Acoustical and physical dynamics of phagocytosed microbubble contrast agents

Ultrasound contrast agents are captured and phagocytosed by activated neutrophils adherent to the venular wall in regions of inflammation. In order to determine the physical and acoustical behavior of phagocytosed microbubbles, we assessed the responses of both phagocytosed and free microbubbles with direct optical and acoustical observation. A high-speed camera was used for optical analysis of bubble radial oscillations during insonation. Observations demonstrated that phagocytosed microbubbles remain acoustically active and capable of large volumetric oscillations during an acoustic pulse despite viscous damping due to the cytoplasm. Comparison of the echoes acquired during insonation of free and phagocytosed microbubbles demonstrated that phagocytosed microbubbles produce an echo with a higher mean frequency than free microbubbles in response to a rarefaction-first single cycle pulse. This frequency difference is predicted using a Rayleigh-Plesset equation modified to describe mechanical properties of thin shells. By fitting theoretical radius-time curves to experimental data, we estimate the effective viscosity of the neutrophil cytoplasm to be approximately 12 cPs. With the treatment of neutrophil cytoplasm as a power-law fluid, this value is in agreement with cytoplasmic viscosity measurements by previous researchers. We conclude that contrast-enhanced ultrasound can acoustic signals from microbubbles inside of neutrophils and may provide a unique tool to identify activated neutrophils at sites of inflammation