Institute of Fundamental Technological Research

This letter analyses the influence of the transducer bandwidth on the compression and the axial resolution of an ultrasound image. The distortion of an electrical signal visible in the final image is a major problem in ultrasonography. To solve this problem, the bit length in Golay-complementary sequences was elongated, narrowing the fractional bandwidth of the coded sequences. Therefore, more energy of the burst signal could be transferred through the ultrasound transducer. The experimental results obtained for transmission of the complementary Golaycoded sequences with two different bit lengths—one-cycle and two-cycles—have been compared, and the efficiency of the pulse compression and its influence on the axial resolution for two fractional bandwidths have been discussed. The results are presented for two transducers having a fractional bandwidth of 25% and 80% and operating at a 6-MHz frequency. The results obtained show that the elongation of the Golay single bit length (doubled in our case) compensate for the limited transducer bandwidth. Twodimensionalultrasoundimagesofatissue-mimickingphantomare presented and demonstrate the benefits of the use of two-cycle bit length.

Coded excitation, Golay sequences, synthetic aperture method, transducer bandwidth, ultrasound imaging

A technique using pulsed High Intensity Focused Ultrasound (HIFU) to destroy deep-seated solid tumors is a promising noninvasive therapeutic approach. A main purpose of this study was to design and test a HIFU transducer suitable for preclinical studies of efficacy of tested, anti-cancer drugs, activated by HIFU beams, in the treatment of a variety of solid tumors implanted to various organs of small animals at the depth of the order of 1–2 cm under the skin. To allow focusing of the beam, generated by such transducer, within treated tissue at different depths, a spherical, 2-MHz, 29-mm diameter annular phased array transducer was designed and built. To prove its potential for preclinical studies on small animals, multiple thermal lesions were induced in a pork loin ex vivo by heating beams of the same: 6 W, or 12 W, or 18 W acoustic power and 25 mm, 30 mm, and 35 mm focal lengths. Time delay for each annulus was controlled electronically to provide beam focusing within tissue at the depths of 10 mm, 15 mm, and 20 mm. The exposure time required to induce local necrosis was determined at different depths using thermocouples. Location and extent of thermal lesions determined from numerical simulations were compared with those measured using ultrasound and magnetic resonance imaging techniques and verified by a digital caliper after cutting the tested tissue samples. Quantitative analysis of the results showed that the location and extent of necrotic lesions on the magnetic resonance images are consistent with those predicted numerically and measured by caliper. The edges of lesions were clearly outlined although on ultrasound images they were fuzzy. This allows to conclude that the use of the transducer designed offers an effective noninvasive tool not only to induce local necrotic lesions within treated tissue without damaging the surrounding tissue structures but also to test various chemotherapeutics activated by the HIFU beams in preclinical studies on small animals.

spherical annular phased array transducer, pulsed HIFU beam, electronically adjustable focal length, local tissue heating, thermal ablation, necrotic lesion

Manipulating particles in the blood pool with noninvasive methods has been of great interest in therapeutic delivery. Recently, it was demonstrated experimentally that red blood cells can be forced to translate and accumulate in an ultrasound field. This acoustic response of the red blood cells has been attributed to sonophores, gas pockets that are formed under the influence of a sound field in the inner-membrane leaflets of biological cells. In this paper, we propose a simpler model: that of the compressible membrane. We derive the spatio-temporal cel dynamics for a spherically symmetric single cell, whilst regarding the cell bilayer membrane as two monolayer Newtonian viscous liquids, separated by a thin gas void. When applying the newly-derived equations to a red blood cell, it is observed that the void inside the bilayer expands to multiples of its original thickness, even at clinically safe acoustic pressure amplitudes. For causing permanent cell rupture during expansion, however, the acoustic pressure amplitudes needed would have to surpass the inertial cavitation threshold by a factor 10. Given the incompressibility of the inner monolayer, the radial oscillations of a cell are governed by the same set of equations as those of a forced antibubble. Evidently, these equations must hold for liposomes under sonication, as well.

spatio-temporal cell dynamics, Rayleigh-Plesset equation, spherical cell, red blood cell, erythrocyte

A combined hydrogen peroxide evaporation and hydrogen low-pressure plasma treatment process for sterilization is introduced and investigated. The combination of hydrogen peroxide evaporation followed by hydrogen plasma treatment offers an advantage regarding sterilization in complex metal geometries or in sealed sterile bags, where plasma treatment alone faces challenges. Within this contribution, the droplet size and film homogeneity after condensation is investigated by optical diagnostics. Sterilization tests with common challenge organisms show the sterilization capabilities of the combined proces in a process challenge device mimicking the worst-casescenario for plasma treatment in a small metal box. Furthermore, sterilization in sealed sterile bags clearly demonstrates the advantage of the combined process, showing full spore inactivation solely for the combined process

bacterial spores, capacitively coupled, low-pressure discharges, sterilization

Hydrophobic food ingredients sensitive to degradation can be protected from their environment by microencapsulation. In an O/W1/W2 system, these hydrophobic compounds are dissolved in oil droplets, dispersed within a gelled matrix microbead (W1), which forms a barrier. The stability and degree of protection delivered by the gel matrix depends on its structure and strength, which in turn depend on the gelling process. For heat sensitive ingredients this process is typically a cold-set gelling process.

We investigated the effect of variations in matrix polymer (alginate and WPI aggregates), gelling agent (acid and calcium), and method of gelation (internal and external), on the ability of microbeads to retain oil droplets, and retain a spherical shape during the extraction from the oil phase.

External gelation with CaCl2 nanoparticles gave the smoothest and strongest microbeads for both protein and alginate, which we attribute to the formation of a shell at the interface of the bead during gelation. Microbeads produced by internal calcium gelation (induced with CaCO3 nanoparticles and GDL) containing the same amount of calcium showed less integrity and gave a mixture of smooth and rough beads. About half of the microbeads produced by acid induced gelation of WPI aggregates (using GDL to lower the internal pH) remained intact. When the pH was brought further from the isoelectric point, fewer beads remained intact. The method of gelation proved to be more important for the microbead integrity than type of matrix polymer, and external gelling was clearly superior to internal and acid induced gelation.

Cold-set gelation, Microbeads, Encapsulation, Strength, Gelation methods

Background:
The primary aim of our study was to evaluate the safety and potential toxicity of gemcitabine combined with microbubbles under sonication in inoperable pancreatic cancer patients. The secondary aim was to evaluate a novel image-guided microbubble-based therapy, based on commercially available technology, towards improving chemotherapeutic efficacy, preserving patient performance status, and prolonging survival.

Methods:
Ten patients were enrolled and treated in this Phase I clinical trial. Gemcitabine was infused intravenously over 30 min. Subsequently, patients were treated using a commercial clinical ultrasound scanner for 31.5 min. SonoVue® was injected intravenously (0.5 ml followed by 5 ml saline every 3.5 min) during the ultrasound treatment with the aim of inducing sonoporation, thus enhancing therapeutic efficacy.

Results:
The combined therapeutic regimen did not induce any additional toxicity or increased frequency of side effects when compared to gemcitabine chemotherapy alone (historical controls). Combination treated patients (n = 10) tolerated an increased number of gemcitabine cycles compared with historical controls (n = 63 patients; average of 8.3 ± 6.0 cycles, versus 13.8 ± 5.6 cycles, p = 0.008, unpaired t-test). In five patients, the maximum tumour diameter was decreased from the first to last treatment. The median survival in our patients (n = 10) was also increased from 8.9 months to 17.6 months (p = 0.011).

Conclusions:
It is possible to combine ultrasound, microbubbles, and chemotherapy in a clinical setting using commercially available equipment with no additional toxicities. This combined treatment may improve the clinical efficacy of gemcitabine, prolong the quality of life, and extend survival in patients with pancreatic ductal adenocarcinoma.

Ultrasound, Microbubbles, Sonoporation, Pancreatic cancer, Image-guided therapy, Clinical trial

Attenuation of ultrasound in tissue can be estimated from the propagating pulse center frequency downshift. This method assumes that the envelope of the emitted pulse can be approximated by a Gaussian function and that the attenuation linearly depends on frequency. The resulting downshift of the mean frequency depends not only on attenuation but also on pulse bandwidth and propagation distance. This kind of approach is valid for narrowband pulses and shallow penetration depth. However, for short pulses and deep penetration, the frequency downshift is rather large and the received spectra are modified by the limited bandwidth of the receiving system. In this paper, the modified formula modeling the mean frequency of backscattered echoes is presented. The equation takes into account the limitation of the bandwidth due to bandpass filtration of the received echoes. This approach was applied to simulate the variation of the mean frequency of the pulse propagating for both weakly and strongly attenuating media and for narrowband and wideband pulses. The behavior of both the standard and modified estimates of attenuation has been validated using RF data from a tissue-mimicking phantom. The ultrasound attenuation of the phantom, determined with a corrected equation, was close to its true value, while the result obtained using the original formula was lower by as much as 50% at a depth of 8 cm.

Tissue attenuation, frequency downshift, bandwidth limitation

Oil-soluble components can be encapsulated in an O/W1/W2 microsystem, in which they are dissolved in oil droplets dispersed in a gelled microbead (W1), which forms a barrier between the oil droplets and the aqueous continuous phase (W2). We investigated the rate and mechanism of breakdown of protein microbeads in a simulated gastric system, and studied the influence of microbead protein concentration, gelling method (cold-set, slow and fast heat-set), and further processing (freeze-drying), on the breakdown process. Breakdown rate decreased with increasing protein content of the beads, for the same method of production. Due to the porosity of the slowly-heated heat-set beads, breakdown occurred evenly throughout the entire bead. Cold-set microbeads of 10% protein broke down slightly slower than the heat-set microbeads of 15%. The denser surface of the 10% beads slowed down the diffusion of the enzymes into the bead's interior, causing the beads to be broken down from the outside inward. All these beads broke down within one hour. Increasing the rate of temperature increase during the heating step dramatically slowed breakdown. There was no significant breakdown of rapidly heated beads within 138 minutes, even though no difference in microstructure between rapidly and slowly heated beads was visible with electron microscopy. Freeze-drying of the beads also slowed their breakdown. After 132 minutes more than half the measured particle volume of were intact beads. Freeze-drying changed the microstructure of the beads irreversibly: rehydrating the dried beads did not result in a breakdown behaviour similar to that of unprocessed beads.

From the 1st to the 5th of June, 2015, the XVIII International Conference for Young Researchers: Wave Electronics and its Applications in the Information and Telecommunication Systems took place. The conference was held on the Mikhail Sholokhov, an inland waterways passenger ship built in 1985, whilst sailing from St. Petersburg to Kizhi, Petrozavodsk, Valaam, and back to St. Petersburg.

Wave electronics, Information and telecommunication systems

In research and industrial processes, it is increasingly common practice to combine multiple measurement modalities. Nevertheless, experimental tools that allow the co-linear combination of optical and ultrasonic transmission have rarely been reported. The aim of this study was to develop and characterise a water-matched ultrasound transducer architecture using standard components, with a central optical window larger than 10 mm in diameter allowing for optical transmission. The window can be used to place illumination or imaging apparatus such as light guides, miniature cameras, or microscope objectives, simplifying experimental setups.

