Evaluation of Stone Features That Cause the Color Doppler Ultrasound Twinkling Artifact.

The color Doppler ultrasound twinkling artifact is a rapid color shift that appears on 43%-96% of kidney stones. Surface microbubbles on kidney stones are theorized to cause twinkling as exposure to elevated static pressures of 0.41-1.13 MPa (approximately 0.5-1 times diagnostic ultrasound pressure and 5-10 times ambient pressure) reduced twinkling. However, it is unclear what external and internal stone features support bubbles. Thirteen ex vivo kidney stones were scanned with color Doppler ultrasound at 2.5, 5 and 18.5 MHz. Select stones were imaged with environmental scanning electron microscopy or underwater micro-computed tomography to evaluate features that may cause twinkling. Results revealed that the lower frequencies produced larger volumes of twinkling. Condensation first occurred in the smallest (∼1 µm diameter) surface pores and may be indicative of where bubbles form. Gas pockets were seen inside two of three tested stones that may contribute to twinkling. Overall, these results provide evidence of cavity structures both externally and internally and their correlation to the twinkling artifact. This indicates that microbubbles may be present on and within kidney stones and may contribute to the twinkling artifact.

Ultrasound-Guided Cytosolic Protein Delivery via Transient Fluorous Masks.

The inability to spatiotemporally guide proteins in tissues and efficiently deliver them into cells remains a key barrier to realizing their full potential in precision medicine. Here, we report ultrasound-sensitive fluoro-protein nanoemulsions which can be acoustically tracked, guided, and activated for on-demand cytosolic delivery of proteins, including antibodies, using clinically relevant diagnostic ultrasound. This advance is accessed through the discovery of a family of fluorous tags, or FTags, that transiently mask proteins to mediate their efficient dispersion into ultrasound-sensitive liquid perfluorocarbons, a phenomenon akin to dissolving an egg in liquid Teflon. We identify the biochemical basis for protein fluorous masking and confirm FTag coatings are shed during delivery, without disrupting the protein structure or function. Harnessing the ultrasound sensitivity of fluorous emulsions, real-time imaging is used to simultaneously monitor and activate FTag-protein complexes to enable controlled cytosolic antibody delivery in vitro and in vivo. These findings may advance the development of image-guided, protein-based biosensing and therapeutic modalities.