Microbubble ultrasound (US) contrast agents have been used to monitor the progression of anti-angiogenic chemotherapies. However, US backscatter measurements used in contrast imaging are inherently variable, given the presence of many microbubbles of random position and size. A model was developed to investigate the influence of US scanner and microbubble characteristics on these variable measurements. The Coefficient of Variation was used to measure variability. It was found that an optimum excitation frequency exists that minimizes this variability. In the case of DefinityTM, a 2.25 MHz centre-frequency pulse yielded a less variable measurement than at 5 MHz. Conversely, decreasing microbubbble concentration was found to significantly increase variability. Evidence suggests that microbubbles are no longer Rayleigh scatterers at sufficient low concentrations. Post-processing was found to aid in reducing measurement variability by averaging samples where microbubble positions are uncorrelated. As well, reduction can be achieved by averaging about a region-of-interest of uniform perfusion.

The utility of microbubble agents in a variety of diagnostic and therapeutic ultrasound techniques has been widely demonstrated, most notably in Contrast Enhanced Ultrasound (CEUS) imaging. Unfortunately, the underlying mechanisms of their response to ultrasound excitation are poorly understood, restricting the development of promising techniques, such as quantitative perfusion imaging. A significant reason for this is that current microbubble characterisation techniques suffer from one or more of the following limitations: i) large experimental uncertainties, ii) physical restrictions on microbubble response and iii) failure to provide large data sets suitable for statistical analysis. This thesis presents a new technique to overcome these limitations. A co-axial microfluidic device is used to hydrodynamically confine microbubbles through the focal region of a laser and ultrasound field. The magnitude of light scattered by isolated microbubbles during ultrasound excitation is converted to radius using Mie Scattering theory. This technique is capable of obtaining large samples (>10³/min) of microbubbles to be efficiently characterised. The response of a commercial contrast agent, SonoVue®, is first investigated for a range of ultrasound exposure parameters; frequency (2 MHz - 4.5 MHz), peak negative pressure (6 kPa - 400 kPa) and pulse length (3 cycles - 8 cycles). Second the device is used to investigate the effect of composition and fabrication on microbubble response to similar ultrasound conditions. The results demonstrate a very large variability in microbubble response independent of initial size, indicating a significant lack of uniformity of coating properties. This is further supported by quantitative fluorescence imaging and quasi-static pressure chamber measurements. The implications of the findings for CEUS imaging and the development of microbubble contrast agents are discussed, as well as the limitations and suggested improvements of the characterisation technique.

O presente trabalho teve como objetivo avaliar as interações entre bolhas com distintos tamanhos (diâmetros) e partículas minerais e o efeito de combinações destas bolhas na flotação de minérios, em escala de bancada. As bolhas empregadas nesses estudos foram: i) Nanobolhas – NBs, diâmetro médio de 150 – 200 nm; ii) Microbolhas – MBs, diâmetro médio de 70 μm; e iii) Macrobolhas – MaBs, diâmetro médio de 1 mm. As dispersões de NBs e MBs foram geradas a partir da despressurização de água saturada com ar e cavitação hidrodinâmica em válvula agulha. As pressões de saturação foram, respectivamente, 2,5 e 4,0 bar. Os estudos de interação bolhas-partícula mineral foram desenvolvidos utilizando um sistema fotográfico que permitiu avaliar a adesão de bolhas às partículas de quartzo (grão de alta pureza) e apatita em função do tipo de bolhas injetadas. Os resultados mostraram que a maior adesão de bolhas no grão de apatita ocorreu com a combinação de NBs + MBs + MaBs e, no caso do quartzo, uma expressiva concentração de MaBs aderidas foi obtida após “condicionamento” do mineral com NBs. As NBs ficaram confinadas às superfícies dos grãos minerais devido à dissipação da energia livre superficial dos sólidos em função de sua rugosidade e, desta forma, aumentaram o mecanismo de adesão de outras bolhas (MBs e/ou MaBs). O estudo de avaliação das associações de bolhas na flotação foi realizado com os sistemas minerais quartzo e minério fosfático. No caso do