Four design variations of a basic architecture were fabricated and characterised with the objective to assess whether the variations influence the acoustic output. The basic architecture consisted of a piezoelectric ring and a glass disc, with an aluminium casing. The designs differed in piezoelectric element dimensions: inner diameter, ID = 10 mm, outer diameter, OD = 25 mm, thickness, TH = 4 mm or ID = 20 mm, OD = 40 mm, TH = 5 mm; glass disc dimensions OD = 20–50 mm, TH = 2–4 mm; and details of assembly.

The transducers’ frequency responses were characterised using electrical impedance spectroscopy and pulse-echo measurements, the acoustic propagation pattern using acoustic pressure field scans, the acoustic power output using radiation force balance measurements, and the acoustic pressure using a needle hydrophone. Depending on the design and piezoelectric element dimensions, the resonance frequency was in the range 350–630 kHz, the −6 dB bandwidth was in the range 87–97%, acoustic output power exceeded 1 W, and acoustic pressure exceeded 1 MPa peak-to-peak.

3D stress simulations were performed to predict the isostatic pressure required to induce material failure and 4D acoustic simulations. The pressure simulations indicated that specific design variations could sustain isostatic pressures up to 4.8 MPa.The acoustic simulations were able to predict the behaviour of the fabricated devices. A total of 480 simulations, varying material dimensions (piezoelectric ring ID, glass disc diameter, glass thickness) and drive frequency indicated that the emitted acoustic profile varies nonlinearly with these parameters.

Ultrasound transducer, De-fouling, Optical window, Acoustic field simulation

Experimental research of ultrasound to induce or improve delivery has snowballed in the past decade. In our work, we investigate the use of low-intensity ultrasound in combination with clinically approved microbubbles to enhance the therapeutic efficacy of chemotherapy. Ten voluntary patients with locally advanced or metastatic pancreatic adenocarcinoma were consecutively recruited. Following standard chemotherapy protocol (intravenous infusion of gemcitabine over 30 min), a clinical ultrasoundscanner was targeted at the largest slice of the tumour using modified non-linear contrastimaging settings (1.9 MHz center frequency, 0.27 MPa peak-negative pressure), and SonoVue® was injected intravenously. Ultrasound and microbubble treatment duration was 31.5 min. The combined therapy did not induce any additional toxicity or increase side effect frequency when compared to chemotherapy alone. Combination treated patients were able to tolerate an increased amount treatment cycles when compare historical controls (n = 63); average of 8.3±6.0 cycles, versus 13.8±5.6 cycles. The median survival also increased from 7.0 months to 17.6 months (p = 0.0044). In addition, five patients showed a primary tumor diameter decrease. Combined treatment of ultrasound,microbubbles, and gemcitabine does not increase side effects and may have the potential to increase the therapeutic efficacy of chemotherapy in patients with pancreatic adenocarcinoma.

Antibubbles are gas bubbles containing a liquid droplet core and, typically, a stabilising outer shell. It has been hypothesised that acoustically driven antibubbles can be used for active leakage detection from subsea production facilities. This paper treats the dynamics of spherically symmetric microscopic antibubbles, building on existing models of bubble dynamics. A more complete understanding of microbubble dynamics demands that the effects of the translational dynamics is included into the Rayleigh-Plesset equation, which has been the primary aim of this paper. Moreover, it is a goal of this paper to derive a theory that is not based on ad-hoc parameters due to the presence of a shell, but rather on material properties. To achieve a coupled set of differential equations describing the radial and translational dynamics of an antibubble, in this paper Lagrangian formalism is used, where a Rayleigh-Plesset-like equation allows for the shell to be modelled from first principles. Two shell models are adopted; one for a Newtonian fluid shell, and the other for a Maxwell fluid shell. In addition, a zero-thickness approximation of the encapsulation is presented for both models. The Newtonian fluid shell can be considered as a special case of the Maxwell fluid shell. The equations have been linearised and the natural and damped resonance frequencies have been presented for both shell models.

microbubbles, spatio–temporal bubble dynamics, Rayleigh-Plesset equation

The purpose of scientific research is to create, expand, and apply knowledge of Life, the Universe, and Everything. All over the world, Universities play a role in creating and transferring knowledge onto the next generation. Scientific merit is typically rewarded with academic titles, the first for understanding the basics of the field (BSc, Vordiplom), the second for mastering more complicated material and understanding how scientific research is done (MSc, Diplom), the third for proving independent thinking and research (PhD, kandidat), and the fourth for proving to initiate and transfer novel research (Habilitation, doktor nauka).

I don’t know how lectures are given outside our beautiful field of physics, but the structure of lectures in our field is very similar, no matter at which University or in which country we are. In short, physics lectures expand on a textbook or a syllabus; derivations that have been skipped in the written material are worked out in detail by the lecturer. Where applicable, exercises and assignments are used to make the students get used to apply the material presented and to prepare the students for the upcoming exam. In addition to such lectures, postgraduate physics students are subjected to seminars where they give presentations summarising a peer-reviewed paper on a given topic after having searched for background material themselves.
Yet, every physicist who has visited an international symposium must have noticed the tremendous difference in presentation style by scientists representing Universities from different countries. I am not referring to the differences between a smooth sales talk and a presentation with more content, but purely to differences in the style of presenting similar scientific work.

Physics presentations, USSR

Microbubble oscillation at specific ultrasound settings leads to permeabilization of surrounding cells. This phenomenon, referred to as sonoporation, allows for the in vitro and in vivo delivery of extracellular molecules, including plasmid DNA. To date, the biological and physical mechanisms underlying this phenomenon are not fully understood. The aim of this study was to investigate the interactions between microbubbles and cells, as well as the intracellular routing of plasmid DNA and microbubbles, during and after sonoporation. High-speed imaging and fluorescence confocal microscopy of HeLa cells stably expressing enhanced green fluorescent protein fused with markers of cellular compartments were used for this investigation. Soft-shelled microbubbles were observed to enter cells during sonoporation using experimental parameters that led to optimal gene transfer. They interacted with the plasma membrane in a specific area stained with fluorescent cholera subunit B, a marker of lipid rafts. This process was not observed with hard-shelled microbubbles, which were not efficient in gene delivery under our conditions. The plasmid DNA was delivered to late endosomes after 3 h post-sonoporation, and a few were found in the nucleus after 6 h. Gene transfer efficacy was greatly inhibited when cells were treated with chlorpromazine, an inhibitor of the clathrin-dependent endocytosis pathway. In contrast, no significant alteration was observed when cells were treated with filipin III or genistein, both inhibitors of the caveolin-dependent pathway. This study emphasizes that microbubble–cell interactions do not occur randomly during sonoporation; microbubble penetration inside cells affects the efficacy of gene transfer at specific ultrasound settings; and plasmid DNA uptake is an active mechanism that involves the clathrin-dependent pathway.

Ultrasound, Microbubble, Sonoporation, Gene delivery, Endocytosis, Clathrin-mediated endocytosis pathway

The bilayer sonophore model suggests that ultrasound induces a pulsating structure in the intra-membrane hydrophobic space between the two lipid monolayer lea ets of the cell membrane, assembled by dissolved gas of the surrounding area, which absorbs acoustic energy and transforms it by creating intra-cellular structural changes. This void has been referred to as a bilayer sonophore. The bilayer sonophore in ates and de ates periodically when exposed to ultrasound and may itself radiate acoustic pressure pulses in the surrounding medium in the same way a gas bubble does: once exposed to ultrasound the bilayer sonophore becomes a mechanical oscillator and a source of intracellular cavitation activity. In this paper, we describe observations of the clustering behaviour of living cells and several other particles in a standing sound eld generated inside a ring transducer. Upon sonication, blood cells and monodisperse polystyrene particles were observed to have been trapped in the same locations, corresponding to nodes of the ultrasound eld. Because polystyrene is hydrophobic, it behaves like a particle trapped inside a thin gas shell. In fact, the sonophore model treats biological cells in a similar way. Microbubbles that form the ultrasound contrast agent Quantison behave di erently, however. These microbubbles accumulated in circles faster than blood cells and polystyrene particles. In addition, they form tightly packed clusters at the nodes, indicating very strong secondary Bjerknes forces. Cluster formation is not to be expected in cells with predicted sonophore sizes on the order of 10 - 100 nm.

In this study, we analyse the behaviour of antibubbles when subjected to an ultrasonic pulse. Speci cally, we derive oscillating behaviour of acoustic antibubbles with a negligible outer shell, resulting in a Rayleigh Plesset equation of antibubble dynamics. Furthermore, we compare theoretical behaviour of antibubbles to behaviour of regular gas bubbles. We conclude that antibubbles and regular bubbles respond to an acoustic wave in a very similar manner if the antibubble's liquid core radius is less than half the antibubble radius. For larger cores, antibubbles demonstrate highly harmonic behaviour, which would make them suitable vehicles in ultrasonic imaging and ultrasound-guided drug delivery.

Studying the effects of ultrasound on biological cells requires extensive knowledge of both the physical ultrasound and cellular biology. Translating knowledge between these fields can be complicated and time consuming. With the vast range of ultrasonic equipment available, nearly every research group uses different or unique devices. Hence, recreating the experimental conditions and results may be expensive or difficult. For this reason, we have developed devices to combat the common problems seen in state-of-the-art biomedical ultrasound research. In this paper, we present the design, fabrication, and characterisation of an open-source device that is easy to manufacture, allows for parallel sample sonication, and is highly reproducible, with complete acoustic calibration. This device is designed to act as a template for sample sonication experiments. We demonstrate the fabrication technique for devices designed to sonicate 24-well plates and OptiCell™ using three-dimensional (3D) printing and low-cost consumables. We increased the pressure output by electrical impedance matching of the transducers using transmission line transformers, resulting in an increase by a factor of 3.15. The devices cost approximately €220 in consumables, with a major portion attributed to the 3D printing, and can be fabricated in approximately 8 working hours. Our results show that, if our protocol is followed, the mean acoustic output between devices has a variance of <1%. We openly provide the 3D files and operation software allowing any laboratory to fabricate and use these devices at minimal cost and without substantial prior know-how.

Sonoporation, experimentation devices, rapid prototyping, ultrasound transducers

An antibubble consists of a liquid droplet, surrounded by a gas, often with an encapsulating shell. Antibubbles of microscopic sizes suspended in fluids are acoustically active in the ultrasonic range. In this study, a Rayleigh-Plesset-like model is derived for micron-sized antibubbles encapsulated by Newtonian fluids. The theoretical behaviour of an encapsulated antibubble is compared to that of an antibubble without an encapsulating shell, a free gas bubble, and an encapsulated gas bubble. Antibubbles, with droplet core sizes in the range of 60– 90% of the equilibrium antibubble inner radius were studied. Acoustic pressures of 100kPa and 300kPa were studied. The antibubble resonance frequency, the phase difference of the radial oscillations with respect to the incident acoustic pulse, and the presence of higher harmonics are strongly dependent of the core droplet size. The contribution to the radial dynamics from a zero-thickness shell is negligible for the bubble size studied, at high acoustic amplitudes, antibubbles oscillate highly nonlinearly independent of core droplet size. This may allow for active leakage detection using harmonic imaging methods.

