Design and implementation of capacitive micromachined ultrasonic transducers for high intensity focused ultrasound

High intensity focused ultrasound (HIFU) is a medical procedure for noninvasive treatment of cancers. High intensity focused ultrasound is used to heat and destroy the diseased tissue. Piezoelectricity has been the core mechanism for generation of ultrasound waves in the treatment. Focusing can be done by using spherically curved transducers or using a lens or electronically steering sound waves by using phased arrays. Current research in HIFU technology targets the development of MR-guided miniaturized ultrasonic probes for treatment of cancerous tumors. Capacitive micromachined ultrasonic transducer (CMUT) is an alternative technology to generate and detect ultrasound. CMUT consists of a suspended membrane The advances in CMUT technology, enables fabricating tiny transducer arrays with wide bandwidth makes them a strong candidate for the application. In this thesis, a new methodology is proposed to design and operate CMUTs to generate high pressures under continuous wave excitation. An accurate nonlinear circuit model of CMUT is developed and the model is carried into a SPICE (Simulation Program with Integrated Circuit Emphasis) simulator for fast simulations. The model includes the radiation impedance of the array, thus the operation in a fluid environment can be simulated. The model is verified by doing FEM simulations. The circuit model provides a novel optimization tool for CMUT operating in non-collapse mode. The optimized CMUT parameters are presented and a sample fabrication is done using anodic bonding process. With the process, a 100 m thick silicon wafer is bonded to a glass substrate. A new driving scheme is proposed without a need of DC voltage. Thus, the charge trapping problem in CMUT operation is eliminated. The fabricated device provides 1.8 MPa surface pressure with -28dB second harmonic for a maximum 125V drive voltage at 1.44 MHz which is currently a state of art performance of a CMUT under continuous wave excitation

How sonoporation disrupts cellular structural integrity: morphological and cytoskeletal observations

Posters: no. 1Control ID: 1672429OBJECTIVES: In considering sonoporation for drug delivery applications, it is essential to understand how living cells respond to this puncturing force. Here we seek to investigate the effects of sonoporation on cellular structural integrity. We hypothesize that the membrane morphology and cytoskeletal behavior of sonoporated cells under recovery would inherently differ from that of normal viable cells. METHODS: A customized and calibrated exposure platform was developed for this work, and the ZR-75-30 breast carcinoma cells were used as the cell model. The cells were exposed to either single or multiple pulses of 1 MHz ultrasound (pulse length: 30 or 100 cycles; PRF: 1kHz; duration: up to 60s) with 0.45 MPa spatial-averaged peak negative pressure and in the presence of lipid-shelled microbubbles. Confocal microscopy was used to examine insitu the structural integrity of sonoporated cells (identified as ones with exogenous fluorescent marker internalization). For investigations on membrane morphology, FM 4-64 was used as the membrane dye (red), and calcein was used as the sonoporation marker (green); for studies on cytoskeletal behavior, CellLight (green) and propidium iodide (red) were used to respectively label actin filaments and sonoporated cells. Observation started from before exposure to up to 2 h after exposure, and confocal images were acquired at real-time frame rates. Cellular structural features and their temporal kinetics were quantitatively analyzed to assess the consistency of trends amongst a group of cells. RESULTS: Sonoporated cells exhibited membrane shrinkage (decreased by 61% in a cell’s cross-sectional area) and intracellular lipid accumulation (381% increase compared to control) over a 2 h period. The morphological repression of sonoporated cells was also found to correspond with post-sonoporation cytoskeletal processes: actin depolymerization was observed as soon as pores were induced on the membrane. These results show that cellular structural integrity is indeed disrupted over the course of sonoporation. CONCLUSIONS: Our investigation shows that the biophysical impact of sonoporation is by no means limited to the induction of membrane pores: e.g. structural integrity is concomitantly affected in the process. This prompts the need for further fundamental studies to unravel the complex sequence of biological events involved in sonoporation.postprin

Developmental delays and subcellular stress as downstream effects of sonoporation

