Photoacoustic computed tomography (PACT) detects acoustic waves that are generated by both ballistic and diffused photons and retrieves the optical absorption distribution through an inverse algorithm, allowing for high-resolution imaging at a depth of up to several centimeters 1. The penetration depth of OR-PAM is typically limited to ~ 1–2 mm because its high spatial resolution depends on the tight optical focus of ballistic photons. Recently, OR-PAM has been used for high-speed, high-resolution mapping of the cortical blood vessel network and to study the hemodynamics in the mouse brain cortex 7. By scanning a tightly focused laser beam in the optical ballistic regime, a PAT system can realize optical-resolution photoacoustic microscopy (OR-PAM) 5, 6. PAT can operate in both the optical ballistic and diffusive regimes, thereby providing multiscale imaging solutions. PAT inherits the advantage of high optical contrast in optical imaging methods while breaking the optical diffusion limit on the penetration of high-resolution optical imaging by detecting ultrasound. The rapid thermoelastic expansion of the stressed tissue generates acoustic waves that propagate in the tissue-with orders of magnitude weaker scattering than light scattering on a per-unit-path-length basis in the acoustic frequency of interest-and are detected by an ultrasonic transducer or a transducer array 3, 4. In PAT, nanosecond-pulsed laser illumination of absorbing molecules generates volume expansion due to the transient local temperature rise. Photoacoustic tomography (PAT) is a nonionizing, hybrid imaging modality that combines optical excitation and acoustic detection to realize high optical contrast and high spatial resolution at depths inside biological tissues 1, 2. An improvement in spatial resolution by a factor of 6 has been realized in vivo by the droplet localization technique. 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. The in vivo resolution enhancement was demonstrated by continuously imaging the cortical layer of a mouse brain during droplet injection. 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 droplets were prepared by dissolving hydrophobic absorbing dye in oil, followed by mixing with water. 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. However, the spatial resolution of PACT is limited by acoustic diffraction. 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.
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