Molecular Acoustic Angiography: A New Technique for High-resolution Superharmonic Ultrasound Molecular Imaging

Ultrasound molecular imaging utilizes targeted microbubbles to bind to vascular targets such as integrins, selectins and other extracellular binding domains. After binding, these microbubbles are typically imaged using low pressures and multi-pulse imaging sequences. In this article, we present an alternative approach for molecular imaging using ultrasound that relies on superharmonic signals produced by microbubble contrast agents. Bound bubbles were insonified near resonance using a low frequency (4 MHz) element and superharmonic echoes were received at high frequencies (25-30 MHz). Although this approach was observed to produce declining image intensity during repeated imaging in both in vitro and in vivo experiments because of bubble destruction, the feasibility of superharmonic molecular imaging was demonstrated for transmit pressures, which are sufficiently high to induce shell disruption in bound microbubbles. This approach was validated using microbubbles targeted to the αvβ3 integrin in a rat fibrosarcoma model (n = 5) and combined with superharmonic images of free microbubbles to produce high-contrast, high-resolution 3-D volumes of both microvascular anatomy and molecular targeting. Image intensity over repeated scans and the effect of microbubble diameter were also assessed in vivo, indicating that larger microbubbles yield increased persistence in image intensity. Using ultrasound-based acoustic angiography images rather than conventional B-mode ultrasound to provide the underlying anatomic information facilitates anatomic localization of molecular markers. Quantitative analysis of relationships between microvasculature and targeting information indicated that most targeting occurred within 50 μm of a resolvable vessel (>100 μm diameter). The combined information provided by these scans may present new opportunities for analyzing relationships between microvascular anatomy and vascular targets, subject only to limitations of the current mechanically scanned system and microbubble persistence to repeated imaging at moderate mechanical indices.