Four-dimensional visualization of rising microbubbles.
ABSTRACT: Four-dimensional imaging, which indicates imaging in three spatial dimensions as a function of time, provides useful evidence to investigate the interactions of rising bubbles. However, this has been largely unexplored for microbubbles, mostly due to problems associated with strong light scattering and shallow depth of field in optical imaging. Here, tracking x-ray microtomography is used to visualize rising microbubbles in four dimensions. Bubbles are tracked by moving the cell to account for their rise velocity. The sizes, shapes, time-dependent positions, and velocities of individual rising microbubbles are clearly identified, despite substantial overlaps between bubbles in the field of view. Our tracking x-ray microtomography affords opportunities for understanding bubble-bubble (or particle) interactions at microscales - important in various fields such as microfluidics, biomechanics, and floatation.
Project description:The acoustically-driven dynamics of isolated particle-like objects in microfluidic environments is a well-characterised phenomenon, which has been the subject of many studies. Conversely, very few acoustofluidic researchers looked at coated microbubbles, despite their widespread use in diagnostic imaging and the need for a precise characterisation of their acoustically-driven behaviour, underpinning therapeutic applications. The main reason is that microbubbles behave differently, due to their larger compressibility, exhibiting much stronger interactions with the unperturbed acoustic field (primary Bjerknes forces) or with other bubbles (secondary Bjerknes forces). In this paper, we study the translational dynamics of commercially-available polymer-coated microbubbles in a standing-wave acoustofluidic device. At increasing acoustic driving pressures, we measure acoustic forces on isolated bubbles, quantify bubble-bubble interaction forces during doublet formation and study the occurrence of sub-wavelength structures during aggregation. We present a dynamic characterisation of microbubble compressibility with acoustic pressure, highlighting a threshold pressure below which bubbles can be treated as uncoated. Thanks to benchmarking measurements under a scanning electron microscope, we interpret this threshold as the onset of buckling, providing a quantitative measurement of this parameter at the single-bubble level. For acoustofluidic applications, our results highlight the limitations of treating microbubbles as a special case of solid particles. Our findings will impact applications where knowing the buckling pressure of coated microbubbles has a key role, like diagnostics and drug delivery.
Project description:<h4>Rationale</h4>Microbubbles conjugated with targeting ligands are used as contrast agents for ultrasound molecular imaging. However, they often contain immunogenic (strept)avidin, which impedes application in humans. Although targeting bubbles not employing the biotin-(strept)avidin conjugation chemistry have been explored, only a few reached the stage of ultrasound imaging in vivo, none were reported/evaluated to show all three of the following properties desired for clinical applications: (i) low degree of non-specific bubble retention in more than one non-reticuloendothelial tissue; (ii) effective for real-time imaging; and (iii) effective for acoustic quantification of molecular targets to a high degree of quantification. Furthermore, disclosures of the compositions and methodologies enabling reproduction of the bubbles are often withheld.<h4>Objective</h4>To develop and evaluate a targeting microbubble based on maleimide-thiol conjugation chemistry for ultrasound molecular imaging.<h4>Methods and results</h4>Microbubbles with a previously unreported generic (non-targeting components) composition were grafted with anti-E-selectin F(ab')2 using maleimide-thiol conjugation, to produce E-selectin targeting microbubbles. The resulting targeting bubbles showed high specificity to E-selectin in vitro and in vivo. Non-specific bubble retention was minimal in at least three non-reticuloendothelial tissues with inflammation (mouse heart, kidneys, cremaster). The bubbles were effective for real-time ultrasound imaging of E-selectin expression in the inflamed mouse heart and kidneys, using a clinical ultrasound scanner. The acoustic signal intensity of the targeted bubbles retained in the heart correlated strongly with the level of E-selectin expression (|r|?0.8), demonstrating a high degree of non-invasive molecular quantification.<h4>Conclusions</h4>Targeting microbubbles for ultrasound molecular imaging, based on maleimide-thiol conjugation chemistry and the generic composition described, may possess properties (i)-(iii) desired for clinical applications.