minério fosfático, constituído por 35% de partículas finas (< 37 μm), os estudos avaliaram a combinação de i) NBs + MaBs; ii) MBs + MaBs; e iii) NBs + MBs + MaBs, nos parâmetros cinéticos e de separação na flotação, em relação ao ensaio standard, que emprega o uso de apenas MaBs. Os efeitos foram avaliados em quatro situações que compreenderam variação de porcentagem de sólidos (28 e 35% em peso) e concentração de reagentes (1000 g.t-1 de coletor + 600 g.t-1 de depressor; e 500 g.t-1 de coletor + 300 g.t-1 de depressor; sendo que o coletor de apatita empregado foi óleo de soja saponificado com hidróxido de sódio e o depressor das partículas de ganga - magnetita, carbonatos, micas, quartzo, diopsídio e perovskita/anatásio - foi amido de milho gelatinizado com hidróxido de sódio). O emprego combinado de bolhas apresentou incrementos na recuperação de P2O5, com pequena queda no teor de P2O5 e elevada cinética, em relação aos ensaios STD em praticamente todas as condições estudadas, com valores variando entre 0,2 e 8.9%. Os incrementos quando a porcentagem de sólidos foi de 35% p/p (até 2,7%) foram consideravelmente menores que os obtidos com 28% de sólidos (até 8,9%). Em algumas condições, o uso concomitante de MBs + MaBs e NBs + MBs + MaBs foi capaz de superar, já na primeira coleta (1,5 min de ensaio), a recuperação total de fosfato obtida no ensaio standard. No caso do quartzo, os ensaios de microflotação (realizados em tubo de Hallimond modificado) com e sem a presença de NBs mostraram que a associação de NBs às MaBs aumentou a recuperação de quartzo (até 39%) em praticamente todas as condições estudadas, mostrando sua efetividade tanto com partículas finas quanto com as mais grossas. Já os ensaios realizados em célula mecânica com e sem adição de NBs, mostraram que o emprego de NBs foi responsável pelo aumento de 23 pontos percentuais na recuperação de quartzo. Ainda, a cinética de flotação aumentou significativamente e já no primeiro min do ensaio com NBs obteve-se cerca do dobro da recuperação de quartzo do ensaio standard. Os resultados obtidos na dissertação mostram o elevado potencial das NBs combinadas a outras distribuições de tamanhos de bolhas na melhoria do processo de adesão bolha-partícula e na flotação de minérios fosfáticos finos e de frações granulométricas distintas de quartzo, principalmente em tempos curtos. / The objective of this work was to evaluate the interactions between bubbles with different sizes (diameters) and mineral particles and the effect of the combinations of these bubbles on the flotation of ores, on a bench scale. The bubbles used in these studies were: i) Nanobubbles - NBs, mean diameter of 150-200 nm; ii) Microbubbles - MBs, average diameter of 70 μm; and iii) Macrobubbles - MaBs, average diameter of 1 mm. Dispersions of NBs and MBs were generated after depressurizing-cavitation of the saturated water in air. The saturation pressures were, respectively, 2.5 and 4.0 bar. The bubble-particle mineral interaction studies were developed using a photographic system that allowed to evaluate the adhesion of bubbles to the particles of quartz (high purity grain) and apatite according to the type of bubbles injected. The results showed that the highest adhesion of bubbles in the apatite grain occurred with the combination of NBs + MBs + MaBs and, in the case of quartz, an expressive concentration of adhered MaBs was obtained after "conditioning" of the mineral with NBs. The NBs were confined to the surfaces of the mineral grains as a result of the dissipation of the free surface energy of the solids due to their roughness and, therefore, it increased the adhesion mechanism of other bubbles (MBs and / or MaBs). The study of the evaluation of the associations of bubbles in the flotation was carried out with the mineral systems quartz and phosphate ore. In the case of phosphate ore, consisting of 35% fine particles (<37 μm), the studies evaluated the combination of i) NBs + MaBs; ii) MBs + MaBs; and iii) NBs + MBs + MaBs, in the kinetics and flotation separation parameters, in relation to the standard test, which employs the use of only MaBs. The effects were evaluated in four situations involving percentage of solids (28 and 35% by weight) and concentration of reagents (1000 g·t-1 collector + 600 g·t-1 depressant and 500 g·t-1 collector + 300 g·t-1 of depressor; the apatite collector used was a saponified soybean oil with sodium hydroxide and the depressant of the ganga - magnetite, carbonates, micas, quartz, diopside and perovskite / anatase particles - was a gelatinized