Posters: no. 2Control ID: 1672434OBJECTIVES: The biological impact of sonoporation has often been overlooked. Here we seek to obtain insight into the cytotoxic impact of sonoporation by gaining new perspectives on anti-proliferative characteristics that may emerge within sonoporated cells. We particularly focused on investigating the cell-cycle progression kinetics of sonoporated cells and identifying organelles that may be stressed in the recovery process. METHODS: In line with recommendations on exposure hardware design, an immersion-based ultrasound platform has been developed. It delivers 1 MHz ultrasound pulses (100 cycles; 1 kHz PRF; 60 s total duration) with 0.45 MPa peak negative pressure to a cell chamber that housed HL-60 leukemia cells and lipid-shelled microbubbles at a 10:1 cell-tobubble ratio (for 1e6/ml cell density). Calcein was used to facilitate tracking of sonoporated cells with enhanced uptake of exogenous molecules. The developmental trend of sonoporated cells was quantitatively analyzed using BrdU/DNA flow cytometry that monitors the cell population’s DNA synthesis kinetics. This allowed us to measure the temporal progression of DNA synthesis of sonoporated cells. To investigate whether sonoporation would upset subcellular homeostasis, post-exposure cell samples were also assayed for various proteins using Western blot analysis. Analysis focus was placed on the endoplasmic reticulum (ER): an important organelle with multi-faceted role in cellular functioning. The post-exposure observation time spanned between 0-24 h. RESULTS: Despite maintaining viability, sonoporated cells were found to exhibit delays in cell-cycle progression. Specifically, their DNA synthesis time was lengthened substantially (for HL-60 cells: 8.7 h for control vs 13.4 h for the sonoporated group). This indicates that sonoporated cells were under stress: a phenomenon that is supported by our Western blot assays showing upregulation of ER-resident enzymes (PDI, Ero1), ER stress sensors (PERK, IRE1), and ER-triggered pro-apoptotic signals (CHOP, JNK). CONCLUSIONS: Sonoporation, whilst being able to facilitate internalization of exogenous molecules, may inadvertently elicit a cellular stress response. These findings seem to echo recent calls for reconsideration of efficiency issues in sonoporation-mediated drug delivery. Further efforts would be necessary to improve the efficiency of sonoporation-based biomedical applications where cell death is not desirable.postprin

A study on the change in plasma membrane potential during sonoporation

Posters: no. 4Control ID: 1680329OBJECTIVES: There has been validated that the correlation of sonoporation with calcium transients is generated by ultrasound-mediated microbubbles activity. Besides calcium, other ionic flows are likely involved in sonoporation. Our hypothesis is the cell electrophysiological properties are related to the intracellular delivery by ultrasound and microbubbles. In this study, a real-time live cell imaging platform is used to determine whether plasma membrane potential change is related to the sonoporation process at the cellular level. METHODS: Hela cells were cultured in DMEM supplemented with 10% FBS in Opticell Chamber at 37 °C and 5% CO2, and reached 80% confluency before experiments. The Calcein Blue-AM, DiBAC4(3) loaded cells in the Opticell chamber filled with PI solution and Sonovue microbubbles were immerged in a water tank on a inverted fluorescence microscope. Pulsed ultrasound (1MHz freq., 20 cycles, 20Hz PRF, 0.2-0.5MPa PNP) was irradiated at the angle of 45° to the region of interest for 1s.The real-time fluorescence imaging for different probes was acquired by a cooled CCD camera every 20s for 10min. The time-lapse fluorescence images were quantitatively analyzed to evaluate the correlation of cell viability, intracellular delivery with plasma membrane potential change. RESULTS: Our preliminary data showed that the PI fluorescence, which indicated intracellular delivery, was immediately accumulated in cells adjacent to microbubbles after exposure, suggesting that their membranes were damaged by ultrasound-activated microbubbles. However, the fluorescence reached its highest level within 4 to 6 minutes and was unchanged thereafter, indicating the membrane was gradually repaired within this period. Furthermore, using DIBAC4(3), which detected the change in the cell membrane potential, we found that the loss of membrane potential might be associated with intracellular delivery, because the PI fluorescence accumulation was usually accompanied with the change in DIBAC4 (3) fluorescence. CONCLUSIONS: Our study suggests that there may be a linkage between the cell membrane potential change and intracellular delivery mediated by ultrasound and microbubbles. We also suggest that other ionic flows or ion channels may be involved in the cell membrane potential change in sonoporation. Further efforts to explore the cellular mechanism of this phenomenon will improve our understanding of sonoporation.postprin

Evaluation of harmonic motion elastography and acousto-optic imaging for monitoring lesion formation by high intensity focused ultrasound

