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Oxygen is a crucial parameter for organotypic in vitro-cultures which are still performed under ambient atmosphere supplemented with 5 % CO2. This can lead to hyperoxia with its associated effects on signaling cascades and their effects on cellular behavior. Here, we describe a platform that enables real-time, label-free, optical oxygen (gradient) measurements in organotypic 3D-/organoid cultures thus allowing for physioxic culture and assay conditions in e. g. mito stress tests and substance testing. [ABSTRACT FROM AUTHOR]

A cross-reactive array in a micro titer plate (MTP) format is described that is based on a versatile and highly flexible scheme. It makes use of rather unspecific metal ions probes having almost identical fluorescence spectra, thus enabling (a) interrogation at identical analytical wavelengths, and (b) imaging of the probes contained in the wells of the MTP using a CCD camera and an array of blue-light-emitting diodes as a light source. The unselective response of the indicators in the presence of mixtures of five divalent cations generates a characteristic pattern that was analyzed by chemometric tools. The fluorescence intensity of the indicators was transferred into a time-dependent parameter applying a scheme called dual lifetime referencing. In this method, the fluorescence decay profile of the indicator is referenced against the phosphorescence of an inert reference dye added to the system. The intrinsically referenced measurements also were performed using blue LEDs as light sources and a CCD camera without intensifiers as the detector. The best performance was observed if each well was excited by a single LED. The assembly allows the detection of dye concentrations in the nanomoles-per-liter range without amplification and the acquisition of 96 wells simultaneously. The pictures obtained form the basis for evaluation by pattern recognition algorithms. Support vector machines are capable of predicting the presence of significant concentrations of metal ions with high accuracy.

We present a referenced scheme for fluorescence intensity measurements that is useful for imaging applications. It is based on the conversion of the fluorescence intensity information into a time-dependent parameter. A phosphorescent dye is added in the form of ~10-[micro]m particles to the sample containing the pH-sensitive fluorescent indicator. Both the reference dye and the pH probe are excited simultaneously by a blue LED, and an overall luminescence is measured. In the time-resolved imaging method presented here, two images taken at different time gates were recorded using a CCD camera. The first image is recorded during excitation and reflects the luminescence signal of both the fluorophore (pH) and the phosphor (reference). The second image, which is measured after a certain delay (after switching off the light source), is solely caused by the long-lived phosphorescent dye. Because the intensity of the fluorophore contains the information on pH, whereas phosphorescence is pHindependent, the ratio of the images displays a referenced intensity distribution that reflects the pH at each picture element (pixel). The scheme is useful for LED light sources and CCD cameras that can be gated with square pulses in the microsecond range. The fundamentals and potential of this new method, to which we refer as time domain dual lifetime referencing (t-DLR), are demonstrated.

In many wetland plants, belowground transport of O2 via aerenchyma tissue and subsequent O2 loss across root surfaces generates small oxic root zones at depth in the rhizosphere with important consequences for carbon and nutrient cycling. This study demonstrates how roots of the intertidal salt-marsh plant Spartina anglica affect not only O2, but also pH and CO2 dynamics, resulting in distinct gradients of O2, pH, and CO2 in the rhizosphere. A novel planar optode system (VisiSens TDR , PreSens GmbH) was used for taking high-resolution 2D-images of the O2, pH, and CO2 distribution around roots during alternating light-dark cycles. Belowground sediment oxygenation was detected in the immediate vicinity of the roots, resulting in oxic root zones with a 1.7 mm radius from the root surface. CO2 accumulated around the roots, reaching a concentration up to threefold higher than the background concentration, and generally affected a larger area within a radius of 12.6 mm from the root surface. This contributed to a lowering of pH by 0.6 units around the roots. The O2, pH, and CO2 distribution was recorded on the same individual roots over diurnal light cycles in order to investigate the interlinkage between sediment oxygenation and CO2 and pH patterns. In the rhizosphere, oxic root zones showed higher oxygen concentrations during illumination of the aboveground biomass. In darkness, intraspecific differences were observed, where some plants maintained oxic root zones in darkness, while others did not. However, the temporal variation in sediment oxygenation was not reflected in the temporal variations of pH and CO2 around the roots, which were unaffected by changing light conditions at all times. This demonstrates that plant-mediated sediment oxygenation fueling microbial decomposition and chemical oxidation has limited impact on the dynamics of pH and CO2 in S. anglica rhizospheres, which may in turn be controlled by other processes such as root respiration and root exudation. [ABSTRACT FROM AUTHOR]

