<i>In Vivo</i> Lifetime Imaging of the Internal O₂ Dynamics in Corals with near-Infrared-Emitting Sensor Nanoparticles

Mapping of O₂ with luminescent sensors within intact animals is challenging due to attenuation of excitation and emission light caused by tissue absorption and scattering as well as interfering background fluorescence. Here we show the application of luminescent O₂ sensor nanoparticles (∼50-70 nm) composed of the O₂ indicator platinum(II) tetra(4-fluoro)phenyltetrabenzoporphyrin (PtTPTBPF) immobilized in poly(methyl methacrylate-<i>co</i>-methacrylic acid) (PMMA-MA). We injected the sensor nanoparticles into the gastrovascular system of intact colony fractions of reef-building tropical corals that harbor photosynthetic microalgae in their tissues. The sensor nanoparticles are excited by red LED light (617 nm) and emit in the near-infrared (780 nm), which enhances the transmission of excitation and emission light through biological materials. This enabled us to map the internal O₂ concentration via time-domain luminescence lifetime imaging through the outer tissue layers across several coral polyps in flowing seawater. After injection, nanoparticles dispersed within the coral tissue for several hours. While luminescence intensity imaging showed some local aggregation of sensor particles, lifetime imaging showed a more homogeneous O₂ distribution across a larger area of the coral colony. Local stimulation of symbiont photosynthesis in corals induced oxygenation of illuminated tissue areas and formation of lateral O₂ gradients toward surrounding respiring tissues, which were dissipated rapidly after the onset of darkness. Such measurements are key to improving our understanding of how corals regulate their internal chemical microenvironment and metabolic activity, and how they are affected by environmental stress such as ocean warming, acidification, and deoxygenation. Our experimental approach can also be adapted for <i>in vivo</i> O₂ imaging in other natural systems such as biofilms, plant and animal tissues, as well as in organoids and other cell constructs, where imaging internal O₂ conditions are relevant and challenging due to high optical density and background fluorescence.