[ES] Los ultrasonidos se han empleado desde los años 90 para el tratamiento de múltiples patologías gracias a su carácter no invasivo y no ionizante, desde el tratamiento localizado del cáncer hasta terapias neurológicas. La focalización de estas ondas de presión y la conformación del haz ha sido un problema que desde varias perspectivas se ha intentado abordar, con el uso de lentes focalizadoras, transductores focalizados y los más sofisticados sistemas phased-array compuestos por múltiples transductores con control electrónico de amplitud y fase. Estos sistemas presentan varios inconvenientes, como el escaso control del haz que ofrecen los transductores focalizados, con un foco fijo y sin control de las posibles distorsiones del campo que puede introducir el medio, o el elevado coste derivado de la compleja electrónica de los sistemas phased-arrays, aunque proporcionan un mejor control del foco y compensación de aberraciones. La revolucionaria idea de los hologramas acústicos como elementos pasivos impresos con tecnología 3D llegó como una alternativa de bajo coste a los previos sistemas. En primer lugar se describió su uso en medios homogéneos para generar las más diversas imágenes acústicas, pero pronto se empezó a estudiar su viabilidad para focalizar haces de ultrasonidos en el interior del cerebro, resultando ser muy útiles en la corrección de las aberraciones que el cráneo introduce en el frente de ondas. Las lentes holográficas son capaces de codificar tanto el campo que se desea generar como las distorsiones de fase que puede introducir el medio en el que se propagan los ultrasonidos. En esta tesis se estudia el diseño de hologramas acústicos y su aplicación en el ámbito biomédico. La tesis puede ser dividida en tres grandes partes: una primera en la cual se describen nuevos métodos para el diseño de lentes holográficas, una segunda en la que se emplean los hologramas ultrasónicos para generación de hipertermia, y una tercera en la que se estudia su uso para terapias transcraneales. En la primera parte de la tesis se investiga el diseño de lentes holográficas para transductores con geometría esférica como alternativa a las lentes planas ya descritas previamente. Además, se estudia un nuevo método para codificar el campo acústico en las lentes de forma que se mejore la imagen producida por ellas, ajustándose más a la deseada. En la segunda parte se estudia cómo es el patrón térmico que genera el campo acústico producido por una lente holográfica cuando se aplica sobre un material con absorción acústica y cómo afecta la difusión térmica a éste. Esta difusión tiene como efecto que el patrón térmico con el tiempo no se parezca al acústico, y que las lentes deban tener en cuenta este proceso en su diseño para aplicaciones térmicas, especialmente si se desean regiones uniformes de calentamiento. Se demuestra cómo la hipertermia generada por ultrasonidos es más dañina en esferoides de células tumorales que la hipertermia tradicional. En la tercera parte se demuestra la viabilidad de los hologramas ultrasónicos para tratamientos neurológicos, aplicados desde la ventana temporal para reducir el eventual calentamiento del hueso que se produce. Además, se estudia en experimentos ex-vivo el campo acústico producido por lentes holográficas a través de un cráneo de macaco, aplicando técnicas de proyección holográfica para obtener aún más información de estas medidas y se diseña un sistema para aplicar estos hologramas en experimentos in-vivo con macacos y comprobar la viabilidad de la apertura de la barrera hematoencefálica de forma localizada. Esta tesis se enfoca a un mejor diseño y entendimiento de las emergentes lentes holográficas, así como a estudiar su validez en aplicaciones biomédicas de gran interés como son la hipertermia, la neuromodulación y la apertura de la barrera hematoencefálica para la administración de fármacos en el cerebro. / [CA] Els ultrasons s'han emprat des dels anys 90 per al tractament de múltiples patologies gràcies al seu caràcter no invasiu i no ionitzant, des del tractament localitzat del càncer fins a teràpies neurològiques. La focalització d'estes ones de pressió i la conformació del feix acústic ha sigut un problema que des de diverses perspectives s'ha intentat abordar, amb l'ús de lents focalizadores, transductors focalitzats i els més sofisticats sistemes phased-array compostos per múltiples transductors amb control individual d'amplitud i fase. Estos sistemes presenten diversos inconvenients, com l'escàs control del feix que ofereixen els transductors focalitzats, amb un focus fix i sense control de les possibles distorsions del camp que pot introduir el medi, o l'elevat cost derivat de la complexa electrònica dels sistemes phased-array, encara que proporcionen un millor control del focus i compensació d'aberracions. La revolucionària idea dels hologrames acústics com a elements passius impresos amb tecnologia 3D va arribar com una alternativa de baix cost als previs sistemes. En primer lloc es va descriure el seu ús en medis