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Herein, a novel pH‐responsive phosphorescent probe based on cyclometalated iridium(III) complex is reported. To prevent oxygen quenching of phosphorescence and to improve the probe biocompatibility, the complex is covalently conjugated with a water‐soluble block‐copolymer that also increases its pH sensitivity. The resulting polymeric nanoprobe demonstrates a strong response of the phosphorescence lifetime onto pH variations in physiological range. Cellular experiments with Chinese hamster ovary (CHO‐K1) cells show the predominant internalization of the probe in acidified cell compartments, endosomes and lysosomes. The analysis of phosphorescence lifetime imaging microscopy data confirms applicability of the sensor for monitoring of intra‐ and extracellular pH in cell cultures. [ABSTRACT FROM AUTHOR]

Abstract The encapsulation and/or surface modification can stabilize and protect the phosphorescence bio‐probes but impede their intravenous delivery across biological barriers. Here, a new class of biocompatible rhenium (ReI) diimine carbonyl complexes is developed, which can efficaciously permeate normal vessel walls and then functionalize the extravascular collagen matrixes as in situ oxygen sensor. Without protective agents, ReI‐diimine complex already exhibits excellent emission yield (34%, λem = 583 nm) and large two‐photon absorption cross‐sections (σ2 = 300 GM @ 800 nm) in water (pH 7.4). After extravasation, remarkably, the collagen‐bound probes further enhanced their excitation efficiency by increasing the deoxygenated lifetime from 4.0 to 7.5 µs, paving a way to visualize tumor hypoxia and tissue ischemia in vivo. The post‐extravasation functionalization of extracellular matrixes demonstrates a new methodology for biomaterial‐empowered phosphorescence sensing and imaging.

The encapsulation and/or surface modification can stabilize and protect the phosphorescence bio‐probes but impede their intravenous delivery across biological barriers. Here, a new class of biocompatible rhenium (ReI) diimine carbonyl complexes is developed, which can efficaciously permeate normal vessel walls and then functionalize the extravascular collagen matrixes as in situ oxygen sensor. Without protective agents, ReI‐diimine complex already exhibits excellent emission yield (34%, λem = 583 nm) and large two‐photon absorption cross‐sections (σ2 = 300 GM @ 800 nm) in water (pH 7.4). After extravasation, remarkably, the collagen‐bound probes further enhanced their excitation efficiency by increasing the deoxygenated lifetime from 4.0 to 7.5 µs, paving a way to visualize tumor hypoxia and tissue ischemia in vivo. The post‐extravasation functionalization of extracellular matrixes demonstrates a new methodology for biomaterial‐empowered phosphorescence sensing and imaging. [ABSTRACT FROM AUTHOR]

Simultaneous imaging of intracellular and blood oxygen levels in tissues provides valuable information on the dynamic behavior of molecular oxygen (O 2 ) in normal and diseased tissues. Here, to achieve this goal, we developed green-emitting intracellular O 2 probes based on the Ir(III) complex, PPY (tris(2-phenylpyridinato)iridium(III)), and investigated the possibility of multicolor O 2 imaging by co-staining tissues with a red-emitting intravascular probe BTP-PEG 48 . The newly synthesized complexes possess modified 2-phenylpyridinato ligand(s) with a cationic or hydrophilic substituent, such as a dimethylamino group, triphenylphosphonium cation, or hydroxy group, in order to enhance cellular uptake efficiency. The photophysical and cellular properties of these complexes were systematically investigated to evaluate their ability as O 2 probes. Among these complexes, PPYDM and PPY2OH, which have a dimethylamino group and two hydroxy groups, respectively, exhibited much higher cellular uptake efficiencies compared with PPY and showed high O 2 sensitivity in HeLa cells. Phosphorescence lifetime imaging microscopy (PLIM) measurements of HeLa cells co-stained with PPYDM and hydrophilic BTP-PEG 48 allowed for the evaluation of intracellular and extracellular O 2 levels in cell culture. We took PLIM images of the pancreas following intravenous administration of PPYDM and BTP-PEG 48 into anesthetized mice. The PLIM measurements using these probes allowed simultaneous O 2 imaging of acinar cells and capillaries in the pancreas with cellular-level resolution. From the phosphorescence lifetimes of PPYDM and BTP-PEG 48 and the calibration curves evaluated in rat pancreatic acinar cells and blood plasma, we found that the average oxygen partial pressures of acinar cells and capillaries were almost equal at about 30 mmHg.

Multicellular spheroids are useful models of mammalian tissue for studies of cell proliferation, differentiation, replacement therapies and drug action. Having a size of 100-500 μm they mimic in vivo micro-environment and characteristic gradients of O2, pH and nutrients. We describe the use of cell-penetrating O2 probes based on phosphorescent Pt-porphyrins to perform high-resolution 2D and 3D mapping of O2 in spheroid structures by live cell fluorescence imaging technique. Optimised procedures for preparation of neurospheres from cortical neural cells isolated from embryonic rat brain, their staining with the phosphorescent O2 probes NanO2 and MM2 and subsequent analysis of oxygenation on different live cell imaging platforms, including widefield and confocal phosphorescence lifetime imaging microscopy (PLIM), conventional confocal and two-photon ratiometric intensity based O2 detection are presented. This is followed by a series of physiological experiments in which oxygenation patterns of the neurospheres are correlated with culturing conditions (atmospheric hypoxia and hyperoxia, size, growth factors), distribution of stem cells, mature neurons and astrocytes, HIF-2α stabilisation and responses to metabolic stimulation. The O2 imaging method allows multiplexing with many conventional fluorescent probes to perform multi-parametric imaging analysis of cells in 3D microenvironment. It can be applied to other types of spheroids and 3D tissue models., (Copyright © 2013 Elsevier Ltd. All rights reserved.)