corn starch with sodium hydroxide). The combined use of bubbles presented increases in the recovery of P2O5, with a small drop in P2O5 content and high kinetics, in relation to the STD tests in practically all the studied conditions, with values varying between 0.2 and 8.9%. The increments when the percentage of solids was 35% w/w (up to 2.7%) were considerably lower than those obtained with 28% solids (up to 8.9%). In some conditions, the concomitant use of MBs + MaBs and NBs + MBs + MaBs was able to overcome, in the first min and a half of the test, the total phosphate recovery obtained in the standard tests. Regarding the quartz, the microflotation tests (performed in modified Hallimond tube) with and without the presence of NBs showed that the association of NBs with MaBs increased the recovery of quartz (up to 39%) in practically all the studied conditions, showing its effectiveness with both fine and coarse particles. However, the tests performed in mechanical cell with and without addition of NBs showed that the use of NBs was responsible for the increase of 23 percentage points in quartz recovery. Also, flotation kinetics increased significantly and ealier in the first min of the NBs test, about twice the quartz recovery of the standard test was achieved. The results obtained in the dissertation show the high potential of NBs combined with other bubble size distributions in the improvement of the bubble-particle adhesion process and the flotation of fine phosphatic or quartz granulometric fractions, especially in short times.

O presente trabalho teve como objetivo avaliar as interações entre bolhas com distintos tamanhos (diâmetros) e partículas minerais e o efeito de combinações destas bolhas na flotação de minérios, em escala de bancada. As bolhas empregadas nesses estudos foram: i) Nanobolhas – NBs, diâmetro médio de 150 – 200 nm; ii) Microbolhas – MBs, diâmetro médio de 70 μm; e iii) Macrobolhas – MaBs, diâmetro médio de 1 mm. As dispersões de NBs e MBs foram geradas a partir da despressurização de água saturada com ar e cavitação hidrodinâmica em válvula agulha. As pressões de saturação foram, respectivamente, 2,5 e 4,0 bar. Os estudos de interação bolhas-partícula mineral foram desenvolvidos utilizando um sistema fotográfico que permitiu avaliar a adesão de bolhas às partículas de quartzo (grão de alta pureza) e apatita em função do tipo de bolhas injetadas. Os resultados mostraram que a maior adesão de bolhas no grão de apatita ocorreu com a combinação de NBs + MBs + MaBs e, no caso do quartzo, uma expressiva concentração de MaBs aderidas foi obtida após “condicionamento” do mineral com NBs. As NBs ficaram confinadas às superfícies dos grãos minerais devido à dissipação da energia livre superficial dos sólidos em função de sua rugosidade e, desta forma, aumentaram o mecanismo de adesão de outras bolhas (MBs e/ou MaBs). O estudo de avaliação das associações de bolhas na flotação foi realizado com os sistemas minerais quartzo e minério fosfático. No caso do minério fosfático, constituído por 35% de partículas finas (< 37 μm), os estudos avaliaram a combinação de i) NBs + MaBs; ii) MBs + MaBs; e iii) NBs + MBs + MaBs, nos parâmetros cinéticos e de separação na flotação, em relação ao ensaio standard, que emprega o uso de apenas MaBs. Os efeitos foram avaliados em quatro situações que compreenderam variação de porcentagem de sólidos (28 e 35% em peso) e concentração de reagentes (1000 g.t-1 de coletor + 600 g.t-1 de depressor; e 500 g.t-1 de coletor + 300 g.t-1 de depressor; sendo que o coletor de apatita empregado foi óleo de soja saponificado com hidróxido de sódio e o depressor das partículas de ganga - magnetita, carbonatos, micas, quartzo, diopsídio e perovskita/anatásio - foi amido de milho gelatinizado com hidróxido de sódio). O emprego combinado de bolhas apresentou incrementos na recuperação de P2O5, com pequena queda no teor de P2O5 e elevada cinética, em relação aos ensaios STD em praticamente todas as condições estudadas, com valores variando entre 0,2 e 8.9%. Os incrementos quando a porcentagem de sólidos foi de 35% p/p (até 2,7%) foram consideravelmente menores que os obtidos com 28% de sólidos (até 8,9%). Em algumas condições, o uso concomitante de MBs + MaBs e NBs + MBs + MaBs foi capaz de superar, já na primeira coleta (1,5 min de ensaio), a recuperação total de fosfato obtida no ensaio standard. No caso do quartzo, os ensaios de microflotação (realizados em tubo de Hallimond modificado) com e sem a presença de NBs mostraram que a associação de NBs às MaBs aumentou a