Large-scale super-resolution optoacoustic imaging facilitated by FeNP/ICG-loaded coreless polyelectrolyte microcapsules

<b>Rationale:</b> Localization optoacoustic tomography (LOT) enhances imaging of deep-tissue microvasculature by leveraging flowing contrast particles. However, achieving high-resolution, large-scale imaging requires contrast agents with strong per-particle signal, good biocompatibility, and prolonged circulation time. This study introduces coreless polyelectrolyte microcapsules (MCs) encapsulating indocyanine green (ICG) and iron oxide nanoparticles (FeNP) to overcome current limitations in LOT imaging. <b>Methods:</b> MCs were engineered using a layer-by-layer technique by depositing polyelectrolytes, FeNP and ICG on a CaCO₃ core, which was eventually dissolved. Their optical, morphological, and biocompatibility properties were characterized via UV-Vis-NIR spectroscopy, SEM, and toxicity assays <i>In vitro</i> and <i>In vivo</i>. Optoacoustic tomography (OAT), motion contrast optoacoustic imaging (MC-OA), directional motion contrast optoacoustic imaging (DMC-OA), localization optoacoustic tomography (LOT), and velocity mapping were conducted in mice to evaluate cerebral, testicular, and tumor vasculature. A raster scanning approach enabled large-scale brain imaging with 9-position coverage. <b>Results:</b> MCs displayed strong optoacoustic contrast, low cytotoxicity, and long circulation times (>45 min). <i>In vivo</i> LOT imaging revealed super-resolved microvascular networks in brain, testis, and tumor, with up to 2.5-fold enhancement in vessel visualization parameters. Velocity maps enabled quantification of cerebral blood flow, and oxygenation maps were further rendered by integrating LOT with spectral unmixing. Extended imaging was enabled by persistent MC signal, facilitating full-cortex vascular imaging.