Monitoring cell and tissue oxygenation is important for the analysis of cell development and differentiation, mitochondrial function, and common (patho)physiological conditions such as ischemia, cancer, neurodegenerative disorders. A number of materials for sensing cellular oxygen (O2) by optical means have been described in recent years, but the diverse range of biological models and measurement tasks demands more versatile, flexible, and simple O2 sensors. A new cell-penetrating phosphorescent nanosensor material called MM2 probe is presented. In it, the highly photostable phosphorescent reporter dye Pt(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin (PtTFPP; emission at 650 nm) and poly(9,9-dioctylfluorene) (PFO) fluorophore act as Förster resonance energy transfer (FRET) donor and two-photon antennae are embedded in cationic hydrogel nanoparticles. Such probe formulation provides efficient delivery into the cell and subsequent sensing and high-resolution imaging of cellular O2 in different detection modalities, including ratiometric intensity and phosphorescence lifetime-based sensing under one-photon and two photon excitation. MM2 probe combines high brightness, photo- and chemical stability, low toxicity, and ease of fabrication and use. Its versatility and analytical performance are demonstrated in physiological experiments with adherent cells and neurospheres representing 2D and 3D respiring objects and detection on time-resolved fluorescent readers, confocal and multiphoton microscopes, and customized microsecond fluorescence/phosphorescence lifetime imaging microscopy (FLIM) systems.
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