Challenges of conducting quantitative ultrasound with a multimodal optical imaging system

High-frequency quantitative ultrasound is a potential non-invasive source of imaging cell-tissue scale biomarkers for major diseases such as heart disease, cancer, and preterm birth. However, one of the barriers to developing such biomarkers is that it is labor-intensive to compare quantitative ultrasound images to optical images of the tissue structure. We have previously developed a multiscale imaging system that can obtain registered qualitative ultrasound and optical images, but there are further technical challenges to obtaining quantitative data: System-specific details of obtaining and processing data with Verasonics high-frequency transducers; the need for high-frequency reference phantoms; and off-axis clutter from imaging above a glass coverslip. This paper provides a characterization of the Verasonics ultrasound system with the 18.5 MHz L22-14v and 28.5 MHz L38-22v transducers, describes the construction of high-frequency reference phantoms, and details methods for reducing off-axis clutter. The paper features a demonstration multiscale image of a wild type mouse mammary gland that incorporates quantitative ultrasound with both transducers and second harmonic generation microscopy. These advances demonstrate a way to obtain, on a single system with a cohesive and integrated pipeline, quantitative ultrasound data that is correlated with optical imaging without the need for extensive sample preparation.