Active leakage detection, Antibubble, Bubble resonance, Microbubble, Nonlinear dynamics

Purpose
Adenocarcinoma of the pancreas remains one of the most lethal human cancers. The high mortality rates associated with this form of cancer are subsequent to late-stage clinical presentation and diagnosis, when surgery is rarely possible and of modest chemotherapeutic impact. Survival rates following diagnosis with advanced pancreatic cancer are very low; typical mortality rates of 50 % are expected within 3 months of diagnosis. However, adjuvant chemotherapy improves the prognosis of patients even after palliative surgery, and successful newer neoadjuvant chemotherapeutical modalities have recently been reported. For patients whose tumours appear unresectable, chemotherapy remains the only option. During the past two decades, the nucleoside analogue gemcitabine has become the first-line chemotherapy for pancreatic adenocarcinoma. In this study, we aim to increase the delivery of gemcitabine to pancreatic tumours by exploring the effect of sonoporation for localised drug delivery of gemcitabine in an orthotopic xenograft mouse model of pancreatic cancer.

Experimental Design
An orthotopic xenograft mouse model of luciferase expressing MIA PaCa-2 cells was developed, exhibiting disease development similar to human pancreatic adenocarcinoma. Subsequently, two groups of mice were treated with gemcitabine alone and gemcitabine combined with sonoporation; saline-treated mice were used as a control group. A custom-made focused ultrasound transducer using clinically safe acoustic conditions in combination with SonoVue® ultrasound contrast agent was used to induce sonoporation in the localised region of the primary tumour only. Whole-body disease development was measured using bioluminescence imaging, and primary tumour development was measured using 3D ultrasound.

Results
Following just two treatments combining sonoporation and gemcitabine, primary tumour volumes were significantly lower than control groups. Additional therapy dramatically inhibited primary tumour growth throughout the course of the disease, with median survival increases of up to 10 % demonstrated in comparison to the control groups.

Conclusion
Combined sonoporation and gemcitabine therapy significantly impedes primary tumour development in an orthotopic xenograft model of human pancreatic cancer, suggesting additional clinical benefits for patients treated with gemcitabine in combination with sonoporation.

Sonoporation, Pancreatic cancer, Ultrasound, Chemotherapy, 3D ultrasound, Bioluminescence

Single clouds of cavitation bubbles, driven by 254 kHz focused ultrasound at pressure amplitudes in the range of 0.48–1.22 MPa, have been observed via high-speed shadowgraphic imaging at 1 × 106 frames per second. Clouds underwent repetitive growth, oscillation and collapse (GOC) cycles, with shock-waves emitted periodically at the instant of collapse during each cycle. The frequency of cloud collapse, and coincident shock-emission, was primarily dependent on the intensity of the focused ultrasound driving the activity. The lowest peak-to-peak pressure amplitude of 0.48 MPa generated shock-waves with an average period of 7.9 ± 0.5 μs, corresponding to a frequency of f0/2, half-harmonic to the fundamental driving. Increasing the intensity gave rise to GOC cycles and shock-emission periods of 11.8 ± 0.3, 15.8 ± 0.3, 19.8 ± 0.2 μs, at pressure amplitudes of 0.64, 0.92 and 1.22 MPa, corresponding to the higher-order subharmonics of f0/3, f0/4 and f0/5, respectively. Parallel passive acoustic detection, filtered for the fundamental driving, revealed features that correlated temporally to the shock-emissions observed via high-speed imaging, p(two-tailed) < 0.01 (r = 0.996, taken over all data). Subtracting the isolated acoustic shock profiles from the raw signal collected from the detector, demonstrated the removal of subharmonic spectral peaks, in the frequency domain. The larger cavitation clouds (>200 μm diameter, at maximum inflation), that developed under insonations of peak-to-peak pressure amplitudes >1.0 MPa, emitted shock-waves with two or more fronts suggesting non-uniform collapse of the cloud. The observations indicate that periodic shock-emissions from acoustically driven cavitation clouds provide a source for the cavitation subharmonic signal, and that shock structure may be used to study intra-cloud dynamics at sub-microsecond timescales.

Acoustic cavitation, Subharmonic, Cloud dynamics, Collapse, Shock-wave

Understanding the propagation of acoustic waves through a liquid-perfused porous solid frame- work such as cancellous bone is an important pre-requisite to improve the diagnosis of osteoporosis by ultrasound. In order to elucidate the propagation dependence upon the material and structural properties of cancellous bone, several theoretical models have been considered to date, with Biot-based models demonstrating the greatest potential. This paper describes the fundamental basis of these models and reviews their performance.

Acoustic, Propagation, Bone, Theoretical Model

Purpose:
The purpose of this study was to investigate the ability and efficacy of inducing sonoporation in a clinical setting, using commercially available technology, to increase the patients’ quality of life and extend the low Eastern Cooperative Oncology Group performance grade; as a result increasing the overall survival in patients with pancreatic adenocarcinoma.

Methods:
Patients were treated using a customized configuration of a commercial clinical ultrasound scanner over a time period of 31.5 min following standard chemotherapy treatment with gemcitabine. SonoVue® ultrasound contrast agent was injected intravascularly during the treatment with the aim to induce sonoporation.

Results:
Using the authors’ custom acoustic settings, the authors’ patients were able to undergo an increased number of treatment cycles; from an average of 9 cycles, to an average of 16 cycles when comparing to a historical control group of 80 patients. In two out of five patients treated, the maximum tumor diameter was temporally decreased to 80 ± 5% and permanently to 70 ± 5% of their original size, while the other patients showed reduced growth. The authors also explain and characterize the settings and acoustic output obtained from a commercial clinical scanner used for combined ultrasound microbubble and chemotherapy treatment.

Conclusions:
It is possible to combine ultrasound, microbubbles, and chemotherapy in a clinical setting using commercially available clinical ultrasound scanners to increase the number of treatment cycles, prolonging the quality of life in patients with pancreatic adenocarcinoma compared to chemotherapy alone.

Ultrasound, Microbubbles, Sonoporation, Chemotherapy

Microbubbles first developed as ultrasound contrast agents have been used to assist ultrasound for cellular drug and gene delivery. Their oscillation behavior during ultrasound exposure leads to transient membrane permeability of surrounding cells, facilitating targeted local delivery. The increased cell uptake of extracellular compounds by ultrasound in the presence of microbubbles is attributed to a phenomenon called sonoporation. In this review, we summarize current state of the art concerning microbubble–cell interactions and cellular effects leading to sonoporation and its application for gene delivery. Optimization of sonoporation protocol and composition of microbubbles for gene delivery are discussed.

Ultrasound, Microbubbles, Physical gene delivery method, Gene therapy

Electron beam lithography (EBL) was used to fabricate microchannels to produce microbubbles with highly homogeneous size distributions. Using the EBL technique, microchannels can be prototyped at a fast and cost effective rate allowing for evaluation of various microbubble shell materials.

Microbubbles, EBL, Targeted drug delivery

Introduction
High-intensity focused ultrasound is finding increasing therapeutic use. However, the frequencies at which it operates are typically limited to below 5 MHz, preventing research into therapy with ultrahigh spatial precision. A reason for this is that the design and fabrication of high-frequency biomedical ultrasound transducers to produce high intensities is an engineering challenge, especially for operating frequencies above 30 MHz, primarily because of the acoustic impedance mismatch and the high attenuation of water of 6dB/cm at 50 MHz leading to a low penetration depth. Commonly used materials such as PZT do not have the ability to produce a high enough intensity, due to de-poling or cracking. A potential application of high-intensity high-frequency ultrasound is non-invasive microsurgery.

Methods
To overcome these problems, we used Y-36o Lithium Niobate (LiNbO3). This crystal has a high Curie temperature and is much more difficult to de-pole at high-power inputs. In addition, Y-36o LiNbO3 has a resonant frequency of 3.3 MHz mm-1, thus allowing for much thicker elements at higher frequencies compared to PZT. A bowl transducer was manufactured using a total of 7 0.5-mm thick elements (4 hexagonal and 5 pentagonal) with a maximum width of 25 mm. The bowl had a curvature radius of 50 mm. The transducer was microballoon-backed in order to simplify the manufacturing process. The pentagonal elements were linked and driven by a 50-dB amplifier, whereas the hexagonal elements were linked and driven by a 55-dB amplifier. To test the available working frequency; single element transducers were manufactured with element thickness ranging from 500 μm to 200 μm, having working frequencies between 6.6 MHz and 20 MHz.

Results
The multi-element focused transducer generated a modulated sound field with an enveloped wavelength of 550 kHz at a frequency of 6.6 MHz with a maximum peak-to-peak pressure of 24.3 MPa; equivalent to mechanical index of 4.7. The modulation could be varied by changing the phase of either the pentagonal or hexagonal linked elements. The microballoon-backed transducers had a 5% reduced acoustic output compared to the air-backed transducer. Single- element transducers produced a maximum peak-to-peak pressure of 14 MPa at 6.3 MHz in the acoustic focus at 12 mm. These transducers were capable of producing over 6 MPa and 4 MPa at the 3rd and 5th harmonics, respectively, corresponding to frequencies of 21 MHz and 35 MHz.

Conclusion
We have established that manufacturing a high frequency, high intensity, multi-element, focused ultrasound transducer using LiNbO3 is feasible. We have also shown it is possible to use the resonant frequency and up to the 5th harmonic to achieve higher working frequencies.

High-frequency ultrasound, Ultrasound transducer, MR-guided Focussed Ultrasound Surgery

Having rst been developed for ultrasound imaging, nowadays, microbubbles are proposed as tools for ultrasound-assisted gene delivery, too. Their behavior during ultrasound exposure causes transient membrane permeability of surrounding cells, facilitating targeted local delivery. The increased cell uptake of extracellular compounds by ultrasound in the presence of microbubbles is attributed to a phenomenon called sonoporation. Sonoporation has been successfully applied to deliver nucleic acids in vitro and in vivo in a variety of therapeutic applications. However, the biological and physical mechanisms of sonoporation are still not fully understood. In this review, we discuss recent data concerning microbubble–cell interactions leading to sonoporation and we report on the progress in ultrasound-assisted therapeutic gene delivery in different organs. In addition, we outline ongoing challenges of this novel delivery method for its clinical use.

Ultrasound, Gene delivery, Sonoporation

Focused ultrasound surgery (FUS) is usually based on frequencies below 5 MHz—typically around 1 MHz. Although this allows good penetration into tissue, it limits the minimum lesion dimensions that can be achieved. In this study, we investigate devices to allow FUS at much higher frequencies, in principle, reducing the minimum lesion dimensions. Furthermore, FUS can produce deep-sub-millimeter demarcation between viable and necrosed tissue; high-frequency devices may allow this to be exploited in super cial applications which may include dermatology, ophthalmology, treatment of the vascular system, and treatment of early dysplasia in epithelial tissue. In this paper, we explain the methodology we have used to build high-frequency high-intensity transducers using Y-36°-cut lithium niobate. This material was chosen because its low losses give it the potential to allow very-high- frequency operation at harmonics of the fundamental operating frequency. A range of single-element transducers with center frequencies between 6.6 and 20.0 MHz were built and the transducers’ e ciency and acoustic power output were measured. A focused 6.6-MHz transducer was built with multiple elements operating together and tested using an ultrasound phantom and MRI scans. It was shown to increase phantom temperature by 32°C in a localized area of 2.5 × 3.4 mm in the plane of the MRI scan. Ex vivo tests on poultry tissue were also performed and shown to create lesions of similar dimensions. This study, therefore, demonstrates that it is feasible to produce high-frequency transducers capable of high-resolution FUS using lithium niobate.