Project description:The amount of gas in ultrasound contrast agents is related to their acoustic activity. Because of this relationship, gas volume has been used as a key variable in normalizing the in vitro and in vivo acoustic behavior of lipid shell-stabilized bubbles with different sizes and shell components. Despite its importance, bubble gas volume has typically only been theoretically calculated based on bubble size and concentration that is typically measured using the Coulter counter for microbubbles and nanoparticle tracking analysis (NTA) for nanoscale bubbles. However, while these methods have been validated for the analysis of liquid or solid particles, their application in bubble analysis has not been rigorously studied. We have previously shown that resonant mass measurement (RMM) may be a better-suited technique for sub-micron bubble analysis, as it can measure both buoyant and non-buoyant particle size and concentration. Here, we provide validation of RMM bubble analysis by using headspace gas chromatography/mass spectrometry (GC/MS) to experimentally measure the gas volume of the bubble samples. This measurement was then used as ground truth to test the accuracy of theoretical gas volume predictions based on RMM, NTA (for nanobubbles), and Coulter counter (for microbubbles) measurements. The results show that the headspace GC/MS gas volume measurements agreed well with the theoretical predictions for the RMM of nanobubbles but not NTA. For nanobubbles , the theoretical gas volume using RMM was 10% lower than the experimental GC/MS measurements; meanwhile, using NTA resulted in an 82% lower predicted gas volume. For microbubbles, the experimental gas volume from the GC/MS measurements was 27% lower compared to RMM and 72% less compared to the Coulter counter results. This study demonstrates that the gas volume of nanobubbles and microbubbles can be reliably measured using headspace GC/MS to validate bubble size measurement techniques. We also conclude that the accuracy of theoretical predictions is highly dependent on proper size and concentration measurements.
Project description:Microbubbles are micron-sized bubbles generated using a fluidic oscillator. They have the potential to decrease the cost of biotechnology processes through decreasing the energy required for aeration and mixing. However, little is known about microbial physiology when grown on microbubbles. This project compared Saccharomyces Cerevisiae growth and transcriptomic effects when cells were grown with microbubble or regular bubble aeration
Project description:The main constituent of an ultrasound contrast agent (UCA) is gas-filled microbubbles. An average UCA contains billions per ml. These microbubbles are excellent ultrasound scatterers due to their high compressibility. In an ultrasound field they act as resonant systems, resulting in harmonic energy in the backscattered ultrasound signal, such as energy at the subharmonic, ultraharmonic and higher harmonic frequencies. This harmonic energy is exploited for contrast enhanced imaging to discriminate the contrast agent from surrounding tissue. The amount of harmonic energy that the contrast agent bubbles generate depends on the bubble characteristics in combination with the ultrasound field applied. This paper summarizes different strategies to characterize the UCAs. These strategies can be divided into acoustic and optical methods, which focus on the linear or nonlinear responses of the contrast agent bubbles. In addition, the characteristics of individual bubbles can be determined or the bubbles can be examined when they are part of a population. Recently, especially optical methods have proven their value to study individual bubbles. This paper concludes by showing some examples of optically observed typical behavior of contrast bubbles in ultrasound fields.
Project description:The growth of surface plasmonic microbubbles in binary water/ethanol solutions is experimentally studied. The microbubbles are generated by illuminating a gold nanoparticle array with a continuous wave laser. Plasmonic bubbles exhibit ethanol concentration-dependent behaviors. For low ethanol concentrations (fe) of ?67.5%, bubbles do not exist at the solid-liquid interface. For high fe values of ?80%, the bubbles behave as in pure ethanol. Only in an intermediate window of 67.5% ? fe ? 80% do we find sessile plasmonic bubbles with a highly nontrivial temporal evolution, in which as a function of time three phases can be discerned. (1) In the first phase, the microbubbles grow, while wiggling. (2) As soon as the wiggling stops, the microbubbles enter the second phase in which they suddenly shrink, followed by (3) a steady reentrant growth phase. Our experiments reveal that the sudden shrinkage of the microbubbles in the second regime is caused by a depinning event of the three-phase contact line. We systematically vary the ethanol concentration, laser power, and laser spot size to unravel water recondensation as the underlying mechanism of the sudden bubble shrinkage in phase 2.
