Acoustic Molecular Imaging Beyond the Diffraction Limit In Vivo

Ultrasound molecular imaging (USMI) is a technique used to noninvasively estimate the distribution of molecular markers <em>in vivo</em> by imaging microbubble contrast agents (MCAs) that have been modified to target receptors of interest on the vascular endothelium. USMI is especially relevant for preclinical and clinical cancer research and has been used to predict tumor malignancy and response to treatment. In the last decade, methods that improve the resolution of contrast-enhanced ultrasound by an order of magnitude and allow researchers to noninvasively image individual capillaries have emerged. However, these approaches do not translate directly to molecular imaging. In this work, we demonstrate super-resolution visualization of biomarker expression <em>in vivo</em> using superharmonic ultrasound imaging (SpHI) with dual-frequency transducers, targeted contrast agents, and localization microscopy processing. We validate and optimize the proposed method <em>in vitro</em> using concurrent optical and ultrasound microscopy and a microvessel phantom. With the same technique, we perform a proof-of-concept experiment <em>in vivo</em> in a rat fibrosarcoma model and create maps of biomarker expression co-registered with images of microvasculature. From these images, we measure a resolution of 23 &mu;m, a nearly fivefold improvement in resolution compared to previous diffraction-limited molecular imaging studies.