Summary: Root‐mediated CO2 uptake, O2 release and their effects on O2 and CO2 dynamics in the rhizosphere of Lobelia dortmanna were investigated. Novel planar optode technology, imaging CO2 and O2 distribution around single roots, provided insights into the spatiotemporal patterns of gas exchange between roots, sediment and microbial community. In light, O2 release and CO2 uptake were pronounced, resulting in a distinct oxygenated zone (radius: c. 3 mm) and a CO2‐depleted zone (radius: c. 2 mm) around roots. Simultaneously, however, microbial CO2 production was stimulated within a larger zone around the roots (radius: c. 10 mm). This gave rise to a distinct pattern with a CO2 minimum at the root surface and a CO2 maximum c. 2 mm away from the root. In darkness, CO2 uptake ceased, and the CO2‐depleted zone disappeared within 2 h. By contrast, the oxygenated root zone remained even after 8 h, but diminished markedly over time. A tight coupling between photosynthetic processes and the spatiotemporal dynamics of O2 and CO2 in the rhizosphere of Lobelia was demonstrated, and we suggest that O2‐induced stimulation of the microbial community in the sediment increases the supply of inorganic carbon for photosynthesis by building up a CO2 reservoir in the rhizosphere. [ABSTRACT FROM AUTHOR]

ABSTRACT Bone tissue engineering (BTE) utilizing biomaterial scaffolds and human mesenchymal stem cells (hMSCs) is a promising approach for the treatment of bone defects. The quality of engineered tissue is crucially affected by numerous parameters including cell density and the oxygen supply. In this study, a novel oxygen-imaging sensor was introduced to monitor the oxygen distribution in three dimensional (3D) scaffolds in order to analyze a new cell-seeding strategy. Immortalized hMSCs, pre-cultured in a monolayer for 30-40% or 70-80% confluence, were used to seed demineralized bone matrix (DBM) scaffolds. Real-time measurements of oxygen consumption in vitro were simultaneously performed by the novel planar sensor and a conventional needle-type sensor over 24 h. Recorded oxygen maps of the novel planar sensor revealed that scaffolds, seeded with hMSCs harvested at lower densities (30-40% confluence), exhibited rapid exponential oxygen consumption profile. In contrast, harvesting cells at higher densities (70-80% confluence) resulted in a very slow, almost linear, oxygen decrease due to gradual achieving the stationary growth phase. In conclusion, it could be shown that not only the seeding density on a scaffold, but also the cell density at the time point of harvest is of major importance for BTE. The new cell seeding strategy of harvested MSCs at low density during its log phase could be a useful strategy for an early in vivo implantation of cell-seeded scaffolds after a shorter in vitro culture period. Furthermore, the novel oxygen imaging sensor enables a continuous, two-dimensional, quick and convenient to handle oxygen mapping for the development and optimization of tissue engineered scaffolds. Biotechnol. Bioeng. 2017;114: 894-902. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

One of the major challenges in radiation therapy is the interference with tissue repair processes due to hypoxic characteristics and pH dysregulation. In this study, we present dual imaging of pH and oxygenation in vitro based on luminescent biocompatible sensor foils that allow studying the effects of irradiation on different cell types in culture. Different sensitivities of fibroblast and oral squamous carcinoma cells were observed by complementing oxygen and pH differences with proliferation assays. This study highlights especially the distinct role of oxygen after irradiation and the difference in proliferation processes of irradiated normal dermal cells in contrast to irradiated tumor cells. [ABSTRACT FROM AUTHOR]

In Salmonella infection, the S almonella pathogenicity island-2 ( SPI-2)-encoded type three secretion system ( T3SS2) is of key importance for systemic disease and survival in host cells. For instance, in the streptomycin-pretreated mouse model SPI-2-dependent S almonella replication in lamina propria CD11c− CXCR1− monocytic phagocytes/macrophages ( MΦ) is required for the development of colitis. In addition, containment of intracellular S almonella in the gut critically depends on the antimicrobial effects of the phagocyte NADPH oxidase ( PHOX), and possibly type 2 nitric oxide synthase ( NOS2). For both antimicrobial enzyme complexes, oxygen is an essential substrate. However, the amount of available oxygen upon enteroinvasive S almonella infection in the gut tissue and its impact on S almonella- MΦ interactions was unknown. Therefore, we measured the gut tissue oxygen levels in a model of S almonella enterocolitis using luminescence two-dimensional in vivo oxygen imaging. We found that gut tissue oxygen levels dropped from ∼78 Torr (∼11% O2) to values of ∼16 Torr (∼2% O2) during infection. Because in vivo virulence of S almonella depends on the S almonella survival in MΦ, S almonella- MΦ interaction was analysed under such low oxygen values. These experiments revealed an increased intracellular replication and survival of wild-type and t3ss2 non-expressing S almonella. These findings were paralleled by blunted nitric oxide and reactive oxygen species ( ROS) production and reduced S almonella ROS perception. In addition, hypoxia enhanced SPI-2 transcription and translocation of SPI-2-encoded virulence protein. Neither pharmacological blockade of PHOX and NOS2 nor impairment of T3SS2 virulence function alone mimicked the effect of hypoxia on S almonella replication under normoxic conditions. However, if t3ss2 non-expressing S almonella were used, hypoxia did not further enhance S almonella recovery in a PHOX and NOS2-deficient situation. Hence, these data suggest that hypoxia-induced impairment of antimicrobial activity and S almonella virulence cooperate to allow for enhanced S almonella replication in MΦ. [ABSTRACT FROM AUTHOR]