Project description:Mezcal is a traditional Mexican spirit, obtained from the distillation of fermented agave juices. Its preparation has been conducted for centuries in an artisanal manner. The method used to determine the correct alcohol content is of particular interest: a stream of the liquor is poured into a small vessel to induce surface bubbles. These bubbles, known as pearls by the Mezcal artisans, remain stable for tenths of seconds only if the alcohol content is close to 50%. For higher or lower alcohol content, the bubbles burst rapidly. The long bubble lifetime is the result of surfactant-induced surface tension changes. However, the precise mechanism and its relation to alcohol content remain unexplained. In this investigation, the extended lifetime of pearls was studied both experimentally and numerically. It was found that changes in surface tension, density, viscosity (resulting from mixing ethanol and water), and the presence of surfactants are all relevant to extend the bubble lifetime. The dimensionless bubble lifetime was found to reach its maximum value when the Bond number was close to unity, corresponding to 2 mm Mezcal bubbles. These findings show that the traditional empirical method does work. Beyond this, the understanding of the process provides physical insight to many other natural and industrial problems for which the stability of surface bubbles is of importance, such as bio-foams, froth floatation, and volcanic flows.
Project description:Microbubbles are currently in clinical use as ultrasound contrast agents and under active investigation as mediators of ultrasound therapy. To improve the theranostic potential of microbubbles, nanoparticles can be attached to the bubble shell for imaging, targeting and/or enhancement of acoustic response. Existing methods for fabricating particle-loaded bubbles, however, require the use of polymers, oil layers or chemical reactions for particle incorporation; embed/attach the particles that can reduce echogenicity; impair biocompatibility; and/or involve multiple processing steps. Here, we describe a simple method to embed nanoparticles in a phospholipid-coated microbubble formulation that overcomes these limitations. Magnetic nanoparticles are used to demonstrate the method with a range of different microbubble formulations. The size distribution and yield of microbubbles are shown to be unaffected by the addition of the particles. We further show that the microbubbles can be retained against flow using a permanent magnet, can be visualised by both ultrasound and magnetic resonance imaging (MRI) and can be used to transfect SH-SY5Y cells with fluorescent small interfering RNA under the application of a magnetic field and ultrasound field.
Project description:We demonstrate that the active thermocapillary stresses induced by multiple microbubbles offer simple routes to directed self-assembly and complex but controllable micromanipulation of mesoscopic colloidal particles embedded in a liquid. The microbubbles are nucleated on a liquid-glass interface using optical tweezers. The flow around a single bubble causes self-assembly of the particles in rings at the bubble-base, while an asymmetric temperature profile generated across the bubble interface breaks the azimuthal symmetry of the flow, and induces simultaneous accumulation and repulsion of particles at different axial planes with respect to the bubble. The flow due to two adjacent bubbles leads to more diverse effects including the sorting of particles, and to local vorticity that causes radial and axial rotation of the particles - the latter being obtained for the first time using optical tweezers. The sorting is enabled by nucleating the bubbles on spatially discrete temperature profiles, while the vorticity is generated by nucleating them in the presence of a temperature gradient which once again causes a strong symmetry-breaking in the azimuthal flow. The flow profiles obtained in the experiments are explained by analytical solutions or qualitative explanations of the associated thermocapillary problem employing the Stokes and heat equations.
Project description:In this article, membrane perforation of endothelial cells with attached microbubbles caused by exposure to single-shot short pulsed ultrasound is described, and the mechanisms of membrane damage and repair are discussed. Real-time optical observations of cell-bubble interaction during sonoporation and successive scanning electron microscope observations of the membrane damage with knowledge of bubble locations revealed production of micron-sized membrane perforations at the bubble locations. High-speed observations of the microbubbles visualized production of liquid microjets during nonuniform contraction of bubbles, indicating that the jets are responsible for cell membrane damage. The resealing process of sonoporated cells visualized using fluorescence microscopy suggested that Ca2+-independent and Ca2+-triggered resealing mechanisms were involved in the rapid resealing process. In an experimental condition in which almost all cells have one adjacent bubble, 25.4% of the cells were damaged by exposure to single-shot pulsed ultrasound, and 15.9% (approximately 60% of the damaged cells) were resealed within 5 s. These results demonstrate that single-shot pulsed ultrasound is sufficient to achieve sonoporation when microbubbles are attached to cells.