recuperação de quartzo (até 39%) em praticamente todas as condições estudadas, mostrando sua efetividade tanto com partículas finas quanto com as mais grossas. Já os ensaios realizados em célula mecânica com e sem adição de NBs, mostraram que o emprego de NBs foi responsável pelo aumento de 23 pontos percentuais na recuperação de quartzo. Ainda, a cinética de flotação aumentou significativamente e já no primeiro min do ensaio com NBs obteve-se cerca do dobro da recuperação de quartzo do ensaio standard. Os resultados obtidos na dissertação mostram o elevado potencial das NBs combinadas a outras distribuições de tamanhos de bolhas na melhoria do processo de adesão bolha-partícula e na flotação de minérios fosfáticos finos e de frações granulométricas distintas de quartzo, principalmente em tempos curtos. / The objective of this work was to evaluate the interactions between bubbles with different sizes (diameters) and mineral particles and the effect of the combinations of these bubbles on the flotation of ores, on a bench scale. The bubbles used in these studies were: i) Nanobubbles - NBs, mean diameter of 150-200 nm; ii) Microbubbles - MBs, average diameter of 70 μm; and iii) Macrobubbles - MaBs, average diameter of 1 mm. Dispersions of NBs and MBs were generated after depressurizing-cavitation of the saturated water in air. The saturation pressures were, respectively, 2.5 and 4.0 bar. The bubble-particle mineral interaction studies were developed using a photographic system that allowed to evaluate the adhesion of bubbles to the particles of quartz (high purity grain) and apatite according to the type of bubbles injected. The results showed that the highest adhesion of bubbles in the apatite grain occurred with the combination of NBs + MBs + MaBs and, in the case of quartz, an expressive concentration of adhered MaBs was obtained after "conditioning" of the mineral with NBs. The NBs were confined to the surfaces of the mineral grains as a result of the dissipation of the free surface energy of the solids due to their roughness and, therefore, it increased the adhesion mechanism of other bubbles (MBs and / or MaBs). The study of the evaluation of the associations of bubbles in the flotation was carried out with the mineral systems quartz and phosphate ore. In the case of phosphate ore, consisting of 35% fine particles (<37 μm), the studies evaluated the combination of i) NBs + MaBs; ii) MBs + MaBs; and iii) NBs + MBs + MaBs, in the kinetics and flotation separation parameters, in relation to the standard test, which employs the use of only MaBs. The effects were evaluated in four situations involving percentage of solids (28 and 35% by weight) and concentration of reagents (1000 g·t-1 collector + 600 g·t-1 depressant and 500 g·t-1 collector + 300 g·t-1 of depressor; the apatite collector used was a saponified soybean oil with sodium hydroxide and the depressant of the ganga - magnetite, carbonates, micas, quartz, diopside and perovskite / anatase particles - was a gelatinized corn starch with sodium hydroxide). The combined use of bubbles presented increases in the recovery of P2O5, with a small drop in P2O5 content and high kinetics, in relation to the STD tests in practically all the studied conditions, with values varying between 0.2 and 8.9%. The increments when the percentage of solids was 35% w/w (up to 2.7%) were considerably lower than those obtained with 28% solids (up to 8.9%). In some conditions, the concomitant use of MBs + MaBs and NBs + MBs + MaBs was able to overcome, in the first min and a half of the test, the total phosphate recovery obtained in the standard tests. Regarding the quartz, the microflotation tests (performed in modified Hallimond tube) with and without the presence of NBs showed that the association of NBs with MaBs increased the recovery of quartz (up to 39%) in practically all the studied conditions, showing its effectiveness with both fine and coarse particles. However, the tests performed in mechanical cell with and without addition of NBs showed that the use of NBs was responsible for the increase of 23 percentage points in quartz recovery. Also, flotation kinetics increased significantly and ealier in the first min of the NBs test, about twice the quartz recovery of the standard test was achieved. The results obtained in the dissertation show the high potential of NBs combined with other bubble size distributions in the improvement of the bubble-particle adhesion process and the flotation of fine phosphatic or quartz granulometric fractions, especially in short times.

