Microbubble Populations Research Articles

Vessel recovery using ultrasound localisation microscopy: An in silico comparative study between minimum variance and delay-and-sum beamformers

The use of particle localisation and tracking algorithms on Contrast Enhanced Ultrasound (CEUS) or other ultrasound mode image data containing sparse microbubble (MB) populations, can produce super-resolved vascularization maps. Typically such data stem from conventional delay and sum (DAS) beamforming that is used widely in ultrasound imaging modes. Recently, adaptive beamforming has shown significant improvement in spatial resolution, but its value to super-resolution image analysis approaches is not fully understood. The in silico study here evaluates the performance of combining minimum variance beamformers (MV BF), established to provide improved lateral resolution, compared to DAS BFs with single particle detection. The isolated effect of a range of simplified image-affecting factors such as flow profile, pulse length, noise, vessel separations and data availability is considered. The study aims to assess the vessel recovery performance using the different beamformers and investigate the link with MB detection and localisation. The MV BF was shown to provide improved microvessel position accuracy compared to conventional DAS BFs. In particular, vessel separations between 0.3–4 λ provided superior localisation uncertainty with the MV. In addition, for a separation of 0.36λ, vessel recovery was achieved with both methods but the use of MV eliminated artifacts that appear as additional vessels. These results were found to be linked to improved MB detection and localisation for the MV BF, which is proposed as suitable for testing in Ultrasound Localisation Microscopy (ULM) imaging using patient data.

Sensing ultrasound localization microscopy for the visualization of glomeruli in living rats and humans

Estimation of glomerular function is necessary to diagnose kidney diseases. However, the study of glomeruli in the clinic is currently done indirectly through urine and blood tests. A recent imaging technique called Ultrasound Localization Microscopy (ULM) has appeared. It is based on the ability to record continuous movements of individual microbubbles in the bloodstream. Although ULM improved the resolution of vascular imaging up to tenfold, the imaging of the smallest vessels had yet to be reported. We acquired ultrasound sequences from living humans and rats and then applied filters to divide the data set into slow-moving and fast-moving microbubbles. We performed a double tracking to highlight and characterize populations of microbubbles with singular behaviors. We decided to call this technique "sensing ULM" (sULM). We used post-mortem micro-CT for side-by-side confirmation in rats. In this study, we report the observation of microbubbles flowing in the glomeruli in living humans and rats. We present a set of analysis tools to extract quantitative information from individual microbubbles, such as remanence time or normalized distance. As glomeruli play a key role in kidney function, it would be possible that their observation yields a deeper understanding of the kidney. It could also be a tool to diagnose kidney diseases in patients. More generally, it will bring imaging capabilities closer to the functional units of organs, which is a key to understand most diseases, such as cancer, diabetes, or kidney failures. This study was funded by the European Research Council under the European Union Horizon H2020 program (ERC Consolidator grant agreement No 772786-ResolveStroke).

Computation of ultrasound propagation in a population of nonlinearly oscillating microbubbles including multiple scattering

In contrast-enhanced echography, the simulation of nonlinear propagation of ultrasound through a population of oscillating microbubbles imposes a computational challenge. Also, the numerical complexity increases because each scatterer has individual properties. To address these problems, the Iterative Nonlinear Contrast Source (INCS) method has been extended to include a large population of nonlinearly responding microbubbles. The original INCS method solves the Westervelt equation in a four-dimensional spatiotemporal domain by generating increasingly accurate field corrections to iteratively update the acoustic pressure. The field corrections are computed by the convolution of a nonlinear contrast source with the Green's function of the linear background medium. Because the convolution integral allows a coarse discretization, INCS can efficiently deal with large-scale problems. To include a population of microbubbles, these are considered as individual contrast point sources with their own nonlinear response. The field corrections are computed as before, but now, in each iteration, the temporal signature of each contrast point source is computed by solving the bubble's Marmottant equation. Physically, each iteration adds an order of multiple scattering. Here, the performance of the extended INCS method and the significance of multiple scattering is demonstrated through various results from different configurations.