Lithium Niobite, Ultrasound Transducer, MRI-Guided ultrasound, Microsurgery

The aim of this study is to deliver genes in Achilles tendons using ultrasound and microbubbles. The rationale is to combine ultrasound-assisted delivery and the stimulation of protein expression induced by US. We found that mice tendons injected with 10 μg of plasmid encoding luciferase gene in the presence of 5 × 10^5 BR14 microbubbles, exposed to US at 1 MHz, 200 kPa, 40% duty cycle for 10 min were efficiently transfected without toxicity. The rate of luciferase expression was 100-fold higher than that obtained when plasmid alone was injected. Remarkably, the luciferase transgene was stably expressed for up to 108 days. DNA extracted from these sonoporated tendons was efficient in transforming competent E. coli bacteria, indicating that persistent intact pDNA was responsible for this long lasting gene expression. We used this approach to restore expression of the fibromodulin gene in fibromodulin KO mice. A significant fibromodulin expression was detected by quantitative PCR one week post-injection. Interestingly, ultrastructural analysis of these tendons revealed that collagen fibrils diameter distribution and circularity were similar to that of wild type mice. Our results suggest that this gene delivery method is promising for clinical applications aimed at modulating healing or restoring a degenerative tendon while offering great promise for gene therapy due its safety compared to viral methods.

Gene delivery, Sonoporation, Tendon

Acoustic cavitation can occur in therapeutic applications of high-amplitude focused ultrasound. Studying acoustic cavitation has been challenging, because the onset of nucleation is unpredictable. We hypothesized that acoustic cavitation can be forced to occur at a specific location using a laser to nucleate a microcavity in a pre-established ultrasound field. In this paper we describe a scientific instrument that is dedicated to this outcome, combining a focused ultrasound transducer with a pulsed laser. We present high-speed photographic observations of laser-induced cavitation and laser- nucleated acoustic cavitation, at frame rates of 0.5×106 frames per second, from laser pulses of energy above and below the optical breakdown threshold, respectively. Acoustic recordings demonstrated inertial cavitation can be controllably introduced to the ultrasound focus. This technique will contribute to the understanding of cavitation evolution in focused ultrasound including for potential therapeutic applications.

Laser-nucleated acoustic cavitation, focussed ultrasound

Ultrasonic imaging is becoming the most popular medical imaging modality, owing to the low price per examination and its safety. However, blood is a poor scatterer of ultrasound waves at clinical diagnostic transmit frequencies. For perfusion imaging, markers have been designed to enhance the contrast in B-mode imaging. These so-called ultrasound contrast agents consist of microscopically small gas bubbles encapsulated in biodegradable shells. In this review, the physical principles of ultrasound contrast agent microbubble behavior and their adjustment for drug delivery including sonoporation are described. Furthermore, an outline of clinical imaging applications of contrast-enhanced ultrasound is given. It is a challenging task to quantify and predict which bubble phenomenon occurs under which acoustic condition, and how these phenomena may be utilised in ultrasonic imaging. Aided by high-speed photography, our improved understanding of encapsulated microbubble behavior will lead to more sophisticated detection and delivery techniques. More sophisticated methods use quantitative approaches to measure the amount and the time course of bolus or reperfusion curves, and have shown great promise in revealing effective tumor responses to anti-angiogenic drugs in humans before tumor shrinkage occurs. These are beginning to be accepted into clinical practice. In the long term, targeted microbubbles for molecular imaging and eventually for directed anti-tumor therapy are expected to be tested.

Ultrasound, Drug delivery systems, Drug targeting, Sonoporation, Contrast media, Liver, Pancreas, Gastrointestinal tract

Introduction
Focussed ultrasound surgery can heat tissue to a temperature that causes protein denaturation and coagulative necrosis. For high-resolution focussed ultrasound microsurgery, high working frequencies are necessary. The manufacture of such equipment is technically challenging. Low acoustic amplitudes have been associated with increased cellular drug and gene uptake.
We studied tissue, microbubbles and cells, using commercial and self-built transducers operating at various frequencies, using very low or very high acoustic amplitudes.

Methods
We manufactured a high-frequency, high-intensity focussed ultrasound transducer, using lithium niobate as the active element.
To study acoustic cavitation, we designed and built a scientific instrument combining a pulsed laser and a high-intensity focussed ultrasound transducer, capable of nucleating at precise locations. The cavitation dynamics were recorded using high-speed cameras. At high acoustic intensities, interacting cavitation clouds were formed.

Results
Clusters formed at a quarter wavelength apart owing to radiation forces. We observed cluster coalescence and translation towards the capillary wall.
Microbubbles under sonication have been observed to create transient pores in adjacent cell membranes, also known as sonoporation. We observed lipid-shelled microbubbles near cancer cells under quasi-continuous low-amplitude sonication. Typically within a second of sonication, microbubbles were seen to enter the cells and dissolve. This new explanation of sonoporation was verified using high-speed photography and confocal fluorescence microscopy.
Our custom-built transducer was capable of creating 2.5×3.4 (mm)2 lesions without affecting surrounding tissue.
Such disruptive effects of ultrasound also have applications outside medicine. Since cyanobacteria contain gas vesicles, we hypothesised that these can be disrupted with the aid of ultrasound. During 1-hour sonication in the clinical diagnostic range, we forced blue-green algae to sink, thus promoting natural decay.

Conclusion
If drug and genes can be successfully coupled to acoustically active vehicles, sonoporation might revolutionise noninvasive therapy as we know it.

Therapeutic ultrasound, Sonoporation

The purpose of this study was to investigate the physical mechanisms of sonoporation, in order to understand and improve ultrasound-assisted drug and gene delivery. Sonoporation is the transient permeabilisation and resealing of a cell membrane with the help of ultrasound and/or an ultrasound contrast agent, allowing for the trans-membrane delivery and cellular uptake of macromolecules between 10 kDa and 3 MDa. The authors studied the behaviour of ultrasound contrast agent microbubbles near cancer cells at low acoustic amplitudes. After administering an ultrasound contrast agent, HeLa cells were subjected to 6?6 MHz ultrasound with a mechanical index of 0?2 and observed with a high-speed camera. Microbubbles were seen to enter cells and rapidly dissolve. The quick dissolution after entering suggests that the microbubbles lose (part of) their shell while entering. The authors have demonstrated that lipid-shelled microbubbles can be forced to enter cells at a low mechanical index. Hence, if a therapeutic agent is added to the shell of the bubble or inside the bubble, ultrasound-guided delivery could be facilitated at diagnostic settings. In addition, these results may have implications for the safety regulations on the use of ultrasound contrast agents for diagnostic imaging.

Sonoporation, Low mechanical index, Microbubbles, Ultrasound contrast agent, HeLa cells, Cell penetration

The ultrasound-induced formation of bubble clusters may be of interest as a therapeutic means. If the clusters behave as one entity, i.e., one mega-bubble, its ultrasonic manipulation towards a boundary is straightforward and quick. If the clusters can be forced to accumulate to a microfoam, entire vessels might be blocked on purpose using an ultrasound contrast agent and a sound source.
In this paper, we analyse how ultrasound contrast agent clusters are formed in a capillary and what happens to the clusters if sonication is continued, using continuous driving frequencies in the range 1– 10 MHz. Furthermore, we show high-speed camera footage of microbubble clustering phenomena.
We observed the following stages of microfoam formation within a dense population of microbubbles before ultrasound arrival. After the sonication started, contrast microbubbles collided, forming small clusters, owing to secondary radiation forces. These clusters coalesced within the space of a quarter of the ultrasonic wavelength, owing to primary radiation forces. The resulting microfoams translated in the direction of the ultrasound field, hitting the capillary wall, also owing to primary radiation forces.
We have demonstrated that as soon as the bubble clusters are formed and as long as they are in the sound field, they behave as one entity. At our acoustic settings, it takes seconds to force the bubble clusters to positions approximately a quarter wavelength apart. It also just takes seconds to drive the clusters towards the capillary wall.
Subjecting an ultrasound contrast agent of given concentration to a continuous low-amplitude signal makes it cluster to a microfoam of known position and known size, allowing for sonic manipulation.

Capillary blocking, Embolisation, Microfoam, Radiation forces, Ultrasound contrast agent

Ultrasound contrast agents consist of encapsulated bubbles in the micrometer size range. At low acoustic amplitudes these microbubbles pulsate linearly, but at high amplitudes they demonstrate highly nonlinear, destructive behaviour. Cellular drug uptake and lysis are increased under sonication, and even more so when a contrast agent is present, owing to the formation of transient porosities in the cell membrane (sonoporation). An overview is given of the physical mechanisms of microbubble behaviour. There are two hypotheses for explaining the sonoporation phenomenon, the first being bubble oscillations near a cell membrane, the second being bubble jetting through the cell membrane. Based on modelling, photography, and cellular uptake measurements, it is concluded that bubble jetting behaviour is unlikely to be the dominant sonoporation mechanism.

Jetting, Sonoporation

The anisotropic pore structure and elasticity of cancellous bone cause wave speeds and attenuation in cancellous bone to vary with angle. Previously published predictions of the variation in wave speed with angle are reviewed. Predictions that allow tortuosity to be angle dependent but assume isotropic elasticity compare well with available data on wave speeds at large angles but less well for small angles near the normal to the trabeculae. Claims for predictions that only include angle-dependence in elasticity are found to be misleading. Audio-frequency data obtained at audio-frequencies in air-filled bone replicas are used to derive an empirical expression for the angle-and porosity-dependence of tortuosity. Predictions that allow for either angle dependent tortuosity or angle dependent elasticity or both are compared with existing data for all angles and porosities.

Ultrasonic imaging is becoming the most popular medical imaging modality. Taking into account all hospital expenses, the costs of ultrasound and of X-ray examinations are roughly the same, whereas MRI and CT scans are approximately three times as expensive. A basic B-mode ultrasound scan typically shows contrast at regions of differing acoustic impedance. However, blood is a poor scatterer of ultrasound waves at clinical diagnostic frequencies, and thus techniques to enhance contrast have naturally emerged over the last few decades. The development of ultrasound contrasts agents, microscopically small bubbles with resonance frequencies in the clinical diagnostic range, has extended the functionality of standard sonography to perfusion assessment and even to molecular imaging.

Bubbles, Ultrasound

Algae are aquatic organisms classified separately from plants. They are known to cause many hazards to humans and the environment. Algae strands contain nitrogen-producing cells that help them float (heterocysts). It is hypothesized that if the membranes of these cells are disrupted by means of ultrasound, the gas may be released analogous to sonic cracking, causing the strands to sink. This is a desirable ecological effect, because of the resulting suppressed release of toxins into the water.
We subjected small quantities of blue-green algae of the Anabaena sphaerica species to ultrasound of frequencies and pressures in the clinical diagnostic range, and observed the changes in brightness of these solutions over time. Blue-green algae were forced to sink at any ultrasonic frequency we studied, supporting our hypothesis that heterocysts release nitrogen under ultrasound insonification in the clinical diagnostic range.
Although the acoustic fields we used to eradicate blue-green algae are perfectly safe in terms of mechanical index, the acoustic pressures surpass the NURC Rules and Procedures by over 35 dB. Therefore, caution should be taken when using these techniques in a surrounding where aquatic or semi-aquatic animals are present.