Malignant or benign tumors may be ablated with high‐intensity focused ultrasound (HIFU). This technique, known as focused ultrasound surgery (FUS), has been actively investigated for decades, but slow to be implemented and difficult to control due to lack of real‐time feedback during ablation. Two methods of imaging and monitoring HIFU lesions during formation were implemented simultaneously, in order to investigate the efficacy of each and to increase confidence in the detection of the lesion. The first, Acousto‐Optic Imaging (AOI) detects the increasing optical absorption and scattering in the lesion. The intensity of a diffuse optical field in illuminated tissue is mapped at the spatial resolution of an ultrasound focal spot, using the acousto‐optic effect. The second, Harmonic Motion Imaging (HMI), detects the changing stiffness in the lesion. The HIFU beam is modulated to force oscillatory motion in the tissue, and the amplitude of this motion, measured by ultrasound pulse‐echo techniques, is influenced by the stiffness. Experiments were performed on store‐bought chicken breast and freshly slaughtered bovine liver. The AOI results correlated with the onset and relative size of forming lesions much better than prior knowledge of the HIFU power and duration. For HMI, a significant artifact was discovered due to acoustic nonlinearity. The artifact was mitigated by adjusting the phase of the HIFU and imaging pulses. A more detailed model of the HMI process than previously published was made using finite element analysis. The model showed that the amplitude of harmonic motion was primarily affected by increases in acoustic attenuation and stiffness as the lesion formed and the interaction of these effects was complex and often counteracted each other. Further biological variability in tissue properties meant that changes in motion were masked by sample‐to‐sample variation. The HMI experiments predicted lesion formation in only about a quarter of the lesions made. In simultaneous AOI/HMI experiments it appeared that AOI was a more robust method for lesion detection.Bernard M. Gordon Center for Subsurface and Imaging Systems (CenSSIS) via the NSF ERC award number EEC‐9986821

Real-time imaging of cellular dynamics during low-intensity pulsed ultrasound exposure

Control ID: 1671584Oral Session 5 - Bioeffects of therapeutic ultrasoundOBJECTIVE: Although the therapeutic potential of low-intensity pulsed ultrasound is unquestionable, the wave-matter interactions involved in the process remain to be vaguely characterized. Here we seek to undertake a series of in-situ cellular imaging studies that aim to analyze the mechanical impact of low-intensity pulsed ultrasound on attached fibroblasts from three different aspects: membrane, cytoskeleton, and nucleus. METHODS: Our experimental platform comprised an in-house ultrasound exposure hardware that was coupled to a confocal microscopy system. The waveguided ultrasound beam was geometrically aligned to the microscope’s fieldof-view that corresponds to the center of a polystyrene dish containing fibroblasts. Short ultrasound pulses (5 cycles; 2 kHz PRF) with 0.8 MPa peak acoustic pressure (0.21 W/cm2 SPTA intensity) were delivered over a 10 min period. Live imaging was performed on both membrane (CellMask) and cytoskeleton (actin-GFP, tubulin-RFP) over the entire observation period (up to 30 min after end of exposure). Also, pre- and post-exposure fixed-cell imaging was conducted on the nucleus (Hoechst 33342) and two cytoskeleton components related to stress fibers: F-actin (phalloidin-FITC) and vincullin (Alexa Fluor 647 conjugated). To study whether mechanotransduction was responsible in mediating ultrasound-cell interactions, some experiments were conducted with the addition of gadolinium that blocks stretch-sensitive ion channels. RESULTS: Cell shrinkage was evident over the course of low-intensity pulsed ultrasound exposure. This was accompanied with contraction of actin and tubulin. Also, an increase in central stress fibers was observed at the end of exposure, while the nucleus was found to have decreased in size. Interestingly, after the exposure, a significant rebound in cell volume was observed over a 30 min. period. These effects were not observed in cases with gadolinium blockage of mechanosensitive ion channels. CONCLUSIONS: Our results suggest that low-intensity pulsed ultrasound would transiently induce remodeling of a cell’s membrane and cytoskeleton, and it will lead to repression of nucleus. This indicates that ultrasound after all represents a mechanical stress on cellular membrane. The post-exposure outgrowth phenomenon is also of practical relevance as it may be linked to the stimulatory effects that have been already observed in low-intensity pulsed ultrasound treatments.postprin

