Enhancing subharmonic response by controlling initial state of monodisperse microbubbles via a multi-gas core

The subharmonic response of microbubbles exposed to ultrasound has shown significant promise for nonlinear imaging and non-invasive blood pressure estimation. Elucidating the underlying mechanisms of subharmonic response of microbubbles is critical for advancing subharmonic-based ultrasound applications. Using a flow-focusing microfluidic device, we controllably generated monodisperse microbubbles (MDMBs) with lipid-packed shells and varying CO₂ volume fractions (0%, 50%, and 80%) in multi-gas (CO₂ and C₃F₈) cores, while maintaining consistent initial surface tension and shell viscoelasticity across MDMB population. Using the measured pressure-dependent attenuation spectra, pressure-dependent resonance-frequency curves were obtained to characterize the bubble state (i.e., elastic, elastic-buckling transition, buckling). With increasing CO₂ volume fraction in the multi-gas core, the initial state of MDMBs after stabilization gradually transitioned from the elastic state to the elastic-buckling transition state. Correspondingly, the surface tension decreased and the shell elasticity increased, demonstrating that regulating the CO₂ fraction in the gas core enables controllable tuning of the initial bubble state. Statistical analysis showed that MDMBs closer to buckling state exhibited a significantly lower (∼12% reduction) threshold peak negative pressure for subharmonic generation and a significantly higher (∼11 dB increment) subharmonic amplitude. The initial state approaching buckling state, achieved by increasing the CO₂ volume fraction in the gas core of fabricated MDMBs, was therefore identified as the key mechanism governing the enhanced subharmonic response. These findings provide direct evidence supporting nonlinear bubble oscillation theory, elucidate subharmonic generation mechanisms, and establish a reliable method for tailoring MDMB states, thereby enhancing subharmonic responses for ultrasound-based medical applications.