Extending the Iterative Nonlinear Contrast Source method to simulate mutual interaction in large populations of microbubbles

There is an ongoing development of ultrasound contrast agents (UCAs) for specific medical diagnostic and therapeutic applications. Recent researches involve all kinds of targeted and loaded microbubbles, monodisperse microbubbles, and phase-change nanodroplets. To design and improve applications that use UCAs, it is necessary to have a thorough understanding of the mutual interaction of ultrasound fields and UCAs. The individual response of microbubbles and nanodroplets is extensively studied and, in the case of bubbles, multiple variants of the Rayleigh-Plesset equation are available to describe their behavior. However, novel applications, such as therapeutic proton beam localization, may strongly depend on the mutual interaction in a population of UCAs. In this presentation, a numerical approach for the analysis of ultrasound-bubble interaction in a population with many (order one million) microbubbles will be discussed. This approach is based on the earlier developed Iterative Nonlinear Contrast Source (INCS) method for simulating nonlinear ultrasound waves. In the current case, the population of microbubbles will be represented by a set of pressure-dependent contrast point sources that are iteratively updated by the INCS method, where each iteration adds an order of multiple scattering. Results will be presented for the linear scattering in populations with different concentrations of microbubbles.

Time-resolved absolute radius estimation of vibrating contrast microbubbles using an acoustical camera.

Ultrasound (US) contrast agents consist of microbubbles ranging from 1 to 10 μm in size. The acoustical response of individual microbubbles can be studied with high-frame-rate optics or an "acoustical camera" (AC). The AC measures the relative microbubble oscillation while the optical camera measures the absolute oscillation. In this article, the capabilities of the AC are extended to measure the absolute oscillations. In the AC setup, microbubbles are insonified with a high- (25 MHz) and low-frequency US wave (1-2.5 MHz). Other than the amplitude modulation (AM) from the relative size change of the microbubble (employed in Renaud, Bosch, van der Steen, and de Jong (2012a). "An 'acoustical camera' for in vitro characterization of contrast agent microbubble vibrations," Appl. Phys. Lett. 100(10), 101911, the high-frequency response from individual vibrating microbubbles contains a phase modulation (PM) from the microbubble wall displacement, which is the extension described here. The ratio of PM and AM is used to determine the absolute radius, R0. To test this sizing, the size distributions of two monodisperse microbubble populations ( R = 2.1 and 3.5 μm) acquired with the AC were matched to the distribution acquired with a Coulter counter. As a result of measuring the absolute size of the microbubbles, this "extended AC" can capture the full radial dynamics of single freely floating microbubbles with a throughput of hundreds of microbubbles per hour.

A Single Short 'Tone Burst' Results in Optimal Drug Delivery to Tumours Using Ultrasound-Triggered Therapeutic Microbubbles.

Advanced drug delivery systems, such as ultrasound-mediated drug delivery, show great promise for increasing the therapeutic index. Improvements in delivery by altering the ultrasound parameters have been studied heavily in vitro but relatively little in vivo. Here, the same therapeutic microbubble and tumour type are used to determine whether altering ultrasound parameters can improve drug delivery. Liposomes were loaded with SN38 and attached via avidin: biotin linkages to microbubbles. The whole structure was targeted to the tumour vasculature by the addition of anti-vascular endothelial growth factor receptor 2 antibodies. Tumour drug delivery and metabolism were quantified in SW480 xenografts after application of an ultrasound trigger to the tumour region. Increasing the trigger duration from 5 s to 2 min or increasing the number of 5 s triggers did not improve drug delivery, nor did changing to a chirp trigger designed to stimulate a greater proportion of the microbubble population, although this did show that the short tone trigger resulted in greater release of free SN38. Examination of ultrasound triggers in vivo to improve drug delivery is justified as there are multiple mechanisms at play that may not allow direct translation from in vitro findings. In this setting, a short tone burst gives the best ultrasound parameters for tumoural drug delivery.

