We present the development and characterization of fluorescent oxygen-sensing microparticles designed for measuring oxygen concentration in microenvironments existing within standard cell culture and transparent three-dimensional (3D) cell scaffolds. The microparticle synthesis employs poly(dimethylsiloxane) to encapsulate silica gel particles bound with an oxygen-sensitive luminophore as well as a reference or normalization fluorophore that is insensitive to oxygen. We developed a rapid, automated and non-invasive sensor analysis method based on fluorescence microscopy to measure oxygen concentration in a hydrogel scaffold. We demonstrate that the microparticles are non-cytotoxic and that their response is comparable to that of a traditional dissolved oxygen meter. Microparticle size (5–40 μm) was selected for microscale-mapping of oxygen concentration to allow measurements local to individual cells. Two methods of calibration were evaluated and revealed that the sensor system enables characterization of a range of hypoxic to hyperoxic conditions relevant to cell and tissue biology (i.e., pO2 10–160 mm Hg). The calibration analysis also revealed that the microparticles have a high fraction of quenched luminophore (0.90 ± 0.02), indicating that the reported approach provides significant advantages for sensor performance. This study thus reports a versatile oxygen-sensing technology that enables future correlations of local oxygen concentration with individual cell response in cultured engineered tissues.
Metastatic cancer cells must traverse a microenvironment ranging from extremely hypoxic, within the tumor, to highly oxygenated, within the host's vasculature. Tumor hypoxia can be further characterized by regions of both chronic and intermittent hypoxia. We present the design and characterization of a microfluidic device that can simultaneously mimic the oxygenation conditions observed within the tumor and model the cell migration and intravasation processes. This device can generate spatial oxygen gradients of chronic hypoxia and produce dynamically changing hypoxic microenvironments in long-term culture of cancer cells. V C 2014 AIP Publishing LLC. [http://dx
Capability of measuring and monitoring local oxygen concentration at the single cell level (tens of microns scale) is often desirable but difficult to achieve in cell culture. In this study, biocompatible oxygen sensing beads were prepared and tested for their potential for real-time monitoring and mapping of local oxygen concentration in 3D micro-patterned cell culture systems. Each oxygen sensing bead is composed of a silica core loaded with both an oxygen sensitive Ru(Ph2phen3)Cl2 dye and oxygen insensitive Nile blue reference dye, and a poly-dimethylsiloxane (PDMS) shell rendering biocompatibility. Human intestinal epithelial Caco-2 cells were cultivated on a series of PDMS and type I collagen based substrates patterned with micro-well arrays for 3 or 7 days, and then brought into contact with oxygen sensing beads. Using an image analysis algorithm to convert florescence intensity of beads to partial oxygen pressure in the culture system, tens of microns-size oxygen sensing beads enabled the spatial measurement of local oxygen concentration in the microfabricated system. Results generally indicated lower oxygen level inside wells than on top of wells, and local oxygen level dependence on structural features of cell culture surfaces. Interestingly, chemical composition of cell culture substrates also appeared to affect oxygen level, with type-I collagen based cell culture systems having lower oxygen concentration compared to PDMS based cell culture systems. In general, results suggest that oxygen sensing beads can be utilized to achieve real-time and local monitoring of micro-environment oxygen level in 3D microfabricated cell culture systems.