Ultrasonic algae eradication, Blue-green algae, Sonic cracking

Ultrasound contrast agents consist of microbubbles with diameters in the micrometer range. Excited by ultrasound, these bubbles exhibit highly nonlinear oscillation. While well developed physical models for microbubble oscillation exist, the efficiency of pulse sequences for sensitive microbubble detection is discussed based on simple mathematical models of general nonlinearity. Typically, Taylor series are used to model microbubble nonlinearity for the development of detection schemes. Recently, pulse sequences were proposed which exploit nonlinear memory of microbubbles, a property that cannot be modeled by a Taylor series but can be explained using a Volterra series. Therefore, this paper discusses and evaluates the usage of Volterra series for the modeling of the scattering behavior of contrast agent microbubbles. A numerically stable linear estimation algorithm is implemented to determine a third order Volterra model for a free gas bubble with a resting radius r0 1⁄4 1 lm. For insonification pressures up to 100 kPa, the identified model allowed for a mean-square error of less than 16 dB with respect to the reference signal. Analysis of the response to narrowband signals showed that the achievable mean-square error is further reduced for the bandwidth available to typical ultrasound transducers used for clinical diagnostics.

Ultrasound contrast agent, Microbubble, Volterra series, Rayleigh–Plesset, System identification, Nonlinear oscillation

It has been proven, that the cellular uptake of drugs and genes is increased, when the region of interest is under ultrasound insonification, and even more when a contrast agent is present. This increased uptake has been attributed to the formation of transient porosities in the cell membrane, which are big enough for the transport of drugs into the cell (sonoporation). Owing to this technique, new ultrasound contrast agents that incorporate a therapeutic compound have become of interest. Combining ultrasound contrast agents with therapeutic substances, such a chemotherapeutics and virus vectors, may lead to a simple and economic method to instantly cure upon diagnosis, using conventional ultrasound scanners. There are two hypotheses for explaining the sonoporation phenomenon, the first being microbubble oscillations near a cell membrane, the second being microbubble jetting through the cell membrane. Based on modeling, high-speed photography, and recent cellular uptake measurements, it is concluded that microbubble jetting behavior is less likely to be the dominant sonoporation mechanism. Ultrasound-directed drug delivery using microbubbles is a promising method that has great potential in the treatment of malignant disorders.

Microbubbles, Ultrasound, Ultrasound contrast agent, Drug delivery, Sonoporation, Therapeutic bubbles

Ultrasound contrast agents consist of microscopically small bubbles encapsulated by an elastic shell. These microbubbles oscillate upon ultrasound insonification, and demonstrate highly nonlinear behavior, ameliorating their detectability. (Potential) medical applications involving the ultrasonic disruption of contrast agent microbubble shells include release-burst imaging, localized drug delivery, and noninvasive blood pressure measurement. To develop and enhance these techniques, predicting the cracking behavior of ultra- sound-insonified encapsulated microbubbles has been of importance. In this paper, we explore microbubble behavior in an ultrasound field, with special attention to the influence of the bubble shell.
A bubble in a sound field can be considered a forced damped harmonic oscillator. For encapsulated microbubbles, the presence of a shell has to be taken into account. In models, an extra damping parameter and a shell stiffness parameter have been included, assuming that Hooke’s Law holds for the bubble shell. At high acoustic amplitudes, disruptive phenomena have been observed, such as microbubble fragmentation and ultrasonic cracking. We analyzed the occurrence of ultrasound contrast agent fragmentation, by simulating the oscillating behavior of encapsulated microbubbles with various sizes in a harmonic acoustic field. Fragmentation occurs exclusively during the collapse phase and occurs if the kinetic energy of the collapsing microbubble is greater than the instantaneous bubble surface energy, provided that surface instabilities have grown big enough to allow for break-up. From our simulations it follows that the Blake critical radius is not a good approximation for a fragmentation threshold.
We demonstrated how the phase angle differences between a damped radially oscillating bubble and an incident sound field depend on shell parameters.

Ultrasound contrast agent, Shell disruption, Fragmentation threshold, Oscillation phase angle, Shell elasticity, Shell friction

New ultrasound contrast agents that incorporate a therapeutic compound have become of interest. Such an ultrasound contrast agent particle might act as the vehicle to carry a drug or gene load to a perfused region of interest. The load could be released with the assistance of ultrasound. Generally, an increase in shell thickness increases the acoustic amplitude needed to disrupt a bubble. High acoustic amplitudes, however, have been associated with unwanted effects on cells. It would be interesting to incorporate a droplet containing drugs or genes inside a microbubble carrier. A liquid core surrounded by a gas encapsulation has been referred to as antibubble. In this paper, the creation of an antibubble with the aid of ultrasound has been demonstrated with high-speed photography.

Antibubble, Ultrasound contrast agent, Drug delivery, High-speed photography

In clinical ultrasound, blood cells cannot be differentiated from surrounding tissue, due to the low acoustic impedance difference between blood cells and their surroundings. Resonant gas bubbles introduced in the bloodstream are ideal markers, if rapid dissolution can be prevented. Ultrasound contrast agents consist of microscopically small bubbles encapsulated by an elastic shell. These microbubbles oscillate upon ultrasound insonification. Microbubbles with thin lipid shells have demonstrated highly nonlinear behavior. To enhance diagnostic ultrasound imaging techniques and to explore therapeutic applications, these medical microbubbles have been modeled. Several detection techniques have been proposed to improve the detectability of the microbubbles. A new generation of contrast agents, with special targeting ligands attached to the shells, may assist the imaging of nonphysical properties of target tissue. Owing to microbubble-based contrast agents, ultrasound is becoming an even more important technique in clinical diagnostics.

Detection, Harmonic imaging, Herring equation, Microbubble, Resonance, RPNNP equation, Scattering, Targeted imaging, Ultrasound, Ultrasound contrast agent

Nitric oxide (NO) has been implicated in smooth muscle relaxation. Its use has been widespread in cardiology. Due to the effective scavenging of NO by hemoglobin, however, the drug has to be applied locally or in large quantities, to have the effect desired. We propose the use of encapsulated microbubbles that act as a vehicle to carry the gas to a region of interest. By applying a burst of high-amplitude ultrasound, the shell encapsulating the gas can be cracked. Consequently, the gas is released upon which its dissolution and diffusion begins. This process is generally referred to as (ultra)sonic cracking.
To test if the quantities of released gas are high enough to allow for NO-delivery in small vessels (ø < 200 lm), we analyzed high-speed optical recordings of insonified stiff-shelled microbubbles. These microbubbles were subjected to ultrasonic cracking using 0.5 or 1.7 MHz ultrasound with mechanical index MI > 0.6. The mean quantity released from a single microbubble is 1.7 fmol. This is already more than the NO production of a 1 mm long vessel with a 50 lm diameter during 100 ms. However, we simulated that the dissolution time of typical released NO microbubbles is equal to the half-life time of NO in whole blood due to scavenging by hemoglobin (1.8 ms), but much smaller than the extravascular half-life time of NO (>90 ms).
We conclude that ultrasonic cracking can only be a successful means for nitric oxide delivery, if the gas is released in or near the red blood cell-free plasma next to the endothelium. A complicating factor in the in vivo situation is the variation in blood pressure. Although our simulations and acoustic measurements demonstrate that the dissolution speed of free gas increases with the hydrostatic pressure, the in vitro acoustic amplitudes suggest that the number of released microbubbles decreases at higher hydrostatic pressures. This indicates that ultrasonic cracking mostly occurs during the expansion phase.

Nitric oxide, Sonic cracking

Ultrasound contrast agents consist of microscopically small encapsulated bubbles that oscillate upon insonification. To enhance diagnostic ultrasound imaging techniques and to explore therapeutic applications, these medical microbubbles have been studied with the aid of high-speed photography. We filmed medical microbubbles at higher frame rates than the ultrasonic frequency transmitted. Microbubbles with thin lipid shells have been observed to act as microsyringes during one single ultrasonic cycle. This jetting phenomenon presumably causes sonoporation. Furthermore, we observed that the gas content can be forced out of albumin-encapsulated microbubbles. These free bubbles have been observed to jet, too. It is concluded that microbubbles might act as a vehicle to carry a drug in gas phase to a region of interest, where it has to be released by diagnostic ultra- sound. This opens up a whole new area of potential applications of diagnostic ultrasound related to targeted imaging and therapeutic delivery of drugs such as nitric oxide.

High-speed photography, Ultrasound contrast agent, Therapeutic microbubbles

We investigated gas release from two hard- shelled ultrasound contrast agents by subjecting them to high-mechanical index (MI) ultrasound and simultaneously capturing high-speed photographs. At an insonifying frequency of 1.7 MHz, a larger percentage of contrast bubbles is seen to crack than at 0.5 MHz. Most of the released gas bubbles have equilibrium diameters between 1.25 and 1.75 m. Their disappearance was observed optically. Free gas bubbles have equilibrium diameters smaller than the bubbles from which they have been released. Coalescence may account for the long dissolution times acoustically observed and published in previous studies. After sonic cracking, the cracked bubbles stay acoustically active.

Sonic cracking

Nanoshelled microbubbles are suitable markers for perfused areas in ultrasonic imaging, and have potential applications in therapy. With radii up to 5 microns, their resonance frequencies are in the lower megahertz range. We explored the physics of nanoshelled microbubbles, with special attention to the influence of the nanoshell on the oscillation offset with respect to the driving phase. Microbubbles above resonance size oscillate π rad out of phase with respect to microbubbles under resonance size. As the damping becomes less, this transition in offset becomes more abrupt. Therefore, the damping due to the friction of the nanoshell can be derived from this abruptness. We support our results with some high-speed optical observations of oscillating microbubbles in an ultrasonic field.

The ultrasonic backscatter coefficient (BSC) of superparamagnetic iron oxide (SPIO) nanoparticles, which are used as a liver MRI contrast agent, was simulated using a Yagi backscattering model. The BSC of SPIO cores that are aggregated in the lysosomes of Kupffer cells is significantly higher (85 dB) than the BSC of non- aggregated SPIO cores. Considering in vivo concentrations, the aggregated SPIO does not elevate the BSC of the liver markedly (9x10-6 dB). Thus, the reported visibility of SPIO in clinical ultrasound cannot be explained by classical scattering theory. Other non-linear effects need to be taken into account.

In intravascular ultrasound, we investigate vessel motion due to pulsating blood flow. Relatively large lateral displacements are observed. To incorporate these, we propose an initially 2D displacement estimation based on an accelerated block matching algorithm for preprocessing. In this paper, an accelerated algorithm based on the Successive Elimination Algorithm is described. Furthermore, we evaluated the algorithm based on intravascular ultrasound data. The proposed algorithm is compared search time and identical motion field.

Purpose:
Ultrasound contrast agents consist of bubbles in the micrometer range encapsulated by nanoshells. These medical microbubbles oscillate linearly upon insonification at low acoustic amplitudes, but demonstrate highly nonlinear, destructive behavior at relatively high acoustic amplitudes. Therefore, medical microbubbles have been investigated for their potential applications in local drug and gene delivery. We used fast-framing photography at more than a million frames per second to investigate medical microbubbles in a diagnostic ultrasonic field. In this presentation, we give an overview of the physical mechanisms of medical microbubble destruction.