homogenis per a generar les més diverses imatges acústiques, però prompte es va començar a estudiar la seua viabilitat per a focalitzar feixos d'ultrasons a l'interior del cervell, resultant ser molt útils en la correcció de les aberracions que el crani introduïx en el front d'ones. Les lents hologràfiques són capaces de codificar tant el camp que es desitja generar com les distorsions de fase que pot introduir el medi en el qual es propaguen els ultrasons. En esta tesi s'estudia el disseny d'hologrames acústics i la seua aplicació en l'àmbit biomèdic. La tesi pot ser dividida en tres grans parts: una primera en la qual se descriuen nous mètodes per al disseny de lents hologràfiques, una segona en la qual s'empren els hologrames ultrasònics per a generació d'hipertèrmia i una tercera en la qual s'estudia el seu ús per a teràpies transcranials. En la primera part de la tesi s'investiga el disseny de lents hologràfiques per a transductors amb geometria esfèrica com a alternativa a les lents planes ja descrites prèviament. A més, s'estudia un nou mètode per a codificar el camp acústic en les lents de manera que es millore la imatge produïda per elles, ajustant-se més a la desitjada. En la segona part s'estudia com és el patró tèrmic que genera el camp acústic produït per una lent hologràfica quan s'aplica sobre un material absorbent i com afecta la difusió tèrmica a aquest. Esta difusió té com a efecte que el patró tèrmic amb el temps no se semble a l'acústic, i les lents hagen de tindre en compte aquest procés en el seu disseny per a aplicacions tèrmiques, especialment si es desitgen regions uniformes de calfament. Es demostra com la hipertèrmia generada per ultrasons és més nociva en esferoides tumorals que la hipertèrmia tradicional. En la tercera part es demostra la viabilitat dels hologrames ultrasònics per a tractaments neurològics, aplicats des de la finestra temporal per a reduir el calfament de l'os que es produïx. A més, s'estudia en experiments ex-vivo el camp acústic produït per lents hologràfiques a través d'un crani de macaco, aplicant tècniques de projecció hologràfica per a obtindre encara més informació d'estes mesures i es dissenya un sistema per a aplicar estos hologrames en experiments in-vivo amb macacos i comprovar la viabilitat de l'obertura de la barrera hematoencefàlica de forma localitzada. Esta tesi s'enfoca a un millor disseny i enteniment de les emergents lents hologràfiques, així com en l'estudi de la seua validesa en aplicacions biomèdiques de gran interès com són la hipertèrmia, la neuromodulació i l'obertura de la barrera hematoencefàlica per a l'administració de fàrmacs en el cervell. / [EN] Ultrasound has been used since the 1990s for the treatment of multiple pathologies thanks to its non-invasive and non-ionising nature, from localised cancer treatment to neurological therapies. The focusing of these pressure waves and beam shaping has been a problem that has been tackled from various perspectives, with the use of focusing lenses, focused transducers and the more sophisticated phased-array systems composed of multiple transducers with electronic amplitude and phase control. These systems have several drawbacks, such as the poor beam control offered by focused transducers, with a fixed focus and no control of possible field distortions introduced by the medium, or the high cost due to the complex electronics of phased-array systems, although they provide better focus control and aberration compensation. The revolutionary idea of acoustic holograms as passive 3D printed elements came as a low-cost alternative to these previous systems. They were first described to be used in homogeneous media to generate a wide range of acoustic images, but their feasibility for focusing ultrasound beams inside the brain was soon studied and proved to be very useful in correcting the aberrations that the skull introduces into the wavefront. Holographic lenses are capable of encoding both the field to be generated and the phase distortions that may be introduced by the medium in which the ultrasound propagates. This thesis studies the design of acoustic holograms and their application in the biomedical field. The thesis can be divided into three main parts: a first one in which new methods for the design of holographic lenses are described, a second one in which ultrasonic holograms are used for hyperthermia generation, and a third one in which their use for transcranial therapies is studied. The first part of the thesis investigates the design of holographic lenses for transducers with spherical geometry as an alternative to the previously described flat lenses. In addition, a new method is studied to encode the acoustic field in the lenses in order to improve the image produced by them, adjusting it more closely to the desired image. The second part studies the thermal pattern generated by the acoustic field produced by a holographic lens when it is applied to an absorbing material and how thermal diffusion affects it. This diffusion has the effect