Using acoustic vortex tweezers (AVT) to spatially accumulate microbubbles (MBs) shows promise for enhancing drug delivery efficiency and reducing off-target effects. The strong echogenicity of accumulated MBs also improves diagnostics via conventional ultrasound (US) B-mode imaging. However, the annular high-pressure distribution of AVT inhibits MBs inflow at the inlet, reducing MBs collection. The spatial resolution of US B-mode imaging further limits theranostic applications of AVT-mediated MBs accumulation. To address these challenges, we integrated an AVT waveform with volumetric super-resolution imaging (VSRI) to monitor the dynamic growth of MBs cluster during accumulation. We used a 5-MHz 2D array transducer for VSRI, employing plane wave pulses interleaved with accumulating pulses to retain MBs at a flow rate of 0.023-0.047 mL/s in a 3-mm vessel phantom. An asymmetrical AVT waveform (AVT*) was produced by modulating the pressure at the MBs inlet compared to the outlet. The effectiveness was validated in rat cerebral vessels for real-time volumetric tracking of MBs clusters. Microscopy observations showed that AVT* could quickly gather flowing MBs into cluster without repelling them at a flow rate of 0.023 mL/s. Statistical results indicated that microscopic data correlated better with VSRI than with B-mode images, suggesting VSRI suffices to detect the dynamics of AVT*-actuated MBs accumulation in real-time. Additionally, VSRI detected a significant increase in MBs cluster size over time during AVT* in the superior sagittal sinus of the rat brain. These findings demonstrate that the proposed strategy can accumulate the flowing MBs at a desired location and simultaneously observe this phenomenon.
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