Here we propose an ultrasound contrast-based imaging method that enables non-invasive quantitative assessment of ambient pressure changes inside the body (such as blood pressure). We subject the microbubbles in the contrast agent to two frequencies: A low-frequency (57 kHz) signal that dynamically manipulates the ambient pressure, and a series of high-frequency (4 MHz) pulses for exciting and imaging the bubble response. The imaging pulses exploit the ambient pressure sensitivity of the subharmonic microbubble response, while the low-frequency signal provided an intrinsic calibration for measurement of ambient pressure changes. We tested this approach in an in vitro setup and show that it can visualize and quantify ambient pressure differences with a sensitivity of 0.5 dB/kPa.

Objective
The response of ultrasound contrast agents is sensitive to ambient pressure, especially via their scattered subharmonic signal, which makes them a promising candidate for non-invasive pressure measurements in vivo. This work aimed to understand the sensitivity to ambient pressure of subharmonic oscillations from single microbubbles.

Methods
The subharmonic oscillation amplitude of single microbubbles in response to varying ambient pressure was studied both experimentally and numerically. In experiment, approximately 2200 single microbubbles from a monodisperse population were measured at a driving frequency close to twice their resonance frequency.

Results
The results of the numerical simulations and experiments show that a pressure change leads to a small size change in the bubble that then changes the lipid packing density, and with that the stiffness of the bubble shell.

Conclusion
The dependency of subharmonic oscillation amplitude to changes in ambient pressure can be explained by a shift in the resonance frequency of the bubble as a function of ambient pressure. The subharmonic response increases with ambient pressure when the resonance frequency shifts toward half the driving frequency and decreases when the resonance frequency shifts away from half the driving frequency. These findings help to understand non-invasive pressure sensing through subharmonic ultrasound imaging.

Ultrasound contrast agents, comprised of phospholipid-coated microbubbles, can be produced as monodisperse populations using a microfluidic flow-focusing device. However, microbubble coalescence remains a significant challenge. High production temperatures (e.g., 55 °C) can be used to suppress coalescence, but it complicates the microfluidic device design and is incompatible with targeting agents and drug conjugates. This study investigates the production of monodisperse microbubbles at room temperature with the addition of the amphiphilic surfactant Pluronic F68. Two 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)-based phospholipid formulations were investigated: F1, containing 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carbonyl-methoxypolyethylene glycol] (DPPE-PEG5000), and F2, which included both DPPE-PEG5000 and polyoxyethylene(40) stearate (PEG40-stearate). We characterized the size stability and acoustic behavior of monodisperse microbubbles produced with various Pluronic F68 concentrations. Adding 5-10 mol % Pluronic F68 was found to effectively suppress coalescence and facilitated the production of monodisperse microbubbles that remained shelf stable for at least 7 days. Acoustic attenuation measurements revealed a shell stiffness ranging from 0.78 to 0.93 N/m for these microbubbles. The 10 mol % Pluronic F68 addition (10PF) demonstrated superior monodispersity and was selected for further experiments. Upon dilution, the size and resonance frequencies of both F1-10PF and F2-10PF decreased over time, though F2-10PF showed better stability compared to F1-10PF for both metrics. Both F1-10PF and F2-10PF exhibited a stronger subharmonic scattering intensity than SonoVue (clinical approved microbubbles), which offers potential for blood pressure sensing. Our study shows that incorporating Pluronic F68 facilitates the production of monodisperse microbubbles at room temperature that are stable long-term and have excellent acoustical properties, with the F2-10PF formulation demonstrating better stability than the F1-10PF.

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.

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.