that the thermal pattern over time does not resemble the acoustic pattern, and lenses must take this process into account in their design for thermal applications, especially if uniform regions of heating are desired. The method is tested on multiple tumour spheroids, and results show that ultrasound-mediated hyperthermia is shown to be more damaging to tumour cell spheroids than traditional hyperthermia. The third part demonstrates the feasibility of ultrasonic holograms for neural treatments, applied from the temporal window to reduce the bone heating that occurs. In addition, the acoustic field produced by holographic lenses through a macaque skull is studied in ex-vivo experiments, applying holographic projection techniques to obtain even more information from these experiments. A system is designed to apply these holograms in in-vivo experiments with macaques to test the feasibility of opening the blood-brain barrier in a localized manner. This thesis focuses on a better design and understanding of the emerging holographic lenses, as well as on studying their validity in biomedical applications of great interest such as hyperthermia, neuromodulation and the opening of the blood-brain barrier for drug delivery to the brain. / Andrés Bautista, D. (2024). Acoustic Holograms for Hyperthermia and Transcranial Ultrasound [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/205788

This thesis consists of 3 studies: 1. Determination of the minimal important difference (MID) of the visual analogue scale for dyspnoea (VASD): Determining the MID of the VASD is essential to interpret the results of trials in patients with malignant pleural effusions (MPEs). Patients undergoing a pleural procedure assessed the change in their VASD and the degree of change in their symptoms on a Likert scale. The mean VASD in patients experiencing a ‘small but just worthwhile’ decrease in their symptoms is the MID for the VASD and was found to be 22mm (95% CI 16 - 27mm). 2. Development of a thoracic ultrasound septation score (TUSS): A TUSS is important for objectively assessing the degree of septation within a pleural effusion. An iterative process was used to demonstrate that degree of septation predicts clinical outcome, to identify candidate factors for inclusion in a TUSS and to determine which factors predicted the degree of septation. The final TUSS consisted of an assessment of the degree of homogeneity of septation distribution and number of septations at the most septated area. 3. Effect of an indwelling pleural catheter (IPC) versus standard care for relieving dyspnoea in patients with MPEs: the TIME2 randomised controlled trial (RCT). The objective of this unblinded RCT was to determine whether IPCs are more effective than chest drains and talc pleurodesis at relieving dyspnoea in patients with MPEs. 106 patients were randomised to either IPC or standard care in a 1:1 ratio. The primary outcome was daily VASD over 42 days post intervention. Dyspnoea improved in both groups with no significant difference in mean dyspnoea in the first 42 days (mean score: IPC 25mm (95% CI 19 – 30), standard care 24mm (95% CI 19 – 29)).

Rotator cuff tendinopathy accounts for the majority of cases of shoulder pain, however, the aetiology and pathophysiology of the condition is poorly understood and treatment interventions for the condition have been introduced on an empirical basis, guided only by unproven theories of biological and structural changes in and around the affected tissue. This thesis explores changes in the provision of surgical interventions for rotator cuff tendinopathy, showing a rapid, sustained increase over the last decade. Investigation into the use of ultrasound as a clinical and research tool led to the development of an independent learning method for surgeons using the modality for shoulder imaging and highlighted the technological limitations of contrast enhanced ultrasound in assessing the microvascularity of the supraspinatus tendon. Development of a novel biopsy method for sampling the supraspinatus tendon permitted collection of tissue samples from several cohorts of individuals with rotator cuff tendinopathy. These studies, for the first time, described tissue changes across the whole spectrum of pathology suggesting the possibility of an early inflammatory phase of the condition followed by tissue senescence and reduced viability as pathology progressed. Paired samples taken before and after treatment identified reduced tissue activity in response to glucocorticoid therapy and a potential healing response from the supraspinatus tendon following surgical repair. Significant deterioration in tissue activity and viability was shown as age increased in a large cohort of individuals, highlighting the major impact of ageing as a factor in the onset and progression of rotator cuff tendinopathy. The techniques introduced provide standardised, reproducible methods for further evaluation of rotator cuff tendinopathy and the development of novel therapeutic interventions.

