In-vivo Lifetime Imaging of the Internal O2 Dynamics in Corals with NIR-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 (~40-50 nm) composed of the O2 indicator platinum(II) tetra(4-fluoro)phenyltetrabenzoporphyrin (PtTPTBPF) immobilized in poly(methyl methacrylate-co-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 ex-cited by red LED light (617 nm) and emit in the near-infrared (780 nm), which enhances 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 over several hours. While luminescence intensity imag-ing showed some local aggregation of sensor particles, lifetime imaging showed a more homogenous 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 dissi-pated rapidly after onset of darkness. Such measurements are key to improve 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 in vivo O2 imaging in other natural systems such as biofilms, plant and animal tissues, as well as in organoids and other cell con-structs, where imaging internal O2 conditions are relevant and challenging due to high optical density and back-ground fluorescence.