An imaging method for oxygen distribution, respiration and photosynthesis at a microscopic level of resolution.

Biological samples are far from homogeneous, with complex compartmentation being the norm. Major physiological processes such as respiration do not therefore occur in a uniform manner within most tissues, and it is currently not possible to image its gradients in living plant tissues. A compact fluorescence ratiometric-based device is presented here, consisting of an oxygen-sensitive foil and a USB (universal serial bus) microscope. The sensor foil is placed on the sample surface and, based on the localized change in fluorescence signal over time, information about the oxygen consumption (respiration) or evolution (photosynthesis) can be obtained. Using this imaging technique, it was possible to demonstrate the spatial pattern of oxygen production and consumption at a c. 20-µm level of resolution, and their visualization in the rhizosphere, stem and leaf, and within the developing seed. The oxygen mapping highlighted the vascular tissues as the major stem sink for oxygen. In the leaf, the level of spatial resolution was sufficient to visualize the gas exchange in individual stomata. We conclude that the novel sensor set-up can visualize gradients in oxygen-consuming and producing processes, thereby facilitating the study of the spatial dynamics of respiration and photosynthesis in heterogeneous plant tissues.

Methodology and Significance of Microsensor-based Oxygen Mapping in Plant Seeds - an Overview.

Oxygen deficiency is commonplace in seeds, and limits both their development and their germination. It is, therefore, of considerable relevance to crop production. While the underlying physiological basis of seed hypoxia has been known for some time, the lack of any experimental means of measuring the global or localized oxygen concentration within the seed has hampered further progress in this research area. The development of oxygen-sensitive microsensors now offers the capability to determine the localized oxygen status within a seed, and to study its dynamic adjustment both to changes in the ambient environment, and to the seed's developmental stage. This review illustrates the use of oxygen microsensors in seed research, and presents an overview of existing data with an emphasis on crop species. Oxygen maps, both static and dynamic, should serve to increase our basic understanding of seed physiology, as well as to facilitate upcoming breeding and biotechnology-based approaches for crop improvement.

Hypoxia in static and dynamic 3D culture systems for tissue engineering of bone.

Tissue engineering of sizeable cell-scaffold constructs is limited by gradients in tissue quality from the periphery toward the center. Because homogenous delivery of oxygen to three-dimensional (3D) cell cultures remains an unsolved challenge, we hypothesized that uneven oxygen supply may impede uniform cellular growth on scaffolds. In this study we challenged static and dynamic 3D culture systems designed for bone tissue engineering applications with a well-growing subclone of MC3T3-E1 preosteoblasts and continuously measured the oxygen concentrations in the center of cell-seeded scaffolds and in the surrounding medium. After as little as 5 days in static culture, central oxygen concentrations dropped to 0%. Subsequently, cells died in central regions of the scaffold but not in its periphery, where oxygen levels were approximately 4%. The use of perfusion bioreactors successfully prevented cell death, yet central oxygen concentrations did not rise above 4%. We conclude that 3D culture in vitro is associated with relevant oxygen gradients, which can be the cause of inhomogeneous tissue quality. Perfusion bioreactors prevent cell death but they do not entirely eliminate 3D culture-associated oxygen gradients. Therefore, we advise continuous oxygen monitoring of 3D culture systems to ensure tissue quality throughout engineered constructs.

Construction and validation of a microprocessor controlled extracorporal circuit in rats for the optimization of isolated limb perfusion.

Although a few experimental approaches to isolated limb perfusion (ILP) are described in the literature, none of these animal models mimics the clinical perfusion techniques adequately to improve the technique of ILP on the basis of valid preclinical data. Therefore, we developed an ILP setup in rats allowing online monitoring of essential perfusion parameters such as temperature (in perfusate, various tissues, and rectum), pH (perfusate), perfusion pressure, and O(2) concentration (in perfusate, tissue), by a tailor-made data acquisition system. This setup permits close supervision of vital parameters during ILP. Various interdependencies, concerning the flow rate and the pressure of perfusate as well as tissue oxygenation were registered. For the measurement of pO(2) values in the perfusate and in different regions of the perfused hind limb, a novel type of microoptode based on quenching of a fluorescent dye was devised. Stable normothermic (37 degrees C) perfusion conditions were maintained at a constant perfusion pressure in the range of 40-60 mm Hg by administration of the spasmo lytic moxaverine (0.5 mg/mL of perfusate as initial dose) at a perfusate flow rate of 0.5 mL/min for 60 min. At the end of an ILP, there were no signs of tissue damage, neither concerning laboratory data (K(+), myoglobin, creatine kinase, lactic dehydrogenase) nor histopathological criteria. The reported ILP model is not only well suited to investigate the effects of hyperthermia but also to assess the efficacy of new antineoplastic approaches, when nude rats, bearing human tumours in the hind limbs, are used.