Master of Science

thesisHemodialysis vascular access, the interface between a dialysis patient and a dialysis machine, is quite literally the lifeblood of a patient's health. Vascular access dysfunction is the leading cause of hospitalization in hemodialysis patients. The occlusive growth of neointimal hyperplasia (NH) in expanded polytetrafluoroethylene (ePTFE) ringed grafts is the primary cause of failure. To further develop a proposed thermal ultrasound treatment to reduce or prevent NH in arteriovenous vascular grafts, the acoustic properties of ePTFE were studied in water and alcohol solutions. Previous reports of ePTFE acoustic properties are critiqued. It was found that the acoustic transmission and attenuation through ePTFE, and therefore the potential for an ultrasound-based therapy for NH, are heavily dependent on the medium in which the graft is immersed, suggesting that the acoustic properties of implanted grafts will change as grafts mature in vivo. The acoustic impedance and attenuation of water-soaked ePTFE were 0.478 ± 1.43 × 10-2 MRayl and 1.78 ± 0.111 Np/cm·MHz, respectively, while the acoustic impedance and attenuation of ePTFE in alcohol were 1.49 ± 0.149 MRayl and 0.77 ± 1.1 × 10-2 Np/cm·MHz, respectively. The use of focused ultrasound to heat implanted ringed ePTFE grafts was numerically modeled from 1.35- and 1.443-MHz transducers for in vitro geometries. Power deposition and heating, in turn, differed by an order of magnitude between various graft acoustic properties. Graft rings were predicted to be substantial absorbing and iv scattering features. In vitro phantom models were constructed: one with and one without thermocouples. At 1 W of acoustic power, the maximum temperature rise was 8˚ C. The thermocouple model containing a water-soaked graft did not experience heating in the far graft wall. The MRTI model confirmed that the graft rings are an absorbing/scattering feature. Heating was not prevented in the presence of water flow through the graft. Water was not heated significantly. Overall, results suggest ultrasound exposure can be used to generate temperature rises corresponding with the potential prevention or inhibition of NH in ringed ePTFE vascular grafts. A hybrid therapeutic/diagnostic transducer design with a therapeutic semi-annular array surrounding a diagnostic linear array is presented. Compared to a solid transducer of the same dimensions, there were only marginal aberrations in the focal plane. Numerical optimization of the element drive configuration indicated that the least distorted focal plane was produced by uniform phase and magnitude at each element

Study on localized thermal expansion gradient formation for acoustic wave generation in a novel thermoacoustic imaging modality

Thermoacoustics is the process of generation of sound by heat or vice versa. Volume generated thermoacoustic signals can be produced by thermal expansion induced volume contraction and rarefaction inside a target body. Thermoacoustic imaging uses this modality to obtain vivid insight into the internal structure of target body, both for non-destructive testing and biomedical imaging. Any penetrating pulsed radiation can be used for such purpose, including microwave where the modality is called thermoacoustics in general or by incident light waves where the same is termed as photoacoustics. The current thesis establishes the theoretical basis for a novel thermoacoustic imaging modality where pulsed ultrasound is used as the incident penetraing source. A formal forward transient theoretical eqution set is derived based on established transient acoustic propagation models and the problem is solved using a commercially available FEM software. The results are then compared with experimental results and considerable agreement has been observed

Design, modelling, characterization and implementation of acoustic lenses for modulation of ultrasound beams.