Sous l’action d’une excitation ultrasonore spécifique, les microbulles d’agents de contraste présentent des oscillations non linéaires. Considérant leur taille et la complexité du phénomène, des expériences et/ou des simulations complexes et onéreuses sont requises. Pour éviter cela, l’utilisation d’une analogie est proposée et validée numériquement. Lorsqu’elles sont soumises aux ultrasons, les microbulles (1-8 µm) présentent une dynamique assez riche et néanmoins complexe dont certains aspects peuvent être décrits par un réseau d’oscillateurs couplés non-linéaires. Dans cette thèse, nous proposons d’étudier le comportement oscillatoire d’une microbulle via un système acousto-mécanique de pendules couplés paramétriquement excités par une force verticale. Il s’agit de comprendre la dynamique d’une bulle unique pour l’étudier ensuite dans des conditions expérimentales et cliniques pour des applications en imagerie et en thérapie. Du point de vue théorique, nous avons montré que les deux systèmes sont décrits par une équation de Mathieu. D’un point de vue expérimental, nous avons développé la chaîne de pendules. Celle-ci consiste en un cercle d’aluminium sur lequel sont fixés des pendules à l’aide de fil de nylon. La chaîne de pendules repose sur le système d’excitation qui génère une excitation sinusoïdale variant de 1 Hz à 5 Hz. Les résultats obtenus (modes de vibration, oscillation sous-harmonique, oscillation radiale) ont été validés par une étude numérique et sont en accord avec les résultats obtenus pour les microbulles. / Under specific ultrasound excitation, contrast microbubbles undergo nonlinear oscillations. Considering the size and the complexity of the phenomenon, expensive and complex experiments and/or simulations are required. To overcome this, the use of an analogy is proposed and validated numerically. When subjected to ultrasound, microbubbles present a fairly rich and complex dynamics of which some aspects can be described by a lattice of nonlinearly coupled oscillators. In this thesis, we propose to study the oscillatory behavior of a microbubble through an acousto-mechanical setup of coupled pendula parametrically excited by a vertical force. The aim of this work is to understand the dynamics of a single bubble, to subsequently study it in experimental and clinical conditions for both imaging and therapy. From the theoretical point of view, we have shown that both systems are described by a Mathieu type equation. From the experimental point of view, we have developped the pendula ring. It consists of an aluminum ring on which pendula are fixed with nylon strings. The pendula chain lies on the excitation system that generate a sinusoidal excitation ranging from 1 Hz to 5 Hz. Results obtained (vibration modes, subharmonic oscillations, radial mode) are in agreement with simulations and are similar to the results obtained for microbubbles.

Microbubbles, currently employed in diagnostic ultrasound as a contrast agent, have a potential new application as vehicles for targeted drug delivery, which could revolutionise medicine by eliminating side-effects. A new device is developed which outperforms all existing devices in terms of minimum microbubble size:channel diameter ratio. A numerical model is established to describe the flow behaviour and it is determined that the flow regime and resulting microbubble size are dependent on the ratio of inner- to outer Weber number.

