Acoustic pH sensor for dynamic ultrasound imaging of cellular acidification

Genetically encoded pH sensors based on fluorescent proteins enable dynamic optical imaging of cellular processes such as endocytosis and exocytosis. To date, light scattering in thick tissue as well as photobleaching of fluorescent proteins prevent deep cellular imaging over sustained periods of time. To visualize intracellular pH variations across opaque organs, we introduce a genetically encoded acoustic pH sensor dubbed pHonon. We modified the outer gas vesicle protein (GvpC) of echogenic protein nanostructures via histidine point mutations. At low pH, engineered gas vesicles exhibit an increased shell stiffness which switched their acoustic response from nonlinear to linear. By combining pHonons with nonlinear ultrasound imaging, we captured dynamic deep tissue images of lysosomal acidification by macrophages in murine liver. The combination of pHonon with nonlinear ultrasound creates the possibility for basic studies of endo- and exocytic activity in deep tissue of living opaque organisms.