Microalgae synthesize and store large volumes of lipids (potentially over 25% of dry weight) which could provide a renewable source of biodiesel. Traditional extraction techniques often produce poor lipid yields particularly from microalgae with robust cell walls. This project investigated the role of power ultrasound as a cell disruption step in lipid extraction from four microalgal species. Nile Red staining was used to assess the time when ultrasound induced increased membrane permeability in each species and lipids were extracted using an ultrasound assisted Bligh and Dyer extraction method. A 20 kHz probe system (40% amplitude, 0.086 W/cm3) caused increased lipid recovery from dry biomass in all cases; D. salina (no cell wall) from 15 to 22.5% of dry biomass after 1 minute (26% when stressed with 35 g/L NaCl). C. concordia (thin cell wall) from 7.5 to 10.5% of dry biomass after 2 minutes (27% with 25% nitrogen reduction in growth media). N. oculata (thick cell wall) from 6.5 to 10% of dry biomass after 16 minutes (31.5% when stressed with 30 g/L NaCl). The stressed cultures yield could be improved to 35% when ultrasound was combined with S070 beating beads. Chlorella sp. (thick cell wall) from 6.3 to 8.7% of dry biomass, after 16 minutes (44% was achieved when harvested at day 9 instead of 15). A Dual Frequency Reactor (16 and 20 kHz, 0.01 W/cm3) flow system with S070 beads demonstrated that high lipid extraction yields could be achieved on a larger level with N. oculata. After 4:48 minutes sonication 24% lipid recovery was achieved. This system could theoretically increase daily microalgal oil production from 3.96 to 5.76 L per day when compared to conventional techniques, at an extra production cost of only 2.9 p/litre (1.5% increase). D. salina, N. oculata and C. concordia resumed normal growth following sonication at 20 kHz after 1-20 days (8 minutes treatment for D. salina, 60 minutes treatment for N. oculata and 16 minutes treatment for C. concordia). It was found that the supernatant of sonicated D. salina and C. concordia when added to established cultures were able to boost their growth.

Doppler ultrasound imaging modalities arguably represent one of the most complex task performed (usually in real time) by ultrasound scanners. At the heart of these techniques lies the ability to detect and estimate soft tissues or blood motion within the human body. As they have become an invaluable tool in a wide range of clinical applications, these techniques have fostered an intensive effort of research in the field of signal processing for more than thirty years, with a push towards more accurate velocity or displacement estimation. Coded excitation has recently received a growing interest in the medical ultrasound community. The use of these techniques, originally developed in the radar field, makes it possible to increase the depth of penetration in B-mode imaging, while complying with safety standards. These standards impose strict limits on the peak acoustic intensity which can be transmitted into the body. Similar solutions were proposed in the early developments of Doppler flow-meters to improve the resolution / sensitivity trade-off from which typical pulsed Doppler systems suffer. This work discusses the potential improvements in resolution, sensitivity and accuracy achievable in the context of modern Doppler ultrasound imaging modalities (taken in its broadest sense, that is, all the techniques involving the estimation of displacements, or velocities). A theoretical framework is provided for discussing this potential improvements, along with simulations for a more quantitative assessment. Colour Flow Imaging (CFI) modalities are taken as the main reference technique for discussion, due to their historical importance, and their relevance in many clinical applications. The potential achievable improvement in accuracy is studied in the context of modern velocity estimation strategies, which can be broadly classified into narrowband estimators (such as the “Kasai” estimator still widely used in CFI) and time shift based wideband strategies (normalised crosscorrelation estimator used, for instance, in applications like strain or strain rate estimation, elastography, etc.). Finally, simulations and theoretical results are compared to experimental data obtained with a simple custom-designed experimental set-up, using a single-element transducer.

