Real-time volumetric imaging of cells and molecules in deep tissues with Takoyaki ultrasound

Acoustic contrast agents and reporter genes play a critical role in allowing ultrasound to visualize blood flow, map molecules and track cellular function in optically opaque living organisms. However, many advanced agents requiring high acoustic pressures have been imaged primarily in 2D, while biological phenomena of interest unfurl in three dimensions. Here, we introduce a method for efficient, dynamic imaging of contrast agents and reporter genes in 3D using multiplexed matrix array transducers. Our "Takoyaki" pulse sequence uses the simultaneous scanning of multiple focal points to excite contrast agents with sufficient acoustic pressure for nonlinear imaging while efficiently covering 3D space. We first characterize and benchmark Takoyaki imaging performance with gas vesicle contrast agents in vitro. Then we establish utility in cellular imaging by visualizing acoustic reporter gene expression in a mouse model of glioblastoma. Finally, we demonstrate real-time volumetric imaging by tracking the dynamics of fluid motion in mouse brain ventricles during and after intraventricular contrast injection. Takoyaki imaging enables a more comprehensive understanding of biological processes by providing spatiotemporal information in 3D within the constraints of accessible multiplexed matrix-array systems.