. 2005. A method for measuring above-and below-ground C stocks in hillside landscapes. Can. J. Soil Sci. 85: 523-530. Information on C stocks in agriculture and forest ecosystems in hillside landscapes is limited. The objective of this study was to develop and test field methods to measure above-and below-ground C stocks in hillside landscapes. Above-ground biomass in agricultural system was determined by measuring weight of residues left after crop harvest. In degraded secondary forests, tree biomass was estimated using allometric equations developed from in situ measurements. Herbs + bushes and litter dry weight were measured in two 0.25-m 2 quadrats located within one 100-m 2 treed plots. Carbon stocks were determined after chemical analysis of plant tissue and soil samples by dry combustion. Geo-referenced cores were taken inside a 1-m-diameter soil sampling clock that allows for spatial and temporal monitoring of soil C changes. The clock was marked with 12 divisions to establish the exact location of present and future sampling points. The below-ground fraction of C (mineral soil and fine roots) amounted to nearly 95% of the total C stock in agricultural systems and between 57 and 82% in the case of forest systems. Soil C stocks in hillside agricultural soils were higher than those found in forested soils with 70% of the C stored below-ground residing in the 0-45 cm of soil. The field method detected differences in C stocks in pools associated with various vegetations and soils in hillside ecosystems. Les auteurs ont déterminé la biomasse aérienne des systèmes agricoles en pesant les débris végétaux après la récolte. Dans les forêts secondaires dégradées, la biomasse des arbres a été estimée au moyen d'équations allométriques élaborées à partir de relevés pris sur le terrain. On a mesuré le poids sec des herbacées et des broussailles ainsi que de la litière dans deux cadrats de 0,25 m 2 situés à l'intérieur d'une parcelle arborée de 100 m 2 . Les réserves de carbone ont été établies après analyse chimique des tissus végétaux et des échantillons de sol par combustion sèche. On a prélevé des carottes géoréférencées dans un cercle d'échantillonnage de un mètre permettant de suivre les variations spatiales et temporelles du C dans le sol. Le cercle a été divisé en 12 secteurs ce qui autorise la localisation exacte des points d'échantillonnage actuels et futurs. La fraction souterraine du C (sol minéral et radicelles) représente près de 95 % des stocks totaux de C des systèmes agricoles et 57 à 82 % des stocks des systèmes forestiers. Les systèmes agricoles en pente possèdent de plus grandes réserves de C que les sols forestiers, 70 % du C souterrain se retrouvant dans la couche de 0 à 45 cm. Cette méthode permet de déceler la fluctuation des réserves de C dans les puits associés aux divers types de végétation et de sol dans les écosystèmes en pente.
Bacterial biofilms are a major obstacle challenging the development of more effective therapies to treat implant infections. Oxygen availability to bacterial cells has been implicated in biofilm formation and planktonic cell detachment; however, there are insufficient tools available to measure oxygen concentrations within complex three-dimensional structures with ~1 µm resolution. Such measurements may complement measures of biofilm structure and cell activity to provide a more comprehensive understanding of biofilm biology. Thus, we developed oxygen-sensing microparticles specifically designed to characterize oxygen transport through the volume of bacterial biofilms. The Stöber method was used to synthesize monodisperse silica microparticles of approximately the same size as a bacterium (~1 µm). Two fluorophores, oxygen-sensitive Ru(Ph2phen3)Cl2, and the reference fluorophore Nile blue chloride were immobilized on the surface of the particles. We demonstrate application of the microparticles toward measuring the oxygen concentration profiles within a live Staphylococcus aureus biofilm.
We present the development and characterization of fluorescent oxygen-sensing microparticles designed for measuring oxygen concentration in microenvironments existing within standard cell culture and transparent three-dimensional (3D) cell scaffolds. The microparticle synthesis employs poly(dimethylsiloxane) to encapsulate silica gel particles bound with an oxygen-sensitive luminophore as well as a reference or normalization fluorophore that is insensitive to oxygen. We developed a rapid, automated and non-invasive sensor analysis method based on fluorescence microscopy to measure oxygen concentration in a hydrogel scaffold. We demonstrate that the microparticles are non-cytotoxic and that their response is comparable to that of a traditional dissolved oxygen meter. Microparticle size (5-40 μm) was selected for microscale-mapping of oxygen concentration to allow measurements local to individual cells. Two methods of calibration were evaluated and revealed that the sensor system enables characterization of a range of hypoxic to hyperoxic conditions relevant to cell and tissue biology (i.e., pO 2 10-160 mm Hg). The calibration analysis also revealed that the microparticles have a high fraction of quenched luminophore (0.90 ± 0.02), indicating that the reported approach provides significant advantages for sensor performance. This study thus reports a versatile oxygen-sensing technology that enables future correlations of local oxygen concentration with individual cell response in cultured engineered tissues.
Several studies about web bligh control were carried out in Panama, the Dominican Republic, and Costa Rica. The pathogen´s genetic variability, bean varietal tolerance, chemical control, and sowing densities were evaluated. In Panama yield was increased through Benomyl applications and a greater sowing density. In the Dominican Republic Rhizoctonia AG 4, AG 2 2 and AG 1 groups were found. Several bean genotypes were evaluated, as well as populations from the web blight tolerant JB X MUS 14 cross. Between 20% and 40 % web blight severity was found in Costa Rica. Coverage and application of chemicals improved bean yield.
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