An Open Access Chamber Designed for the Acoustic Characterisation of Microbubbles

Microbubbles are used as contrast agents in clinical ultrasound for Left Ventricular Opacification (LVO) and perfusion imaging. They are also the subject of promising research in therapeutics as a drug delivery mechanism or for sonoporation and co-administration. For maximum efficacy in these applications, it is important to understand the acoustic characteristics of the administered microbubbles. Despite this, there is significant variation in the experimental procedures and equipment used to measure the acoustic properties of microbubble populations. A chamber was designed to facilitate acoustic characterisation experiments and was manufactured using additive manufacturing techniques. The design has been released to allow wider uptake in the research community. The efficacy of the chamber for acoustic characterisation has been explored with an experiment to measure the scattering of SonoVue® microbubbles at the fundamental frequency and second harmonic under interrogation from emissions in the frequency range of 1.6 to 6.4 MHz. The highest overall scattering values were measured at 1.6 MHz and decreased as the frequency increased, a result which is in agreement with previously published measurements. Statistical analysis of the acoustic scattering measurements have been performed and a significant difference, at the 5% significance level, was found between the samples containing contrast agent and the control sample containing only deionised water. These findings validate the proposed design for measuring the acoustic scattering characteristics of ultrasound contrast agents.

Improved Sensitivity of Ultrasound-Based Subharmonic Aided Pressure Estimation Using Monodisperse Microbubbles.

Subharmonic aided pressure estimation (SHAPE) has been shown effective for noninvasively measuring hydrostatic fluid pressures in a variety of clinical applications. The objective of this study was to explore potential improvements in SHAPE sensitivity using monodisperse microbubbles. Populations of monodisperse microbubbles were created using a commercially available microfluidics device (Solstice Pharmaceuticals). Size distributions were assessed using a Coulter Counter and stability of the distribution following fabrication was evaluated over 24 hours. Attenuation of the microbubble populations from 1 to 10 MHz was then quantified using single element transducers to identify each formulation's resonance frequency. Frequency spectra over increasing driving amplitudes were investigated to determine the nonlinear phases of subharmonic signal generation. SHAPE sensitivity was evaluated in a hydrostatic pressure-controlled water bath using a Logiq E10 scanner (GE Healthcare). Monodisperse lipid microbubble suspensions ranging from 2.4 to 5.3 μm in diameter were successfully created and they showed no discernable change in size distribution over 24 hours following activation. Calculated resonance frequencies ranged from 2.1 to 6.3 MHz and showed excellent correlation with microbubble diameter (R2 > 0.99). When investigating microbubble frequency response, subharmonic signal occurrence was shown to begin at 150 kPa peak negative pressure, grow up to 225 kPa, and saturate at approximately 250 kPa. Using the Logiq E10, monodisperse bubbles demonstrated a SHAPE sensitivity of -0.17 dB/mmHg, which was nearly twice the sensitivity of the commercial polydisperse microbubble currently being used in clinical trials. Monodisperse microbubbles have the potential to greatly improve the sensitivity of SHAPE for the noninvasive measurement of hydrostatic pressures.

Optimization of microbubble-based DNA vaccination with low-frequency ultrasound for enhanced cancer immunotherapy.

Immunotherapy is an important cancer treatment strategy; nevertheless, the lack of robust immune cell infiltration in the tumor microenvironment remains a factor in limiting patient response rates. In vivo gene delivery protocols can amplify immune responses and sensitize tumors to immunotherapies, yet non-viral transfection methods often sacrifice transduction efficiency for improved safety tolerance. To improve transduction efficiency, we optimized a strategy employing low ultrasound transmission frequency-induced bubble oscillation to introduce plasmids into tumor cells. Differential centrifugation isolated size-specific microbubbles. The diameter of the small microbubble population was 1.27 ± 0.89 μm and that of larger population was 4.23 ± 2.27 μm. Upon in vitro insonation with the larger microbubble population, 29.7% of cancer cells were transfected with DNA plasmids, higher than that with smaller microbubbles (18.9%, P <0.05) or positive control treatments with a commercial transfection reagent (12%, P < 0.01). After 48 h, gene expression increased more than two-fold in tumors treated with large, as compared with small, microbubbles. Furthermore, the immune response, including tumor infiltration of CD8+ T cells and F4/80+ macrophages, was enhanced. We believe that this safe and efficacious method can improve preclinical procedures and outcomes for DNA vaccines in cancer immunotherapy in the future.

