In vivo superresolution photoacoustic computed tomography by localization of single dyed droplets

Photoacoustic (PA) computed tomography (PACT) is a noninvasive hybrid imaging technique that combines optical excitation and acoustic detection to realize high contrast, high resolution, and deep penetration in biological tissues. However, the spatial resolution of PACT is limited by acoustic diffraction. Here, we report in vivo superresolution PACT, which breaks the acoustic diffraction limit by localizing the centers of single dyed droplets that are flowing in blood vessels. The droplets were prepared by dissolving hydrophobic absorbing dye in oil, followed by mixing with water. The dyed droplets generate much higher-amplitude PA signals than blood and can flow smoothly in vessels; thus, they are excellent tracers for localization-based superresolution imaging. The in vivo resolution enhancement was demonstrated by continuously imaging the cortical layer of a mouse brain during droplet injection. The droplets that were flowing in the vessels were localized, and their center positions were used to construct a superresolution image that exhibits sharper features and more finely resolved vascular details. An improvement in spatial resolution by a factor of 6 has been realized in vivo by the droplet localization technique.