Methods and Materials:
Three ultrasound contrast agents were studied with high-speed photography during insonification. The agents were inserted through a cellulose capillary with a diameter of 0.2mm. The capillary was positioned below a microscope whose optical focus coincided with the ultrasonic focus. We captured images of insonified medical bubbles at higher frame rates than the ultrasonic frequency transmitted (typically 0.5MHz). The acoustic amplitudes corresponded to mechanical indices between 0.03 and 0.8. To compare theory and experiments, we simulated insonified medical microbubble behavior, based on the behavior of large, unencapsulated bubbles in an acoustic field.

Results:
At low acoustic amplitudes (mechanical index <0.1) bubbles pulsate moderately, as predicted from theory. At high amplitudes (mechanical index >0.6) their elongated expansion phase is followed by a violent collapse. Microbubbles have been observed to coalesce (merge), fragment, crack, and jet (act as a microsyringe) during one single ultrasonic cycle. From our observations of jetting through medical bubbles, we computed that the pressure at the tip of the jet is high enough to penetrate any human cell. One image sequence reveals the temporary formation of a liquid drop inside a microbubble.

Conclusions:
Medical microbubble oscillation and translation can be modeled using large, unencapsulated bubble theory. The number of fragments generated by untrasound-induced microbubble break-up has been related to the energy absorbed by the microbubble. Medical bubbles might be used as vehicles that carry a drug to a region of interest, where the release can be controlled with ultrasound. Liquid jets may act as microsyringes, injecting a drug into target tissue. Microbubble phenomena also have potential applications in imaging and noninvasive pressure measurements.

Microbubble, Ultrasound

We studied the interaction of ultrasound contrast agent bubbles coated with a layer of lipids, driven by 0.5 MHz ultrasound. High-speed photography on the submicrosecond timescale reveals that some bubbles bounce off each other, while others show very fast coalescence during bubble expansion. This fast coalescence cannot be explained by dissipation-limited film drainage rates. We conclude that the lipid shell ruptures upon expansion, exposing clean free bubble interfaces that support plug flow profiles in the film and inertia-limited drainage whose time scales match those of the observed coalescence.

Microbubble coalescence, Ultrasound contrast agent, Film drainage, High-speed photography

When encapsulated microbubbles are subjected to high-amplitude ultrasound, the following phenomena have been reported: oscillation, translation, coalescence, fragmentation, sonic cracking and jetting. In this paper, we explain these phenomena, based on theories that were validated for relatively big, free (not encapsulated) gas bubbles. These theories are compared with high-speed optical observations of insonified contrast agent microbubbles. Furthermore, the potential clinical applications of the bubble-ultrasound interaction are explored. We conclude that most of the results obtained are consistent with free gas bubble theory. Similar to cavitation theory, the number of fragments after bubble fission is in agreement with the dominant spherical harmonic oscillation mode. Remarkable are our observations of jetting through contrast agent microbubbles. The pressure at the tip of a jet is high enough to penetrate any human cell. Hence, liquid jets may act as remote-controlled microsyringes, delivering a drug to a region-of-interest. Encapsulated microbubbles have (potential) clinical applications in both diagnostics and therapeutics.

Encapsulated microbubbles, Ultrasound contrast agent, Radiation forces, Coalescence, Fragmentation, Jets

This paper describes a noninvasive method to measure local hydrostatic pressures in fluid filled cavities. The method is based on the disappearance time of a gas bubble, as the disappearance time is related to the hydrostatic pressure. When a bubble shrinks, its response to ultrasound changes. From this response, the disappearance time, and with it the hydrostatic pressure, can be determined.
We investigated the applicability of the gases Ar, C3F8, Kr, N2, Ne, and SF6, based on their diffusive properties. For pressure measurements with a limited duration, e.g. 150 ms, Kr and Ar bubbles are most suitable, since they are most sensitive to pressure change. If there is also a limitation to bubble size, e.g. a maximum diameter of 6 lm, SF6 is most suitable.
We present improvements of a method that correlates the duration of the decay of the fundamental ultrasound response to the hydrostatic overpressure. We propose to correlate the duration until subharmonic occurrence in combination with its decay, to hydrostatic overpressure, since the subharmonic decays more rapidly than the fundamental response. For a dissolving Ar gas bubble with an initial diameter of 14 lm, the overpressure can be determined 4 times as precise from the decay of the subharmonic response as from the decay of the fundamental response. Overpressures as small as 11 mmHg may be discriminated with this method.

Noninvasive pressure measurement, Blood pressure, Microbubble, Sonic cracking

Ultrasound contrast agents consist of microscopically small encapsulated bubbles that oscillate upon insonification. To enhance diagnostic ultrasound imaging techniques and to explore therapeutic applications, these medical bubbles have been studied with the aid of high-speed photography. We filmed medical bubbles at higher frame rates than the ultrasonic frequency transmitted. Microbubbles have - among others - been observed to fragment and jet during one single ultrasonic cycle. Gas was released from encapsulated microbubbles. It is concluded that bubbles might act as a vehicle to carry a drug in gas phase to a region of interest, where it has to be released by ultrasound whose amplitudes are still in the diagnostic range.

Oscillating bubbles, Targeted drug delivery, Micro-injection needles

Ultrasound contrast agents (UCAs) are used in a clinical setting to enhance the backscattered signal from the blood pool to estimate perfusion and blood flow. The UCAs consist of encapsulated microbubbles, measuring 1–10 m in diameter. Acoustic characterization of UCAs is generally carried out from an ensemble of bubbles. The measured signal is a complicated summation of all signals from the individual microbubbles. Hence, characterization of a single bubble from acoustic measurements is complex.
In this study, 583 optical observations of freely flowing, oscillating, individual microbubbles from an experimental UCA were analyzed. The excursions during ultra- sound exposure were observed through a microscope. Images were recorded with a high frame rate camera operating at 3 MHz. Microbubbles on these images were measured off-line, and maximal excursions were determined. A technique is described to determine the diameters of the bubbles observed. We compared the maximal excursions of microbubbles of the same initial size in an ultrasound field with a 500 kHz center frequency at acoustic amplitudes ranging from 0.06 MPa to 0.85 MPa.
It was concluded that maximal excursions of identical bubbles can differ by 150% at low acoustic pressures (mechanical index or MI 0.2). At a high acoustic pressure (MI = 1.2) an image sequence was recorded on which a bubble collapsed, but an apparently identical bubble survived.

The cover page shows a sequence of microscopic image frames of a freely flowing contrast agent microbubble. The frames were taken during one cycle of ultrasound insonification, with a center frequency of 500 kHz. The peak negative acoustic pressure at the region of interest was 0.85 MPa. Each frame corresponds to a 45 x 27 μm2 area. The exposure time of each frame was 10 ns. Interframe times were 330 ns, except for the time between frames e and f, which was 660 ns. The sequence shows a growing gas encapsulated microbubble of 5.3 μm (a) and 17.6 μm (b), and its maximal growth of 22.9 μm (c). After shrinking to 20.2 μm (d), it ruptured (e). The microbubble had been pushed to the lower left side of the frame, apparently by water that was propelled into the microbubble. A subframe shows the negative of the region of interest. Finally, the deformed mcrobubble re-occurred as an assymetric shape (f). Understanding of microbubble-rupturing behavior is neccessary for developments in medical release burst imaging and ultra- sound-guided drug delivery. This work has been supported by the Technology Foundation STW (RKG.5104) and the Interuniversity Cardiology Institute of The Netherlands.

An antibubble consists of a liquid droplet, surrounded by a gas, often with an encapsulating shell. Antibubbles of microscopic sizes suspended in fluids are acoustically active in the ultrasonic range. Antibubbles have applications in food processing and guided drug delivery. We study the sound generated from antibubbles, with droplet core sizes in the range of 0–90% of the equilibrium antibubble inner radius. The antibubble resonance frequency, the phase difference of the echo with respect to the incident acoustic pulse, and the presence of higher harmonics are strongly dependent of the core droplet size. Antibubbles oscillate highly nonlinearly around resonance size. This may allow for using antibubbles in clinical diagnostic imaging and targeted drug delivery.

Antibubble, Nonlinear, Echo, Rayleigh-Plesset, Targeted drug delivery

Hydroelectric power is a clean source of energy, providing up to 20% of the World's electricity. Nevertheless, hydroelectric power plants are plagued with a common problem: silt. The silt causes damage to turbine blades, which then require repairing or replacing. In this study, we investigated the possibility to filter micron-sized particles from water using ultrasound. We designed a custom-made flow chamber and performed flow simulations and experiments to evaluate its efficacy. We used a 195-kHz ultrasound transducer operating in continuous-wave mode with acoustic output powers up to 12W. Our acoustic simulations showed that it should be possible to force a 200-μm particle over 2cm in flow, using an acoustic pressure of 12 MPa. Our flow simulations showed, that the fluid flow is not drastically decreased with the flow chamber, which was validated by the experimental measurements. The flow was not reduced when the ultrasound was activated. The acoustic filtering was effective between acoustic powers of 2.6 and 6.4W, where the particle concentration in the clean output was statistically significantly lower than the null experiments.

Acoustic filtering

Therapeutic ultrasound has been in use for over 70 years but has primarily been a thermal modality. Sonoporation, the use of ultrasound and stable gas microbubbles in the size range of 2-10 μm to form transient pores in cell membranes, has been of great interest in the past 15 years. This technique could be used to improve the delivery of current drugs in very localised regions. There are several phenomena behind sonoporation that all occur non-exclusively: push, pull, jetting, inertial cavitation, shear and, translation. Pre-clinical work has shown that sonoporation can be used to reduce primary tumour burden and inhibit metastatic development. Our clinical trial showed that ultrasound in combination with microbubbles and chemotherapy can effectively double the number of chemotherapy cycles patients can undergo, meaning that the patients were healthier for a longer period of time. Nevertheless, sonoporation is still in its infancy and there is vast room for improvement in both the areas of microbubbles and ultrasound.

Sonoporation

Clinical diagnostic ultrasound has been known as one of the safest imaging modalities available, yet very little is known about the cellular response to such acoustic conditions. With the increased interest in therapeutic ultrasound it is becoming ever more important to understand the effects of ultrasound on cells.In our work here we investigate the effect of clinical diagnostic ultrasound on several cell signalling proteins (p38 p-Thr180/p-Tyr182, ERK 1/2 p-Thr202/p-Tyr204 and p53 ac-Lys382) on leukaemia cells (MOLM-13) and monocytes. Our results show that leukaemia cells and monocytes react differently to ultrasound and microbubbles. A relatively small increase in p38 signalling was seen in the leukemic cells, and only at higher intensities in combination with microbubbles. In contrast the monocytes showed an increase in p38 signalling at all acoustic intensities with microbubbles and at the high acoustic intensity without microbubbles. Furthermore, the leukemic cells showed an overall increase in ERK 1/2 signalling whereas the monocytes showed a decrease. These results indicate that the leukaemia cells are less sensitive to stress induced by ultrasound and microbubbles when compared to normal monocytes. In conclusion, our results show that clinical diagnostic ultrasound does have a measurable effect on intracellular signalling but may differ drastically between different cell types. This may affect the conditions necessary for therapeutic ultrasound.