Acoustic characterization and stability assessment of size-isolatedprotein-shelled microbubbles

The side size distribution of ultrasound contrast agents has a strong effect on their performance in therapy and imaging. In this work, attenuation spectroscopy was performed to characterize the acoustic response and stability of three size-isolated populations of protein-shelled microbubbles with mean diameters ranging from 1.66 to 3.77 μm.

The iterative nonlinear contrast source method for simulating ultrasound propagation through a polydisperse microbubble population

Multiple scattering of sound by a population of particles attracts scientific interest for many decades. In contrast-enhanced echography, the simulation of ultrasound propagation through a dense cloud of nonlinearly oscillating microbubbles imposes a numerical challenge. This is particularly the case for polydisperse concentrations in which each scatterer has individual and independent properties. To address this problem, the Iterative Nonlinear Contrast Source (INCS) method has been adapted. Originally, this solved the Westervelt equation by letting its nonlinear term represent a contrast source in a “linearized” medium, and iteratively updating the pressure by computing the 4D spatiotemporal convolution between this source and the Green’s function. Because convolution allows a coarse discretization, INCS is suitable to deal with large-scale problems. In this talk, microbubbles are regarded as nonlinearly responding point sources that act as contrast sources. The total scattered pressure is computed iteratively, as in the original method, but now in each iteration the temporal signature of the contrast sources is calculated by solving every bubble’s own Marmottant equation. Physically, each iteration accounts for an order of multiple scattering. Numerical results will also be presented, demonstrating that INCS can accurately and efficiently simulate ultrasound propagation through a 3D population of polydisperse nonlinear microbubbles.

In situ characterisation of size distribution and rise velocity of microbubbles by high-speed photography

Using microbubbles has gained significant interest in many domestic and industrial applications due to bubble stability in solution and increased mass transfer area. The characterisation of microbubble populations is therefore important and aids in the understanding of their behaviour. Microbubble characterisation remains challenging, particularly at high bubble densities. We have developed an in situ and automated method, based on image analysis, to determine bubble size distributions and bubble rise velocity at bubble densities of up to approximately 7 bubbles mm−2. The method uses image analysis of a side-stream viewing slit and was tested using air bubbles in water at diameters between 20 and 150 µm under a range of different conditions. The developed system enables fast, simple and accurate size determination for microbubbles, including continuous sampling and observation.

The effect of size range on ultrasound-induced translations in microbubble populations

Microbubble translations driven by ultrasound-induced radiation forces can be beneficial for applications in ultrasound molecular imaging and drug delivery. Here, the effect of size range in microbubble populations on their translations is investigated experimentally and theoretically. The displacements within five distinct size-isolated microbubble populations are driven by a standard ultrasound-imaging probe at frequencies ranging from 3 to 7 MHz, and measured using the multi-gate spectral Doppler approach. Peak microbubble displacements, reaching up to 10 μm per pulse, are found to describe transient phenomena from the resonant proportion of each bubble population. The overall trend of the statistical behavior of the bubble displacements, quantified by the total number of identified displacements, reveals significant differences between the bubble populations as a function of the transmission frequency. A good agreement is found between the experiments and theory that includes a model parameter fit, which is further supported by separate measurements of individual microbubbles to characterize the viscoelasticity of their stabilizing lipid shell. These findings may help to tune the microbubble size distribution and ultrasound transmission parameters to optimize the radiation-force translations. They also demonstrate a simple technique to characterize the microbubble shell viscosity, the fitted model parameter, from freely floating microbubble populations using a standard ultrasound-imaging probe.