Le tendon est un organe impliqué dans de nombreux troubles musculo squelettiques. Les connaissances des différentes molécules impliquées dans la biologie des tendons permettent d’envisager des traitements par thérapie génique. L'objectif de cette thèse a été d'évaluer la faisabilité de transférer des gènes dans les tendons par deux méthodes non virales : une méthode chimique utilisant des nanoparticules de silice mésoporeuses et une méthode physique reposant sur l’utilisation des ultrasons et des microbulles de gaz (sonoporation). Dans une première étude, nous avons montré que des nanoparticules de silice mésoporeuses sont capables de transférer efficacement des gènes dans le tendon d’Achille de rat. Celles-ci ont été utilisées pour transférer le gène du facteur de croissance PDGF dans des tendons lésés. Nos résultats indiquent une accélération de la réparation du tendon lésé. Dans une deuxième étude, nous avons utilisé la sonoporation, technique jamais appliquée aux tendons. Nous avons déterminé les paramètres acoustiques permettant un transfert de gènes efficace dans le tendon d'Achille de souris. Une expression stable d’un transgène a été maintenue pendant 100 jours suite à une injection locale d’ADN en présence de microbulles de gaz. Nous avons ensuite utilisé cette méthode dans un modèle de souris transgénique invalidée pour le gène de la fibromoduline possédant des fibres de collagène anormales. Une restauration remarquable du phénotype de ces souris a été observée suite au transfert du gène de la fibromoduline. Nous avons ensuite étudié les interactions entre les microbulles, la membrane plasmique et le transfert de gènes dans les cellules sous ultrasons. Ces études ont permis d’identifier pour la première fois, une pénétration des microbulles de gaz dans la cellule et un trafic intracellulaire de l’ADN impliquant la voie d’endocytose clathrine dépendante. En conclusion, les deux méthodes de transfert de gènes non virales ont permis de transfecter des gènes efficacement dans les tendons. Ces résultats prometteurs permettent d’envisager leur exploitation pour des applications thérapeutiques dans les pathologies tendineuses. / Tendons are often involved in many musculoskeletal disorders. Knowledge of the different molecules involved in tendons biology allows to consider treatments based on gene therapy. The goal of my work was to evaluate the feasibility of gene transfer in tendons by using two non-viral methods: mesoporous silica nanoparticles and a physical method based on ultrasound-assisted gas microbubbles (sonoporation). In the first study, we showed that mesoporous silica nanoparticles could efficiently transfer genes in rat Achilles tendon. They were used to transfer plasmid DNA encoding PDGF growth factor gene in injured tendons. Data demonstrated that DNA/MSN complexes have improved the healing of injured tendons. In the second study, we used the sonoporation method which has never been applied to the tendons. First, we determined the acoustic parameters for gene transfer in mice Achilles tendon. A stable expression of the transgene was maintained up to 100 days following one local injection of DNA in the presence of gas microbubbles. We then used this method to restore the fibromodulin knocked out gene in a transgenic mouse model having disorganized collagen fibers in tendons. Following the transfer of fibromodulin gene, a remarkable restoration of collagen fibers in these mice was observed. In the last part, we studied the interactions of microbubbles with the plasma membrane and the plasmid DNA internalization into the cells by sonoporation. These studies have identified for the first time a rapid penetration of gas microbubbles inside the cell and an intracellular routing of DNA involving the clathrinmediated endocytosis pathway. In conclusion, both methods of non viral gene transfer led to an efficient gene transfer in tendons. These promising results obtained during this project allow considering their use for therapeutic applications in tendon diseases.

