Effect of Acoustic Parameters and Microbubble Concentration on the Likelihood of Encapsulated Microbubble Coalescence

Abstract

Microbubble contrast agents are commonly used for therapeutic and diagnostic imaging applications. Under certain conditions, these contrast agents can coalesce on ultrasound application and form larger bubbles than the initial population. The formation of large microbubbles potentially influences therapeutic outcomes and imaging quality. We studied clinically relevant ultrasound parameters related to low-pressure therapy and contrast-enhanced ultrasound imaging to determine their effect on microbubble coalescence and subsequent changes in microbubble size distributions in vitro. Results indicate that therapeutic ultrasound at low frequencies, moderate pressures and high duty cycles are capable of forming bubbles greater than two times larger than the initial bubble distribution. Furthermore, acoustic parameters related to contrast-enhanced ultrasound imaging that are at higher frequency, low-pressure and low-duty cycle exhibit no statistically significant changes in bubble diameter, suggesting that standard contrast ultrasound imaging does not cause coalescence. Overall, this work suggests that the microbubble coalescence phenomenon can readily occur at acoustic parameters used in therapeutic ultrasound, generating bubbles much larger than those found in commercial contrast agents, although coalescence is unlikely to be significant in diagnostic contrast-enhanced ultrasound imaging. This observation warrants further expansion of parameter ranges and investigation of resulting effects.

Keywords: Contrast-enhanced ultrasound; Microbubble coalescence; Ultrasound; Ultrasound contrast agent.

Figure 1:. (Color online) Experimental setup.

A 200μm inner diameter (240 μm outer diameter) capillary tube was placed at the shared focus of an ultrasound transducer (1 or 5 MHz) and the inverted microscope objective. A camera is attached to the microscope for recording high-framerate optical imaging. The ultrasound transducer’s focal zone is indicated within the yellow region.

Figure 2:. (Color online) High-speed optical microscopy showing ultrasound-induced microbubble clustering and coalescence.

Representative high speed optical microscopy images showing microbubble clustering and coalescence at varying ultrasound parameters (A) 1 MHz, 100 kPa, 1% DC, (B) 1 MHz, 400 kPa, and 1% DC, (C) 1 MHz, 100 kPa, 10% DC, (D) 5 MHz, 100 kPa, 1% DC, (E) 5 MHz, 400 kPa, 1% DC, (F) 5 MHz, 100 kPa, 10% DC.

Figure 3:. (Color online) Microbubble clustering dynamics.

Clustering analysis at varying acoustic parameters showing the percentage of clustered bubbles over time since the start of ultrasound application (vertical line) at (A) 1 MHz, 100 kPa, and varying duty cycle; (B) 1 MHz, 1% duty cycle, and varying pressure; (C) 5 MHz, 100 kPa, and varying duty cycle; (D) 5 MHz, 1% duty cycle, and varying pressure.

Figure 4:. Comparison of maximal mean microbubble diameters during ultrasound application.

Maximal mean microbubble diameter (volume-weighted) at a microbubble concentration of 2×10⁸ microbubbles/mL and ultrasound parameters: (A) 1 MHz, 100 kPa, and varying duty cycle; (B) 1 MHz, 1% duty cycle, and varying pressure; (C) 5 MHz, 100 kPa, and varying duty cycle; (D) 5 MHz, 1% duty cycle, and varying pressure. (E) change in maximal mean bubble diameter before vs. during ultrasound application (1 MHz, 100 kPa, and 10% duty cycle) for two concentrations of microbubbles (2×10⁶ and 2×10⁸ microbubbles/mL). Statistical significance was set a priori at p<0.05 and is depicted as (*) for p <0.05, (**) for p<0.01, (***) for p<0.001, and (****) for p<0.0001.