Anxiety and depression are highly prevalent and often comorbid disorders that cause significant personal and economic burdens (Lépine, 2001). Because a significant number of people with depression do not respond to therapy (Fava, 2003), the development of alternative treatment methods may lessen the burden of such mental disorders. Recent research has focused on brain stimulation methods, many of which require invasive surgery or have limited precision in targeting specific areas. Transcranial ultrasound (TUS) is an alternative, noninvasive brain stimulation method that has greater spatial precision than existing methods (Tufail, 2011). TUS has been found to excite neurons in animal brains (Tufail et. al, 2010) and increase positive mood in humans (Sanguinetti et al, 2013). The present study examined TUS, for the first time, as a potential mood intervention. Twenty-four college students with mild to moderate depressive symptoms were randomly assigned to TUS stimulation or TUS sham (no power administered). Participants completed one TUS session each day for five days. Although depression scores did not change differentially for TUS/Sham, trait worry decreased in the stimulation but not the sham condition. Additionally, those in the stimulation condition rated themselves significantly less tense ten minutes after stimulation compared to those in the sham condition. TUS stimulation did not impact a brain electrical activity index associated with approach motivation, frontal asymmetry. These results have significant implications for the potential utility of TUS as an intervention for anxiety disorders or worry-related psychopathology, warranting future investigation of implicated brain electrical activity and mood changes.

The effect of power ultrasound on algae blooms (Microcystis aeruginosa) over a 30 minute period was assessed using 200 and 400 mL suspensions of optical density of 2.0 at 680 nm. The frequencies employed were 20, 40, 580 (40%, 80%, and maximum intensity), 864 (40%, 80% and maximum intensity) and 1146 kHz (40%, 80% and maximum intensity). Ultrasound can induce two different effects on algal cells; inactivation at high power (≥ 0.0022 Wcm-3) and de-agglomeration at low power (≤ 0.0042 Wcm-3). Ultrasonic effects were observed using haemocytometer, optical density, UV-visible spectrometer, fluorospectrometer and flow cytometry. Using a 40 kHz bath (0.0214 Wcm-3) led to de-agglomeration resulting in an overall increase in algae of -0.28% by haemocytometer and -4.20% by optical density. The highest inactivation achieved was 91.54% (haemocytometer) and 44.63% (optical density) using 1146 kHz (maximum intensity, 0.0248 Wcm-3) and 200 mL suspension. In terms of efficiency to achieve inactivation (i.e. inactivation % / power) the best result was observed at 864 kHz (40% power setting, 0.0042 Wcm-3) with 200 mL suspension giving 8226.19 by haemocytometer and 5011.90 by optical density. This initial part of the study allowed a comparison to be made of the ultrasonic parameters that would lead to optimum algae removal in terms of acoustic energy input. The haemocytometer results for cells number were generally higher than those indicated by optical density which is probably due to the fact that the former records only cell numbers remaining whereas the latter is an overall measure of algae concentration (ruptured cells will still register, because their contents remain in suspension). Studies on de-agglomeration and inactivation were also undertaken using small or medium-scale ultrasonic equipment that were models for industrial scale systems. The following volumes of algae suspension and equipment were employed: Sonolator (Sonic Corporation, 5L flow), 16 kHz and 20 kHz Dual Frequency Reactor (DFR, Advanced Sonics LLC, 1L static and 3.5 L flow), 20 kHz Vibrating Tray (Advanced Sonics LLC, 1.5L static) and 20 kHz ultrasonic probe (made at Southeast University, 4L static). The most effective inactivation effects were obtained with the DFR reactor in static mode and 60% power setting for 10 minutes which achieved reductions calculated at 79.25% using haemocytometry and 60.44% by optical density. The third part of this study was to gain a greater understanding of the basic mechanisms of the action of ultrasound on algae and to interpret this in terms of its potential for algal cell removal and control. Algal cell activity was assessed by three methods: using a UV-visible spectrometer (Shimazu, 2450PC), a fluorometer (Shimazu, RF5301) and a flow cytometer (BD FACS Calibur). Ultrasonic damage to Chlorophyll A was revealed through observation of the loss in UV-Vis spectrophotometer peaks around 600 nm together with the decrease in fluorometer results for peaks around 500 and 680 nm. Flow cytometer results were able to identify the number of both intact cells and damaged/ruptured cells thus giving greater insight into the mechanism of ultrasonic inactivation. The direct rupture of cells by power ultrasound was prevalent at low frequencies ≤ 40 kHz due to the mechanical effects of cavitation collapse and inactivation of algal cells by free radicals occurred at high frequencies ≥ 100 kHz and medium powers where mechanical effects are much reduced. In conclusion, this work has shown that power ultrasound can provide a suitable method to control algal growth in small and medium laboratory scales. Scale-up beyond this point is the subject of further research but the results herein clearly demonstrate the importance of choosing the correct ultrasonic parameters in terms of frequency, power and exposure time.