Les accidents de désaturation (ADD) résultent de la formation de microbulles dans les tissus lors de la décompression. De nombreux corps de métiers sont concernés par cette problématique : plongeurs, astronautes, tunneliers, personnel médical hyperbare... On observe que ces accidents peuvent survenir alors même que les tables de décompression sont respectées. La détection et la caractérisation des bulles de décompression présentent un intérêt diagnostic potentiel pour la prévention des ADD. Aujourd’hui, la détection de microbulles est réalisée par des personnels de santé expérimentés en utilisant des systèmes Doppler. Cependant, cette approche présente une grande dépendance à l'opérateur et ne permet pas d'obtenir une information quantitative (nombre, taille) sur la distribution des bulles circulantes. Par ailleurs, elle n'est pas adaptée à la détection de bulles tissulaires.Ces limitations conduisent à mettre en oeuvre une méthode ultrasonore bifréquentielle de détection et de caractérisation par mise en résonance des microbulles. Les contraintes de mesure temps-réel, de polydispersité en taille ([1 à 200 μm]) et de sursaturation des tissus imposent d'utiliser des ondes d'excitation de très faibles puissances mais très large bande.Les solutions mises en oeuvre visent d'une part à réduire la complexité de l'instrumentation et d'autre part à prendre en considération la dynamique de l'excitation. Par ailleurs, une solution originale, ayant fait l’objet d’un dépôt de brevet, est développée. Elle permet de s'affranchir de la mise en résonance tout en conservant un caractèrediscriminant séparant bulles et tissus. / Decompression sicknesses (DCS) are a consequence of microbubbles formation in tissues during decompression. Many fields are affected by this issue: divers, astronauts, tunneling, hyperbaric medical staff... It is observed that these accidents can occur even if the decompression tables are respected. The detection and characterization of decompression bubbles have a diagnostic potential for the prevention of DCS. Today, the detection of microbubbles is performed by experienced health workers using Doppler systems. However, this approach has a high dependence on the operator and does not provide quantitative information (number, size) about the distribution of circulating bubbles. Moreover, it is not suitable for the detection of stationary bubbles (tissue bubbles).These limitations lead to the development of a bi-frequency ultrasonic method for microbubbles detection and characterization by setting them into resonance. The constraints as real-time measurements, size polydispersity ([1 to 200 μm]) and saturation of tissues require the use of very low powerful excitation but high bandwidth waves.The solutions implemented are aimed firstly to reduce the complexity of the instrumentation and secondly to consider the dynamics of the excitation. In addition, an original solution, protected by a patent, has been developed. It allows to overcome the measurement of resonance while maintaining a discriminating character between bubbles and tissues.

The aim of this thesis was to determine the solubilizing capacity of microbubbles based on SonoVue®, and phospholipids SonoVue® is made of, by using the UV-VIS spectrophotometry. The concentrations of solubilized substances within these systems was further determined. In particular, the properties of natrium dipalmitoylphosphatidylglycerole and distearoyl¬phosphatidylcholine were investigated. The microbubbles were produced using these phospholipids with the addition of polyethyleneglycol and palmitic acid. The solubilizing capacity was determined using hydrophobic solutes Sudan Red G, Oil Red O, 4-Di-2-Asp and Nile Red in order to obtain a model system of solubilized drugs or other hydrophobic substances. The behavior of solutes in phospholipids and our prepared microbubbles were examined in a moderately polar medium â physiological saline solution (0.15 M NaCl). The vizualization of prepared microbubbles was performed using optical and fluorescence microscopy. 4-Di-2-Asp, as a fluorescence probe, was not suitable for microbubble vizualization. The size of microbubbles that were produced during the experiment was almost the same as the size of microbubbles of commercially made SonoVue®. The concentration of solubilized hydrophobic solutes inside the liposomes of phospholipids ranged from tens to hundreds of micromoles per liter. With increasing concentration of phospholipids the concentration of solubilized solutes also increased. The results of this experiment can be used for further research focused on the solubilization of drugs in microbubbles, and contrast agents which are used in ultrasonography.

Ultrasound contrast agents composed of micro-bubble filled with gas are introduced to increase the backscattered power from blood. Their intravenously injection results in the improved contrast in the images. The aim of this master thesis project is to manufacture MB suspension at varied temperature and shear forces and to inspect the size distribution and concentration of the PVA-shelled micro-bubble with standard methods according to the developed protocol. A pulser-receiver (Panametrics PR 5072) setup combined with two transducers (2.25 MHz and 5 MHz) was used to investigate the backscattered enhancement of the micro-bubble suspension. Images were collected with transmission optical microscope (OLYMPUS IX71) with the aid of counting chamber. The diameter and concentration of the micro-bubbles were analyzed by Image J. The pulser-receiver setup was used to test the acoustic response. The mean diameter of micro-bubbles was from 2.03 to 4.38 µm with a standard deviation between 0.40 and 1.12 µm and the micro-bubble concentration varied from 0.07× to 5.22× MBs/ml. The enhancement of the ultrasound backscattered power was greater than 20 dB or even reached 30 dB when the energy was increased. / 3MiCRON