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.