Nonlinear Frequency Mixing Ultrasound Imaging of Nanoscale Contrast Agents

Objective: Nanoscale ultrasound contrast agents show promise as alternatives for diagnostics and therapies due to their enhanced stability and ability to traverse blood vessels. Nonetheless, their reduced size limits echogenicity. This study introduces an enhanced nanobubble frequency mixing ultrasound imaging method, by capitalizing on their nonlinear acoustic response to dual-frequency excitation. Methods: A single broadband transducer (L12-3v) controlled by a programmable ultrasound system was used to transmit a dual-frequency single-cycle wavefront. The frequency mixing effect enabled simultaneous transducer capture of nanobubble-generated sum and difference frequencies in real time without the need for additional hardware or post-processing, by substituting the single-frequency wavefront in a standard contrast harmonic pulse inversion imaging protocol, with the dual-frequency wavefront. Results: Optimization experiments were conducted in tissue mimicking phantoms. Among the dual-frequency combinations that were tested, the highest contrast was obtained using 4&8 MHz. The nanobubble contrast improved with increased mechanical index, and achieved a maximal contrast improvement of 8.4 ± 0.5 dB compared to 4 MHz pulse inversion imaging. In imaging of a breast cancer tumor mouse model, after a systemic nanobubble injection, the contrast was improved by 3.4 ± 1.7, 4.8 ± 1.8, and 6.3 ± 1.6 dB for mechanical indices of 0.04, 0.08, and 0.1, respectively. Conclusion: Nonlinear frequency mixing significantly improved the nanobubble contrast, which facilitated their imaging in-vivo. Significance: This study offers a new avenue to enhance ultrasound imaging utilizing nanobubbles, potentially leading to advancements in other diagnostic applications.