PROJECT SUMMARY We aim to renew a highly productive collaboration that has developed revolutionary new ultrasound imaging hardware and processing approaches. Our innovative technologies have enabled high-signal-to-noise and high- resolution contrast-enhanced microvascular imaging. Specifically, we developed the world’s first ultra-broadband co-axial dual-frequency linear arrays, which enable transmission of ultrasound at low frequencies and reception of high-frequency content from microbubbles well beyond their 7th harmonic. These transducers enable superharmonic imaging, which provides high-resolution contrast images of the microvasculature with essentially no tissue background and is used for visualizing vessels on the order of 100 microns in both animal and human tissues. Significantly, we have utilized these tools to image microvascular angiogenesis, known for decades to be a biomarker of cancer, yet not previously possible to image directly with ultrasound. Our ‘acoustic angiography’ imaging readily shows drastic differences in microvasculature density and structure between malignant cancers and healthy tissue, providing superior sensitivity and specificity to early micro-breast tumors in mice genetically predisposed to breast cancer. Furthermore, our excellent superharmonic signal separation enables super-resolution imaging without the cumbersome spatiotemporal filtering approaches otherwise used for this process, which are readily corrupted by tissue motion or slow flow. Our next generation approach will enable outstanding microvascular resolution (<100 um) in deep tissue (4-8 cm), despite slow microvascular flow, and with robustness to tissue motion. Despite our advances in hardware and imaging techniques, challenges remain to make our technology optimal for imaging human cancers. The shallow focal depth of the arrays designed in the prior project period, limited by prototype array and lens designs, and carried over from their original small-animal imaging applications, is far from optimized for the 3-4 cm depth needed to image suspicious breast lesions in humans. In this renewal, we will direct the development and optimization of dual-frequency arrays specifically for deeper clinical imaging (targeting depths up to 8cm). Our loss in resolution due to lower frequency bandwidths will be recovered by superharmonic super-resolution processing. We have demonstrated this approach with excellent results using the prototype dual frequency arrays, enabling recovery of sub-100 micron resolution, even with frequencies that can penetrate 6-8 cm. Furthermore, since microvascular imaging provides the most diagnostic information when acquired in 3D, we will develop the first dual-frequency 2D arrays. Successful completion of these aims will advance ultrasound imaging closer to a practical clinical modality for identifying and assessing angiogenic and molecular biomarkers of cancer with high specificity and sensitivity for future applications such as di...