Phospho-signaling pathways, Ultrasound contrast agent

Glass windowed ultrasound transducers have several potential uses ranging from multi-modal research (ultrasound and optics) to industrial application in oil and gas or chemistry. In our work here we compare four different designs for transparent glass windowed ultrasound transducers. Each design was characterised using field scanning, radiation force measurements, frequency sensitivity measurement and FEM simulations. Field scans showed that small variations in design can greatly affect the size and location of the acoustic focus. The results coincided with those seen in the simulations. Radiation force measurements showed that the devices were able to easily exceed acoustic powers of 10W, with efficiencies of up to 40%. Isostatic simulations shows that the design also affects the physical strength of the devices. Current designs were able to withstand between 300 and 700 psi on the front surface. The devices were cost effective due to the minimal amount of materials necessary and the simple fabrication process. More work needs to be done to improve the power output and stress handling capabilities.

Ultrasound transducer

Separating oil, gas, and water is a slow, and therefore expensive, process, especially if the gas bubbles and oil droplets are very small. Yet, with increasingly strict regulations on filtered sea water quality, it is a process of major importance to most oil and gas industry. Using customised ultrasonic devices, we have been able to drive microbubbles through saturated fluids, forcing the bubbles to cluster and form microfoams at equal distances. These microfoams were then driven out of the fluid. In this presentation highspeed photography footage of this process will be shown. Ultrasound-assisted separation is a cheap technique that may have applications on a much bigger scale.

Ultrasound, Oil, gas and water separation

The purpose of this study was to investigate the physical mechanisms of sonoporation, to understand and ameliorate ultrasound-assisted drug and gene delivery. Sonoporation is the transient permeabilisation of a cell membrane with help of ultrasound and/or an ultrasound contrast agent, allowing for the trans-membrane delivery and cellular uptake of macromolecules between 10 kDa and 3 MDa. We studied the behaviour of ultrasound contrast agent microbubbles near cancer cells at low acoustic amplitudes. After administering an ultrasound contrast agent, HeLa cells were subjected to 6.6-MHz ultrasound with a mechanical index of 0.2 and observed with a high- speed camera. Microbubbles were seen to enter cells and rapidly dissolve. The quick dissolution after entering suggests that the microbubbles lose (part of) their shell whilst entering. We have demonstrated that lipid-shelled microbubbles can be forced to enter cells at a low mechanical index. Hence, if a therapeutic load is added to the bubble, ultrasound-guided delivery could be facilitated at diagnostic settings. However, these results may have implications for the safety regulations on the use of ultrasound contrast agents for diagnostic imaging.

Sonoporation, Ultrasound, Microbubbles, HeLa Cancer cells, Drug delivery, Low Mechanical Index

Ultrasound as a therapeutic means is increasing in popularity, owing to its non-invasiveness and low cost. Biomedical applications include surgery, cyanobacteria eradication, and drug and gene delivery. Recent advances have been associated with the ultrasound-induced formation of transient porosities in cell membranes. These and other applications have been associated with the behaviour of medical microbubbles near cells.
Common methods of studying the dynamic behaviour of microbubbles are high-speed photography and optical microscopy.
Here, we give a brief overview of our fields of study in biomedical ultrasonics, concentrating on bubble–cell interactions.
There are three standards for the safe use of biomedical sound. When introducing bubbles, these safety guidelines may be misleading.

Ultrasound, Sonoporation, Safety guidelines

Focused ultrasound surgery (FUS) is usually based on frequencies below 5 MHz, typically around 1 MHz. Whilst this allows good penetration into tissue, it limits the minimum lesion dimensions that can be achieved. In the study reported here, we investigated devices to allow FUS at much higher frequencies, therefore in principle reducing the minimum lesion dimensions. We explain the methodology we have used to build high-frequency high-intensity transducers using Y-36o cut lithium niobate. This material was chosen as its low losses give it the potential to allow very high-frequency operation at harmonics of the fundamental operating frequency. A range of single element transducers with a centre frequency between 6.6 MHz and 20.0 MHz was built and the transducers’ efficiency and acoustic power output were measured. A focussed 6.6-MHz transducer was built with multiple elements operated together and tested using an ultrasound phantom and MRI scans. It was shown to increase phantom temperature by 32OC in a localised area of 2.5 mm × 3.4 mm in the plane of the MRI scan. This study therefore demonstrates that it is feasible to produce high-frequency transducers capable of high-resolution focused ultrasound surgery using lithium niobate.

FUS, high frequency, lithium niobate, high resolution, transducer manufacture, MRI compatibility

The ultrasound-induced formation of bubble clusters may be of interest as a therapeutic means. If the clusters behave as one entity, i.e., one mega-bubble, its ultrasonic manipulation towards a boundary is straightforward and quick. If the clusters can be forced to accumulate to a microfoam, entire vessels might be blocked on purpose using an ultrasound contrast agent and a sound source. Alternatively, the microfoam could be removed from the blood pool. The latter technique might be applicable in highly toxic ultrasound-guided drug delivery. We analysed how ultrasound contrast agents with different shell compositions form clusters in a capillary and what happens to the clusters if sonication is continued, using continuous driving frequencies in the range 1–10 MHz. We observed the following stages of microfoam formation within a dense population of microbubbles before ultrasound arrival. After the sonication started, contrast microbubbles collided, forming small clusters, owing to secondary radiation forces. These clusters coalesced within the space of a quarter of the ultrasonic wavelength, owing to primary radiation forces. The resulting microfoams translated in the direction of the ultrasound field, hitting the capillary wall, also owing to primary radiation forces.
We have demonstrated that as soon as the bubble clusters are formed and as long as they are in the sound field, they behave as one entity. At our acoustic settings, it takes seconds to force the bubble clusters to positions approximately a quarter wavelength apart. The clusters contain approximately 2,000 ultrasound contrast agent microbubbles. Clusters streaming trough a capillary interact, forming morphing microfoam. Subjecting an ultrasound contrast agent of given concentration to a continuous low-amplitude signal makes it cluster to a microfoam of known position and known size, allowing for sonic manipulation, including the release of its contents.

Ultrasound that is routinely used for imaging is now exploited for therapeutic applications including drug delivery or gene transfer. Today, ultrasound imaging is an established and confident technique for diagnosis. It is mainly based on the development of contrast imaging methods that aim to identify and display the echo from contrast agent as well as rejecting the echo from surrounding tissue offering thus a more resolutive detection. Ultrasound contrast agents or microbubbles (MB) are small gas bubbles encapsulated by a stabilizing shell, with a typical diameter of micron range. Ultrasound pulses are typically applied with a frequency near the resonance frequency of the gas bubble and the bubbles oscillations produce strong echoes from regions of perfused tissue [1-2]. Activation of microbubbles (MB) under specific ultrasound (US) beams induces a transient cell membrane permeabilization with a process known as sonoporation [3-4]. This work aims at evaluating the use of ultrasound and microbubbles for gene transfer in Achilles tendons.

Ultrasound, Sonoporation, Gene transfer

This paper reports progress on the development of a novel rain disdrometer and the methods of noise reduction. The proposed instrument will measure the raindrop size distribution using the sound generated by raindrops landing in a tank of water.
When an incident hydrometeor impacts on the surface of a liquid, two processes create an acoustic signal. The first is a broadband impact pulse which is related to the impact size and velocity. The second is created when pockets of air are trapped underneath the water's surface; this is termed entrainment. One particular solution to disdrometry is to use the acoustic signature of the impacts to classify the parameters of the rain event.
To an extent, entrainment can be predicted, since fluid dynamics dictates that a bubble will oscillate and emit an acoustic signal as a damped sinusoid. In a rain event however, the bubbles can occupy a wide area in both temporal and spectral regions, overpowering the comparably small impact pulse.
We present three methods to remove bubble related noise within acoustic disdrometry. These include the addition of a driving signal to force a bubble to oscillate in a non-resonant way, liquid additives to suppress the formation of bubbles and signal processing methods to filter any remaining bubble noise.
It was found from simulation that driving a bubble does not reduce the entrainment signal. Experimental conditions can hardly be kept constant. Adding an oil film prevented bubble formation, but the maintenance required makes it less suitable to be used in the field. Using signal processing methods proved to be the most sustainable and flexible way of suppressing bubble noise.

Acoustic disdrometry

Algae have been proven to be a severe health hazard to humans, aquatic and semi-aquatic animals. Chemical methods available to control the algae have unwanted side-effects. For this reason, ultrasonic algae control has been under investigation. We measured the eradication effectiveness of ultrasound at three typical centre frequencies. At all three frequencies physical damage to the algae was observed. We conclude that it is possible to eradicate blue-green algae in the clinical diagnostic range. Taking into account the geometry, the low attenuation in water, and the NATO Undersea Research Centre for Human Diver and Marine Mammal Risk Mitigation Rules and Procedures, even at these low voltages, the safe swimming distance is at least several meters away from the sound source.

Ultrasound, Algae eradication

The benefits of ultrasonics in algae control have been well known [1]. The transmit frequencies used to study this application have been as low as 20 kHz and as high as 1.7 MHz. Most commercial equipment operates in the lower ultrasonic range. There have been speculations about the physical mechanism behind the algae eradication, specifically about the role of cavitation. Furthermore, the consequences for swimmers in water subjected to ultrasonic treatment have been unknown. In this study, we investigate the role of cavitation as potential danger for swimmers. Furthermore, we give an estimate of swimmer safety radii, based on current regulations.

Ultrasound, Swimmer safety

The driving of contrast microbubbles towards a boundary by means of primary radiation (Bjerknes) forces has been of interest for ultrasound-assisted drug delivery. Secondary radiation forces, resulting from oscillating microbubbles under ultrasound insonification, may cause the mutual attraction and subsequent coalescence of contrast microbubbles. This phenomenon has been less studied. Microbubbles with a negligible shell can be forced to translate towards each other at relatively low mechanical indices (MI). Thick-shelled microbubbles would require a higher MI to be moved. However, at high MI, microbubble disruption is expected. We investigated if thick-shelled contrast agent microbubbles can be forced to cluster at high-MI. Two thick-shelled contrast agents, inserted through a cellulose capillary, were subjected to 3 MHz, high- MI pulsed ultrasound from a commercial ultrasound machine, and synchronously captured through a high numerical aperture microscope. The agent QuantisonTM did not translate, but showed a small percentage of disrupted bubbles. The agent M1639 showed the ultrasound-induced formation of bubble clusters, and the translation thereof towards the capillary boundary. It is concluded, that forced translation and clustering of thick-shelled contrast microbubbles is feasible.

Ultrasound contrast agents consist of gas-filled microbubbles stabilized by a shell. Under ultrasound insonification, these bubbles oscillate nonlinearly with resonance frequencies being well within the diagnostic range. Currently, different detection methods are proposed, often with a heuristic reasoning based on the bubble nonlinearity being modeled by a time-invariant polynomial characteristic. However, it has been demonstrated [1] that microbubbles exhibit the behavior of a nonlinearity with memory. To optimize detection schemes, we propose to take this into account by ultrasound contrast agent modeling with a Wiener series. With these models, which can be identified from acoustic measurements, nonlinear system theory can be applied to improve detection methods. The feasibility of contrast agent modeling by Wiener series was evaluated on a contrast agent simulation, implemented by a modified Rayleigh-Plesset differential equation. For a sinusoidal input, the Wiener series approximated contrast agent behavior with a mean square error of 7.6% of the power of the contrast agent signal. The Wiener series approach was subsequently validated in an experimental setup where the nonlinear characteristics of a commercially available contrast agent were identified. The model obtained allowed for a mean square prediction error of 2.6% of the power of the measured signal for a pseudo-random multilevel sequence. With these experiments, it has been shown that the modeling of the oscillation behavior of ultrasound contrast agents with a Wiener series is feasible.

