Imaging of oxygen and hypoxia in cell and tissue samples

Molecular oxygen (O₂) is a key player in cell mitochondrial function, redox balance and oxidative stress, normal tissue function and many common disease states. Various chemical, physical and biological methods have been proposed for measurement, real-time monitoring and imaging of O₂ concentration, state of decreased O₂ (hypoxia) and related parameters in cells and tissue. Here, we review the established and emerging optical microscopy techniques allowing to visualize O₂ levels in cells and tissue samples, mostly under in vitro and ex vivo, but also under in vivo settings. Particular examples include fluorescent hypoxia stains, fluorescent protein reporter systems, phosphorescent probes and nanosensors of different types. These techniques allow high-resolution mapping of O₂ gradients in live or post-mortem tissue, in 2D or 3D, qualitatively or quantitatively. They enable control and monitoring of oxygenation conditions and their correlation with other biomarkers of cell and tissue function. Comparison of these techniques and corresponding imaging setups, their analytical capabilities and typical applications are given.     

Bafilomycin A1 activates respiration of neuronal cells via uncoupling associated with flickering depolarization of mitochondria

Bafilomycin A1 (Baf) induces an elevation of cytosolic Ca²⁺ and acidification in neuronal cells via inhibition of the V-ATPase. Also, Baf uncouples mitochondria in differentiated PC12 (_dPC12), _dSH-SY5Y cells and cerebellar granule neurons, and markedly elevates their respiration. This respiratory response in _dPC12 is accompanied by morphological changes in the mitochondria and decreases the mitochondrial pH, Ca²⁺ and ΔΨm. The response to Baf is regulated by cytosolic Ca²⁺ fluxes from the endoplasmic reticulum. Inhibition of permeability transition pore opening increases the depolarizing effect of Baf on the ΔΨm. Baf induces stochastic flickering of the ΔΨm with a period of 20 ± 10 s. Under conditions of suppressed ATP production by glycolysis, oxidative phosphorylation impaired by Baf does not provide cells with sufficient ATP levels. Cells treated with Baf become more susceptible to excitation with KCl. Such mitochondrial uncoupling may play a role in a number of (patho)physiological conditions induced by Baf.     

Imaging oxygen in neural cell and tissue models by means of anionic cell-permeable phosphorescent nanoparticles

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 O₂ levels on a time-resolved fluorescence plate reader and PLIM microscope. PA2 also efficiently stains rat brain slices and permits detailed O₂ 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 O₂ in tissues, which we relate to the localisation of specific progenitor cell populations. Overall, these anionic probes are useful for sensing O₂ levels in various cells and tissues, particularly in neural cells, and facilitate high-resolution imaging of O₂ in 3D tissue models.     

Quantitative analysis of mucosal oxygenation using ex vivo imaging of healthy and inflamed mammalian colon tissue

Colonic inflammation is associated with decreased tissue oxygenation, significantly affecting gut homeostasis. However, the crosstalk between O₂ consumption and supply in the inflamed tissue are not fully understood. Using a murine model of colitis, we analysed O₂ in freshly prepared samples of healthy and inflamed colon tissue. We developed protocols for efficient ex vivo staining of mouse distal colon mucosa with a cell-penetrating O₂ sensitive probe Pt-Glc and high-resolution imaging of O₂ concentration in live tissue by confocal phosphorescence lifetime-imaging microscopy (PLIM). Microscopy analysis revealed that Pt-Glc stained mostly the top 50–60 μm layer of the mucosa, with high phosphorescence intensity in epithelial cells. Measured O₂ values in normal mouse tissue ranged between 5 and 35 μM (4–28 Torr), tending to decrease in the deeper tissue areas. Four-day treatment with dextran sulphate sodium (DSS) triggered colon inflammation, as evidenced by an increase in local IL6 and mKC mRNA levels, but did not affect the gross architecture of colonic epithelium. We further observed an increase in oxygenation, partial activation of hypoxia inducible factor (HIF) 1 signalling, and negative trends in pyruvate dehydrogenase activity and O₂ consumption rate in the colitis mucosa, suggesting a decrease in mitochondrial respiration, which is known to be regulated via HIF-1 signalling and pyruvate oxidation rate. These results along with efficient staining with Pt-Glc of rat and human colonic mucosa reveal high potential of PLIM platform as a powerful tool for the high-resolution analysis of the intestinal tissue oxygenation in patients with inflammatory bowel disease and other pathologies, affecting tissue respiration.