Phospholipid-coated microbubbles with a uniform acoustic response are a promising avenue for functional ultrasound sensing. A uniform acoustic response requires both a monodisperse size distribution and uniform viscoelastic shell properties. Monodisperse microbubbles can be produced in a microfluidic flow focusing device. Here, we investigate whether such monodisperse microbubbles have uniform viscoelastic shell properties and thereby a uniform “mono-acoustic” response. To this end, we visualized phase separation of the DSPC and DPPE-PEG5000 lipid shell components and measured the resonance curves of nearly 2000 single and freely floating microbubbles using a high-frequency acoustic scattering technique. The results demonstrate inhomogeneous phase-separated shell microdomains across the monodisperse bubble population, which may explain the measured inhomogeneous viscoelastic shell properties. The shell viscosity varied over an order of magnitude and the resonance frequency by a factor of two indicating both a variation in shell elasticity and in initial surface tension despite the relatively narrow size distribution.@en

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 0 = 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.

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Monodisperse microbubble ultrasound contrast agents may dramatically increase the sensitivity and efficiency in ultrasound imaging and therapy. They can be produced directly in a microfluidic flow-focusing device, but questions remain as to the interfacial chemistry, such as the formation and development of the phospholipid monolayer coating over time. Here, we demonstrate the synthesis of monodisperse bubbles with radii of 2-10 μm at production rates ranging from 10⁴ to 10⁶ bubbles/s. All bubbles were found to dissolve to a stable final radius 2.55 times smaller than their initial radius, independent of the nozzle size and shear rate, indicating that the monolayer self-assembles prior to leaving the nozzle. The corresponding decrease in surface area by a factor 6.6 reveals that lipid molecules are adsorbed to the gas-liquid interface in the disordered expanded state, and they become mechanically compressed by Laplace pressure-driven bubble dissolution to a more ordered condensed state with near zero surface tension. Acoustic characterization of the stabilized microbubbles revealed that their shell stiffness gradually increased from 0.8 to 2.5 N/m with increasing number of insonations through the selective loss of the more soluble lipopolymer molecules. This work therefore demonstrates high-throughput production of clinically relevant monodisperse contrast microbubbles with excellent control over phospholipid monolayer elasticity and microbubble resonance.

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The sensitivity and efficiency in contrast-enhanced ultrasound imaging and therapy can potentially be increased by the use of resonant monodisperse bubbles. However, bubbles of the same size may respond differently to ultrasound due to differences in their phospholipid shell. In an acoustic bubble sorting chip, resonant bubbles can be separated from the polydisperse agent. Here, a sample of acoustically sorted bubbles is characterized by measuring scattering and attenuation simultaneously using narrowband acoustic pulses at peak negative pressures of 10, 25, and 50 kPa over a 0.7-5.5 MHz frequency range. A second sample is characterized by attenuation measurements at acoustic pressures ranging from 5 to 75 kPa in steps of 2.5 kPa. Scattering and attenuation coefficients were modeled by integration over the pressure and frequency dependent response of all bubbles located within the non-uniform acoustic characterization beam. For all driving pressures and frequencies employed here, the coefficients could be modeled using a single and unique set of shell parameters confirming that acoustically sorted bubbles provide a uniform acoustic response. Moreover, it is shown that it is crucial to include the pressure distribution of the acoustic characterization beam in the modeling to accurately determine shell parameters of non-linearly oscillating bubbles.

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In this study we present a combined optical sizing and acoustical characterization technique for the study of the dynamics of single freely-floating ultrasound contrast agent microbubbles exposed to long burst ultrasound excitations up to the milliseconds range. A co-axial flow device was used to position individual microbubbles on a streamline within the confocal region of three ultrasound transducers and a high-resolution microscope objective. Bright-field images of microbubbles passing through the confocal region were captured using a high-speed camera synchronized to the acoustical data acquisition to assess the microbubble response to a 1-MHz ultrasound burst. Nonlinear bubble vibrations were identified at a driving pressure as low as 50 kPa. The results demonstrate good agreement with numerical simulations based on the shell-buckling model proposed by Marmottant et al. [J. Acoust. Soc. Am. 118, 3499-3505 (2005)]. The system demonstrates the potential for a high-throughput in vitro characterization of individual microbubbles.@en