Measurement of nuclei seeding in hydrodynamic test facilities

Microbubble populations within the test section of a variable-pressure water tunnel have been characterised for various operating conditions. The tunnel was operated with demineralised water and artificially seeded with microbubbles from an array of generators located in a plenum upstream of the tunnel contraction. The generators produce a polydisperse population of microbubbles 10–200 $$\upmu \hbox {m}$$ in the diameter. The microbubbles are generated from supersaturated feed water within a confined turbulent cavitating microjet. The generator and tunnel operating parameters were systematically varied to map the range of nuclei concentrations and size ranges possible in the test section. Microbubbles were measured with Mie Scattering Imaging (MSI), an interferometric sizing technique. A new method was introduced to calibrate the detection volume and extend the dynamic range of the MSI. The acquisition and processing of microbubble measurements with MSI have a fast turn-around such that nuclei concentration measurements are approaching real time. Estimation of the total bubble concentration was within 5% of the sampled concentration after only 100 detections but 10$$^4$$ were necessary for full histogram convergence. The tunnel is operated with water at low dissolved gas content to ensure all injected microbubbles dissolve and do not complete the tunnel circuit. As a result of this, the injected population is altered by dissolution as well as pressure change during the short residence between plenum and test section. The transformation is shown to be complex, changing with tunnel operating conditions. The measured test section nuclei populations were found to follow a power law for the higher concentrations. Test section nuclei concentrations of 0–24 $$\hbox {mL}^{-1}$$ can be achieved through variation of generator and tunnel operating parameters. a A schematic of the experiment. b Sample image data. c Measured concentration of the seeded microbubble cavitation nuclei. d Distribution of bubble concentration by size.

Using the Doppler effect to characterize microbubble shell viscoelasticity

The experimental testing and quality control of microbubble formulations for ultrasound applications requires high-throughput measurement of physicochemical properties, such as shell elasticity and viscosity. Single-bubble optical experiments are slow and expensive. Ultrasound attenuation experiments on microbubble populations can be done quickly, but the measurement is averaged over the ensemble, and it is often difficult to separate effects of microbubble size from shell viscoelasticity. Here, we discuss a relatively simple alternative method of using the Doppler effect to measure ultrasound radiation force-induced displacements of many individual microbubbles within a population. Our method involves insonifying a diluted suspension of freely floating microbubbles with a linear-array ultrasound probe driven by an open scanner. The microbubble displacements along the axis of the insonified region were acquired from the frequency shifts in the measured echo signals using the multi-gate spectral Doppler approach. These measurements were compared with theoretical peak microbubble displacements computed by combining a modified Rayleigh-Plesset equation and a balance of translational forces. The shell elasticity and viscosity producing the measured peak microbubble displacement was determined by assuming a resonant bubble size at each driving frequency. Our initial results are encouraging, and demonstrate a step toward high-throughput microbubble characterization using the Doppler effect.

Microbubble Radiation Force-Induced Translation in Plane-Wave Versus Focused Transmission Modes.

Due to the primary radiation force, microbubble displacement has been observed previously in the focal region of single-element and array ultrasound probes. This effect has been harnessed to increase the contact between the microbubbles and targeted endothelium for drug delivery and ultrasound molecular imaging. In this study, microbubble displacements associated with plane-wave (PW) transmission are thoroughly investigated and compared to those obtained in focused-wave (FW) transmission over a range of pulse repetition frequencies, burst lengths (BLs), peak negative pressures, and transmission frequencies. In PW mode, the displacements, depending upon the experimental conditions, are in some cases consistently higher (e.g., by 28%, when the longest BL was used at PRF = 4 kHz), and the axial displacements are spatially more uniform compared to FW mode. Statistical analysis on the measured displacements reveals a slightly different frequency dependence of statistical quantities compared to transient peak microbubble displacements, which may suggest the need to consider the size range within the tested microbubble population.