Thesis (MMed (Obstetrics and Gynaecology))--University of Stellenbosch, 2010. / Please refer to full text for abstract.

Acousto-electric (AE) effect comes from an interaction between electrical current and acoustic pressure generated when acoustic waves travel through a conducting material. It currently has two main application areas, ultrasound current source density imaging (UCSDI) and AE hydrophone. UCSDI can detect the current direction by modulating the dipole field with ultrasound pulse, and it is now used to form 3D imaging of dipole changing in one period of treatment, such as arrhythmia in the heart and epilepsy in the brain. As ultrasound pulse passes through electrical field, it convolutes or correlates with the inner product of the electric fields formed by the dipole and detector. The polarity of UCSDI is not determined by Doppler effect that exists in pulse echo (PE) signal, but the gradient of lead field potentials created by dipole and recording electrode, making the base-banded AE voltage positive at the anode and negative at cathode. As convolution shifts spectrum lower, the base band frequency for polarity is different from the center frequency of AE signal. The simulation uses the principles of UCSDI, and helps to understand the phenomena in the experiment. 3-D Fast Fourier Transform accelerates the computing velocity to resolve the correlation in the simulation of AE signal. Most single element hydrophones depend on a piezoelectric material that converts pressure changes to electricity. These devices, however, can be expensive, susceptible to damage at high pressure, and/or have limited bandwidth and sensitivity. An AE hydrophone requires only a conductive material and can be constructed out of common laboratory supplies to generate images of an ultrasound beam pattern consistent with more expensive hydrophones. Its sensitivity is controlled by the injected bias current, hydrophone shape, thickness and width of sensitivity zone. The design of this device needs to be the tradeoff of these parameters. Simulations were made to optimize the design with experimental validation using specifically fabricated devices composed of a resistive element of indium tin oxide (ITO).

Therapeutic efficacy of drugs depends on their ability to reach the treatment target. Drugs that exert their effect within cells are constrained by an inability to cross the cell membrane. Methods are being developed to overcome this barrier including biochemical and biophysical strategies. The application of ultrasound with microbubbles increases the permeability of cell membranes allowing molecules, which otherwise would be excluded, to enter the intracellular space of cells; a phenomenon known as sonoporation. This thesis describes studies aimed at improving our understanding of the mechanism underpinning sonoporation and of the exposure parameters affecting sonoporation efficiency. Cancer cells (KHT-C) in suspension were exposed to ultrasound and microbubbles – total of 97 exposure conditions. The effects on cells were assessed through uptake of cell-impermeable molecules (10 kDa to 2 MDa FITC-dextran), cell viability and microscopic observations of the plasma membrane using flow cytometry, colony assay and electron microscopy techniques. Sonoporation was a result of the interaction of ultrasound and microbubbles with the cell membrane. Disruptions (30-100 nm) were generated on the cell membrane allowing cell impermeable molecules to cross the membrane. Molecules up to 2 MDa in size were delivered at high efficiency (~70% permeabilisation). Sonoporation was short lived; cells re-established their barrier function within one minute, which allowed compounds to remain inside the cell. Following uptake, cells remained viable; ~50% of sonoporated cells proliferated. Sonoporation efficiency depended on ultrasound and microbubble exposure conditions. Microbubble disruption was a necessary but insufficient indicator of ultrasound-induced permeabilisation. The exposure conditions can be tailored to achieve a desired effect; cell permeability of ~70% with ~25% cell death versus permeability of ~35% with ~2% cell death. In addition, sonoporation depended on position in the cell cycle. Cells in later stages were more prone to being permeabilised and killed by ultrasound and microbubbles. This study indicated that sonoporation can be controlled through exposure parameters and that molecular size may not be a limiting factor. However, the transient nature may necessitate that the drug be in close vicinity to target cells in sonoporation-mediated therapies. Future work will extend the investigation into in vivo models.