Tesis por compendio[ES] La capacidad de controlar y modificar los haces de energía ha sido motivo de investigación por parte de la comunidad científica desde largo tiempo atrás. En el campo de la acústica, este control energético de las ondas mecánicas tiene numerosas aplicaciones. Desde las aplicaciones industriales, alimentarias, farmacéuticas, etcétera hasta la biomedicina. Esta tesis se basa en la aplicación del control y modulación focal de los ultrasonidos para el uso en este último campo. Se puede modular y controlar los focos de ultrasonidos de diferentes formas. En este caso, se han desarrollado lentes planas que utilizan el principio de la difracción para lograr focalizar los haces. Las ventajas del uso de lentes planas de focalización permiten ser implementadas de forma sencilla en procesos de mecanización e incluso mediante impresión 3D. Se propone utilizar transductores planos que al emitir sobre una lente acústica, se produzca una conformación focal de características controladas. La lente conocida como lente de Fresnel (FZP) ha sido escogida como base de diseño en la implementación de las diferentes soluciones que logran cumplir con los objetivos marcados. Mediante la aplicación de modificaciones en una FZP se puede lograr pasar de una lente con capacidades extraordinarias de focalización a una lente capaz de controlar la resolución lateral y la profundidad de foco e incluso mejorar la ganancia. El objetivo final de aplicación es el uso en transductores de ultrasonidos de alta intensidad conocidos como HIFU. Mejorar la capacidad de resolución hace que se puedan desarrollar mejores terapias oncológicas que supongan un índice mayor de éxito en la lucha contra el cáncer. En la presente tesis se ha propuesto, además, una novedosa lente FZP basada en el cambio de fase que puede resultar un antes y un después en aplicaciones biomédicas. Se ha conseguido no solo mejorar la eficiencia de una FZP, sino que se ha conseguido implementar en materiales compatibles con resonancia magnética. Se han desarrollado modelos numéricos basados en el método de los elementos finitos que emulan la física involucrada. Las medidas han sido realizadas en condiciones controladas por un sistema robotizado de alta precisión. Todos los resultados obtenidos y publicados han sido desarrollados de forma numérica y experimental, validándose el método de trabajo y dando consistencia a las soluciones propuestas.[CA] La capacitat de controlar i modificar els feixos d'energia ha sigut motiu d'investigació per part de la comunitat científica des de llarg temps arrere. En el camp de l'acústica, este control energètic de les ones mecàniques té nombroses aplicacions. Des de les aplicacions industrials, alimentàries, farmacèutiques, etcètera fins la biomedicina. Esta tesi es basa en l'aplicació del control i modulació focal dels ultrasons per a l'ús en este últim camp. Es pot modular i controlar els focus d'ultrasons de diferents formes. En este cas, s'han desenvolupat lents planes que utilitzen el principi de la difracció per a aconseguir focalitzar els feixos. Els avantatges de l'ús de lents planes de focalització permeten ser implementades de forma senzilla en processos de mecanització i inclús per mitjà d'impressió 3D. Es proposa utilitzar transductores plans que a l'emetre sobre una lent acústica, es produïsca una conformació focal de característiques controlades. La lent coneguda com a lent de Fresnel (FZP) ha sigut triada com a base de disseny en la implementació de les diferents solucions que aconseguixen complir amb els objectius marcats. Per mitjà de l'aplicació de modificacions en una FZP es pot aconseguir passar d'una lent amb capacitats extraordinàries de focalització a una lent capaç de controlar la resolució lateral i la profunditat de focus i inclús millorar el guany. L'objectiu final d'aplicació és l'ús en transductores d'ultrasons d'alta intensitat coneguts com HIFU. Millorar la capacitat de resolució fa que es puguen desenvolupar millors teràpies oncològiques que suposen un índex major d'èxit en la lluita contra el càncer. En la present tesi s'ha proposat, a més, una nova lent FZP basada en el canvi de fase que pot resultar un abans i un després en aplicacions biomèdiques. S'ha aconseguit no sols millorar l'eficiència d'una FZP, sinó que s'ha aconseguit implementar en materials compatibles amb ressonància magnètica. S'han desenvolupat models numèrics basats en el mètode dels elements finits que emulen la física involucrada. Les mesures han sigut realitzades en condicions controlades per un sistema robotitzat d'alta precisió. Tots els resultats obtinguts i publicats han sigut desenvolupats de forma numèrica i experimental, validant-se el mètode de treball i donant consistència a les solucions proposades.[EN] The ability to control and modify energy beams has been the subject of research by the scientific community for a long time. In the acoustic field, this energetic control of mechanical waves has numerous applications. From industrial, food, pharmaceutical applications, et cetera, to biomedicine. This thesis is based on the ultrasound control and focal modulation applications. It is possible to modulate and control the ultrasound focii in different ways. In this case, flat lenses were developed based on the principle of diffraction to focus the beams. The advantages of using flat focusing lenses allow them to be easily implemented in machining and drilling processes and even through 3D printing. It was proposed to use planar transducers that when emitting on an acoustic lens, controlled characteristics of focal conformation were produced. The lens known as Fresnel Zone Plane (FZP) was chosen as the implementation design basis for the different solutions that manage to fulfill the objectives set. By applying modifications to an FZP it was possible to go from a lens with extraordinary focusing capabilities to a lens that was capable to control lateral resolution, depth of focus and even improving the gain. The final objective application was the use in high intensity ultrasound transducers known as HIFU. Improving the ability to resolve makes it possible to develop better cancer therapies that represent a higher rate of success in the fight against cancer. In the present thesis, a novel FZP lens based on phase change has also been proposed that can be a before and after in biomedical applications. It has not only been possible to improve the efficiency of an FZP, but it has also been possible to implement it in materials compatible with magnetic resonance imaging. Numerical models based on the finite element method were developed for emulating the involved physics. Measurements were carried out under controlled conditions by a high precision robotic system. All the results obtained and published were developed numerically and experimentally, validating the working method and giving consistency to the proposed solutions.I want to acknowledge the following public funding sources that have made possible this research: Grant BES-2016-077133 (Ministerio de Ciencia, Innovación y Universidades de España) Project TEC2015-70939-R (MINECO/FEDER). Tomsk Polytechnic University within the framework of Tomsk Polytechnic University Competitiveness Enhancement Program.Tarrazó Serrano, D. (2020). Design, modelling, characterization and implementation of acoustic lenses for modulation of ultrasound beams [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/159895TESISCompendi