Ultrasound contrast agents consist of small encapsulated bubbles with diameters below 10 μ m. The encapsulation influences the behavior of these microbubbles when they are insonified by ultrasound. The highly nonlinear behavior of ultrasound contrast agents at relatively high acoustic amplitudes (mechanical index>0.6) has been attributed to nonlinear bubble oscillations and to bubble destruction. For microbubbles with a thin, highly elastic nanoshell, it has been demonstrated that the presence of the nanoshell becomes negligible at high insonifying amplitudes. From our simulations it follows that the Blake critical radius is not valid for microbubble fragmentation. The low maximal excursion observed and simulated for a thick, stiff-shelled microbubble is in agreement with previous acoustic analyses. The ultrasound-induced gas release from stiff-shelled bubbles has been reported. However, we also observed gas release from microbubbles with a thin, elastic shell.

Ultrasound contrast agent, Encapsulated microbubble, Nanoshell

Ultrasound contrast agents have been investigated for their potential applications in local drug and gene delivery. A microbubble might act as the vehicle to carry a drug or gene load to a perfused region of interest. The load has to be released with the assistance of ultrasound. We investigate the suitability of antibubbles for ultrasound-assisted local delivery. As opposed to bubbles, antibubbles consist of a liquid core surrounded by a gas encapsulation. Incorporating a liquid drop containing drugs or genes inside an ultrasound contrast agent microbubble, however, is technically challenging.
An ultrasound-insonified microbubble generates a pressure field that is inversely proportional to the distance from the microbubble. Therefore, an oscillating contrast agent microbubble may create a surface instability with a relatively big bubble at a short distance. For big enough instabilities, a drop may be formed inside the big bubble.
Three different contrast agents were subjected to 0.5 MHz ultrasound, with mechanical indices >0.6. The contrast agents were inserted through an artificial capillary which led through the acoustic focus of the transducer. High-speed photographs were captured at a speed of 3 million frames per second and higher. We observed that ultrasound contrast microbubbles below resonance size may create visible surface instabilities with bubbles above resonance size. With an albumin-shelled contrast agent, we induced a surface instability that was big enough to create an antibubble inside a free (unencapsulated) gas bubble with an 8 micron diameter. The surface instability has been attributed to the presence of a contrast microbubble with a 3 micron diameter. This instability has the form of a re-entrant jet protruding into the gas bubble. The inward protrusion grew and subsequently drained, leaving a droplet with a five micron diameter inside the bubble. In a subsequent recording after 100 ms, only the gas bubble could be detected. Thus, the life- time of the antibubble was less then 100 ms. The presence of a surfactant on the interfaces might lead to an improved stability of an antibubble.

Antibubble, Ultrasound

The disruption of contrast agent microbubbles has been implicated in novel techniques for high-MI imaging and local drug delivery. At MI>0.6, microbubble fragmentation has been observed with thin-shelled agent (≈10nm), and shell rupture with thick-shelled agent (≈250nm). To predict the disruption of these nanoshelled microbubbles, destruction thresholds have been under investigation. In several studies, the Blake threshold pressure was associated with microbubble destruction. The Blake threshold pressure is the peak rarefactional acoustic pressure at which the critical Blake radius is reached, approximately twice the equilibrium radius, above which a bubble behaves like an inertial cavity. We studied the acoustic pressures at which a thin-shelled microbubble fragments and those at which a thick-shelled microbubble cracks. More specifically, we investigated the validity of the Blake threshold for these phenomena. The oscillating and fragmenting behavior of microbubbles with a 10nm shell was simulated at a driving frequency of 0.5–2 MHz, using a modified Rayleigh-Plesset equation and assuming that fragmentation occurs when the kinetic energy of the microbubble surpasses the instantaneous bubble surface energy. For microbubbles with radii between 1 and 6μm, the fragmentation thresholds lie between 20 and 200 kPa. Generally, the critical radius is much smaller than twice the equilibrium radius. The moment of break-up during the collapse phase is in agreement with high- speed optical observations that were presented previously.
Furthermore, the shell rupture behavior of microbubbles with a thick shell was analyzed for quasistatic pressure changes (relatively low ultrasonic frequencies), assuming that the shell obeys Hooke’s Law. The rupture threshold pressure of −80 kPa had been determined from acoustical data. For shells with the typical Young’s modulus 2MPa and Poisson ratio 0.5, this is in agreement with the observation that the maximal excursion upon rupture of such bubbles is smaller that 0.3μm.
In conclusion, we may state that the Blake threshold is neither a good estimator for the fragmentation, nor for the rupture of contrast agent microbubbles.

Shell rupture, Fragmentation threshold, Blake threshold

Predicting the dynamic behavior of ultrasound insonified lipid-shelled microbubbles has been of much clinical interest. For perfusion measurements, a technique named flash- echo has been proposed. A burst of high-MI ultrasound is to destroy the contrast agent bubbles, supposedly resulting in a strong scattering signal that is visible on the B-mode image: the flash. The absence of this strong response in parts of the B-mode image indicates a (too) low perfusion. In this paper, we investigate how microbubbles collapse and fragment. An overview of fragmentation theory is given, followed by some high-speed optical observations of collapsing and fragmenting microbubbles in an ultrasonic field. Fragmentation occurs exclusively during the collapse phase. We hypothesize that fragmentation will only occur if and only if the kinetic energy of the collapsing microbubble is greater than the instantaneous bubble surface energy. In contradiction to the assumption that the Blake critical radius is a good approximation for a fragmentation threshold, our simulations show Rmax/R0 ≪2 for most microbubbles.

Flash echo, Microbubble fragmentation, Ultrasound contrast agent

When gas bubbles collide, the following stages of bubble coalescence have been reported: flattening of the opposing bubble surfaces prior to contact, drainage of the interposed liquid film toward a critical minimal thickness, rupture of the liquid film, and formation of a single bubble. During insonification, expanding contrast agent microbubbles may collide with each other, resulting in coalescence or bounce.
In this study, we investigate the validity of the film drainage formalism for expanding free bubbles, by subjecting rigid-shelled contrast agent microbubbles to ultrasound, in order to release gas, and photograph the coalescence of these free gas bubbles. As with colliding bubbles, bubble surface flattening is related to the Weber number. Only inertial film drainage between free interfaces explains the observed coalescence times. In accordance with theory, smaller bubble fragments coalesce on very small time scales, while larger bubbles bounce off each other.

Understanding the mechanisms of microbubble destruction is needed for the development of ultrasound guided drug and gene delivery methods and for the improvement of diagnostic ultrasonic contrast agent (UCA) detection methods. We performed 482 experiments on the coalescence and collapse mechanisms of a soft- shelled and a hard-shelled contrast agent, by subjecting an experimental lipid-shelled UCA and the hard-shelled UCA QuantisonTM to 500 kHz, high- pressured ultrasound (MI≈1.0), and recording microscopic images of these events with a fast- framing camera. Results showed that bubble fragmentation into smaller bubbles is the primary mechanism for lipid-shelled contrast microbubble destruction during the first cycles after ultrasound arrival. In 28% of our experimental events with a lipid-shelled UCA, we observed bubble coalescence. The coalescence mechanism was observed to be analog to the process desribed for larger gas bubbles. Repetitive coalescence and fragmentation was clearly recorded with a fast-framing camera. We also demonstrated the formation and collapse of large lipid-shelled bubbles and bubble clusters. Furthermore we showed that sonic cracking is feasible for the hard-shelled contrast agent QuantisonTM.

In this study we analyze the behavior of individual experimental ultrasonic contrast bubbles, insonofied by 500 kHz ultrasound, at acoustic pressures between 0.06 and 0.66 MPa. The oscillations were observed under a microscope with a fast framing camera.
It is concluded that apparently identical bubbles can expand to different maximal diameters.

We study the sonication of stable particles that encapsulate a liquid core, so-called antibubbles. Acoustically active antibubbles can potentially be used for ultrasound-guided drug delivery. In this presentation, we derive the oscillating behaviour of acoustic antibubbles with a negligible outer shell, resulting in a Rayleigh-Plesset equation of antibubble dynamics. Furthermore, we compare the theoretical behaviour of antibubbles to that of regular gas bubbles. We conclude that antibubbles and regular bubbles are acoustically active in a very similar way, if the liquid core is less than half the antibubble radius. For larger cores, antibubbles demonstrate highly harmonic behaviour, which would make them suitable vehicles in ultrasonic imaging and ultrasound-guided drug delivery.

Harmonics, Antibubbles

Background: Pancreatic Adenocarcinoma (PDAC) represents one of the most lethal human cancers. Surgery is often unfeasible, and the tumors respond poorly to radiation or chemotherapeutic drugs. Consequently, pancreatic cancer represents a huge burden to society and the need for new therapeutic options is evident. Experimental research using ultrasound to improve therapeutic delivery has soared in the past decade. We aimed toevaluate the safety and potential toxicity of gemcitabine combined with microbubbles under sonication in inoperable pancreatic cancer patients. The secondary goal was to develop a novel image-guided microbubble-based therapy, based on commercially available technology, towards improving chemotherapeutic efficacy, preserving patient performance grade, and prolongation of survival. Methods: Ten patients were enrolled and treated in this Phase I clinical trial. Gemcitabine was infused intravenously over 30 min. Subsequently patients were treated using a commercial clinical ultrasound scanner for 31.5 min. SonoVue was injected intravenously (0.5 ml followed by 5 ml saline every 3.5 min) during the ultrasound treatment with the aim of enhancing therapeutic efficacy. Results: The combined therapeutic regimen did not induce any additional toxicity or increase side effect frequency when compared to gemcitabine chemotherapy alone (historical controls). Combination treated patients (n = 10) tolerated an increased number of gemcitabine cycles compared with historical controls (n = 63 patients; average of 8.3±6.0 cycles, versus 13.8±5.6 cycles). In five patients, the maximum tumor diameter was decreased during treatment. The median survival in our patients was also increased from 7.0 months to 17.6 months (p = 0.0044). Conclusions: We perform the first-in-human study evaluating the toxicity and efficacy of ultrasound and microbubble enhanced chemotherapy. It is possible to combine ultrasound, microbubbles, and chemotherapy in a clinical setting with no additional toxicity. This combined treatment may improve the clinical efficacy of chemotherapeutic agents, prolong the quality of life, and extend survival in patients with PDAC. Clinical trial information: NCT01674556.

Bubbles are emptiness, microscopic clouds surrounded by the world. Born by chance with a violent and brief life, collapsing in a union with the infinite [1]. Remarkably similar to the life of a human being. However, there are some discrepancies when comparing the life of a bubble, and that of a human being. For example, the nature of the acoustically active bubble is described by the language of mathematics, where understanding the behaviour of bubble dynamics has been a goal for physicists for centuries [2]. However, not only physicists have been enchanted by the intrinsic beauty encompassed in the simplicity of a bubble. Throughout history bubbles have been a symbol in art.

Antibubbles