information of these agents in the thrombolysis setting is limited. Therefore, in this study we investigated potential adverse effects of acoustic cavitation generated by UCA on alteplase (t-PA), the drug used for treatment of ischemic stroke patients. A volume of 0.9 mL of alteplase was dispensed into a custom-made polyester sample tube. For treatments in the presence or absence of cavitation either 0.1 mL Optison or phosphate buffer saline was combined with alteplase. Three independent samples of each treatment group were exposed to ultrasound of 2 MHz frequency at three different peak negative acoustic pressures of 0.5, 1.7, and 3.5 MPa for a duration of 60 min. All treatments were carried out in a cavitation detection system which was used to insonify the samples and record acoustic emissions generated within the sample. After ultrasound exposure, the treated samples and three untreated drug samples were tested for their enzymatic activity using a chromogenic substrate. The insonified samples containing Optison demonstrated cavitational activity proportional to acoustic pressure. No significant cavitation activity was observed in the absence of Optison. Enzymatic activity of alteplase in both insonified groups was comparable to that in the control group. These tests demonstrated that exposure of alteplase to 60 min of 2 MHz ultrasound at acoustic pressures ranging from 0.5 MPa to 3.5 MPa, in the presence or absence of Optison had no adverse effects on the stability of this therapeutic compound.     

Multibubble sonoluminescence intensity dependence on liquid temperature at different ultrasound intensities.

It is shown that the influence of liquid temperature on the sonoluminescence (SL) intensity is different depending on the ultrasound intensity. At the ultrasound intensities not much higher than the cavitation appearance threshold the SL intensity increases with the temperature. At the ultrasound intensities considerably exceeding the cavitation threshold the SL intensity decreases with an increase of the temperature. At intermediate ultrasound intensities the SL intensity temperature dependence is extreme: the cavitation activity at first increases with temperature, reaches a maximum and then decreases. Continuous and pulsed modes of irradiation at frequencies 880 and 21.9 kHz were used in experiments.     

流动微泡群瞬态空化强度时域分布的比例反馈调节*

声波、血流环境及流动微泡群的稳定性都会影响焦区内瞬态空化强度(ICI)在超声作用时间内的分布,从而影响基于瞬态空化的治疗效率和生物安全性。本文在搭建仿体中流动微泡群瞬态空化发生和实时测量系统的基础上,设计了基于LabView FPGA的比例反馈控制器,在保持脉冲重复频率和脉冲长度不变的条件下,通过选择适当的比例系数,依据当前周期的声波激励下实时测量的ICI,实时调节下一周期声波信号的峰值负压,以调控ICI在时间上的分布。研究表明,在最优比例系数(1 × 107)下,和开环系统相比,ICI的稳定率提升~2.31 倍,ICI的时域下降速率减小~94.41%;在超声作用时间内总ICI也基本达到期望水平。这些结果表明该比例反馈控制器在调控脉冲超声激励下流动微泡群ICI时域分布的有效性,有望改进瞬态空化在相关疾病治疗中的效率和安全性。     

Chemical effects of continuous and pulsed ultrasound in aqueous solutions

Molecules dissolved in water can undergo pyrolysis and free radical attack when cavitation is produced by ultrasound. Macromolecules are also degraded in the main chain. The yields of these processes depend on the volatility and hydrophobicity of the solutes and on the nature of the gas in the solution. The effects of 1 MHz (quartz) and 20 kHz (horn) are compared; the yield ratio of oxidation to mechanical degradation is strongly dependent on the cavitation conditions. Chemical effects produced by pulsed ultrasound are also described and compared with effects observed in continuous irradiation experiments.     

Stable cavitation induces increased cytoplasmic calcium in L929 fibroblasts exposed to 1-MHz pulsed ultrasound

An increase in cytoplasmic calcium (Ca²⁺ increase) is a second messenger that is often observed under ultrasound irradiation. We hypothesize that cavitation is a physical mechanism that underlies the increase in Ca²⁺ in these experiments. To control the presence of cavitation, the wave type was controlled in a sonication chamber. One wave type largely contained a traveling wave (wave type A) while the other wave type largely contained a standing wave (wave type B). Fast Fourier transform (FFT) analysis of a sound field produced by the wave types ascertained that stable cavitation was present only under wave type A ultrasound irradiation. Under the two controlled wave types, the increase in Ca²⁺ in L929 fibroblasts was observed with fluorescence imaging. Under wave type A ultrasound irradiation, an increase in Ca²⁺ was observed; however, no increase in Ca²⁺ was observed under wave type B ultrasound irradiation. We conclude that stable cavitation is involved in the increase of Ca²⁺ in cells subjected to pulsed ultrasound.