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.

PtPFPP-carbohydrate conjugates are promising O2probes for 3D PLIM imaging of live spheroids and brain explants.

Cell-permeable phosphorescent probes enable the study of cell and tissue oxygenation, bioenergetics, metabolism, and pathological states such as stroke and hypoxia. A number of such probes have been described in recent years, the majority consisting of cationic small molecule and nanoparticle structures. While these probes continue to advance, adequate staining for the study of certain cell types using live imaging techniques remains elusive; this is particularly true for neural cells. Here we introduce novel probes for the analysis of neural cells and tissues: negatively charged poly(methyl methacrylate-co-methacrylic acid)-based nanoparticles impregnated with a phosphorescent Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP) dye (this form is referred to as PA1), and with an additional reference/antennae dye poly(9,9-diheptylfluorene-alt-9,9-di-p-tolyl-9H-fluorene) (this form is referred to as PA2). PA1 and PA2 are internalised by endocytosis, result in efficient staining in primary neurons, astrocytes, and PC12 cells and multi-cellular aggregates, and allow for the monitoring of local O(2) levels on a time-resolved fluorescence plate reader and PLIM microscope. PA2 also efficiently stains rat brain slices and permits detailed O(2) imaging experiments using both one and two-photon intensity-based modes and PLIM modes. Multiplexed analysis of embryonic rat brain slices reveals age-dependent staining patterns for PA2 and a highly heterogeneous distribution of O(2) in tissues, which we relate to the localisation of specific progenitor cell populations. Overall, these anionic probes are useful for sensing O(2) levels in various cells and tissues, particularly in neural cells, and facilitate high-resolution imaging of O(2) in 3D tissue models.

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Proteins present in infant formulas are modified by oxidation and glycation during processing. Modified amino acid residues released from proteins may be absorbed in the gastrointestinal tract, and pose a health risk to infants. In this study, the markers of glycation furosine (1.7–3.5 μg per milligram of protein) and N ε-(carboxymethyl)­lysine (28–81 ng per milligram of protein) were quantitated in infant formulas. The effects of these species, and other amino acid modifications, at the levels detected in infant formulas, on 3T3-L1 (murine preadipocyte) and Caco-2 (human intestinal epithelial) cells were assessed. Incubation of 3T3-L1 cells for 48 h with amino acid side chain oxidation and glycation products (1 and 10 μM) resulted in a loss (up to 40%, p < 0.05) of cell thiols and decreased metabolic activity compared with those of the controls. In contrast, Caco-2 cells showed a stimulation (10–50%, p < 0.05) of cellular metabolism on exposure to these products for 24 or 48 h. A 28% (p < 0.05) increase in protein carbonyls was detected upon incubation with 200 μM modified amino acids for 48 h, although no alteration in transepithelial electrical resistance was detected. Oxidation products were detected in the basolateral compartments of Caco-2 monolayers when modified amino acids were applied to the apical side, consistent with limited permeability (up to 3.4%) across the monolayer. These data indicate that modified amino acids present in infant formulas can induce effects on different cell types, with evidence of bioavailability and induction of cellular stress. This may lead to potential health risks for infants consistently exposed to high levels of infant formulas.

Measurement of cell oxygenation and oxygen consumption is useful for studies of cell bioenergetics, metabolism, mitochondrial function, drug toxicity and common pathophysiological conditions. Here we present a new platform for such applications which uses commercial multichannel biochips (μ-slides, Ibidi) and phosphorescent O2 sensitive probes. This platform was evaluated with both extracellular and intracellular O2 probes, several different cell types and treatments including mitochondrial uncoupling and inhibition, depletion of extracellular Ca(2+) and inhibition of V-ATPase and histone deacetylases. The results show that compared to the standard microwell plates currently used, the μ-slide platform provides facile O2 measurements with both suspension and adherent cells, higher sensitivity and reproducibility, and faster measurement time. It also allows re-perfusion and multiple treatments of cells and multi-parametric analyses in conjunction with other probes. Optical measurements are conducted on standard fluorescence readers and microscopes.

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