Microbubble translation in plane-wave ultrasound transmission

Primary radiation force is capable of translating microbubbles in the focal region of single-element and array ultrasound probes. This effect can be harnessed to enhance the contact between ligand-bearing microbubbles and targeted endothelium for applications in targeted drug delivery and ultrasound molecular imaging. In this study, displacements of lipid-coated microbubbles associated with plane-wave transmission are investigated using the multi-gate Doppler approach, and compared with focused-wave transmission at equivalent peak negative pressures. In plane wave transmission, the radiation force is nearly uniform over the field of view and therefore allows for a more uniform translation of microbubbles compared to focused wave. Statistically determined median displacements are in good agreement with the axial and lateral ultrasound beamplots both in plane-wave and focused-wave transmissions, while peak microbubble displacements reveal a number of discrepancies. Distinct size-isolated microbubble populations (diameters 1–2 μm, 3–4 μm, 4–5 μm, 5–8 μm, and polydisperse) were tested, showing important differences in their displacements and a strong driving frequency dependence thereof. These findings help tune the ultrasound transmission parameters for uniform and size-selective microbubble translations.

The photopolymerization of DC8,9PC in microbubbles

The polymerization of diacetylene lipids is a valuable mechanism that can be exploited for various applications ranging from sensor materials to triggered drug delivery. Therefore, mixtures of diacetylene lipid and saturated phospholipids have been extensively studied in liposome vesicles and Langmuir monolayers. The present study investigates the polymerization of 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) in a new assembly, i.e. in a lipid microbubble shell. Lipid microbubbles have a high degree of echogenicity which allows to probe the properties of the mixed lipid monolayer shell using ultrasound spectroscopy. Microbubbles with varying DC8,9PC content in a 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) matrix were characterized using ultrasound transmission spectroscopy, UV–vis absorption spectroscopy and microscopy imaging. The microbubble populations are characterized before and after UV radiation (254 nm), in order to investigate the effect of introducing DC8,9PC into the microbubble shell, and whether diacetylenes can polymerize in this configuration. Microbubbles were found to be more stable when either DSPC or DC8,9PC dominates the composition. UV radiation induced the formation of a diacetylene polymer in all DC8,9PC containing compositions that were investigated, and the polymerization was found to impair the stability of the microbubbles.

Combining Acoustic Trapping With Plane Wave Imaging for Localized Microbubble Accumulation in Large Vessels.

The capability of accumulating microbubbles using ultrasound could be beneficial for enhancing targeted drug delivery. When microbubbles are used to deliver a therapeutic payload, there is a need to track them, for a localized release of the payload. In this paper, a method for localizing microbubble accumulation with fast image guidance is presented. A linear array transducer performed trapping of microbubble populations interleaved with plane wave imaging, through the use of a composite pulse sequence. The acoustic trap in the pressure field was created parallel with the direction of flow in a model of a vessel section. The acoustic trapping force resultant from the large gradients in the acoustic field was engendered to directly oppose the flowing microbubbles. This was demonstrated numerically with field simulations, and experimentally using an Ultrasound Array Research Platform II. SonoVue microbubbles at clinically relevant concentrations were pumped through a tissue-mimicking flow phantom and exposed to either the acoustic trap or a control ultrasonic field composed of a single-peak acoustic radiation force beam. Under the flow condition at a shear rate of 433 s-1, the use of the acoustic trap led to lower speed estimations ( ) in the center of the acoustic field, and an enhancement of 71% ± 28%( ) in microbubble image brightness.

A flow focusing microfluidic device with an integrated Coulter particle counter for production, counting and size characterization of monodisperse microbubbles.

Flow focusing microfluidic devices (FFMDs) have been investigated for the production of monodisperse populations of microbubbles for chemical, biomedical and mechanical engineering applications. High-speed optical microscopy is commonly used to monitor FFMD microbubble production parameters, such as diameter and production rate, but this limits the scalability and portability of the approach. In this work, a novel FFMD design featuring integrated electronics for measuring microbubble diameters and production rates is presented. A micro Coulter particle counter (μCPC), using electrodes integrated within the expanding nozzle of an FFMD (FFMD-μCPC), was designed, fabricated and tested. Finite element analysis (FEA) of optimal electrode geometry was performed and validated with experimental data. Electrical data was collected for 8-20 μm diameter microbubbles at production rates up to 3.25 × 105 MB s-1 and compared to both high-speed microscopy data and FEA simulations. Within a valid operating regime, Coulter counts of microbubble production rates matched optical reference values. The Coulter method agreed with the optical reference method in evaluating the microbubble diameter to a coefficient of determination of R2 = 0.91.