Microscopic characterization of functionalized paper as a platform for 3D cell cultures

To achieve an understanding and complete description of the functional properties of three dimensional (3D) cell culture systems, a large set of parameters is required, which clearly contrasts this cell cultivation approach from traditional two dimensional (2D), planar cultivation techniques . As an alternative to describe the characteristics of a 3D cell culture system by its physicochemical properties (e. g. stiffness, porosit y, level of crosslinking), the behavior of the cultivated cells can be used as a read-out parameter to characterize the 3D cultivation system . In this work, the cellular parameters membrane dynamics, actin fiber morphology and migration were used to investigate the differences between classic, planar and a collagen based three dimensional cell culture system Membrane dynamics – assessed by FRAP measurements of CAAX -mCherry – as well as actin formation – visualized by in vitro staining with LifeAct -tagRFP – showed distinct differences when investigated in planar or three dimensional systems. FRAP experiments with CAAX - mCherry showed, that even though the overall membrane composition does not appear to be different, mobility of the membrane is significantl y higher in three dimensional cell culture systems than in two dimensional . A view at the actin cytoskeleton revealed the already established difference: stress fibers and cortical actin are more pronounced in planar cell culture systems compared to cells c ultivated in three dimensional systems. Interestingly, cells originall y seeded in collagen hydrogels which migrated towards the glass surface show features in actin cytoskeleton formation resembling both culture conditions: both, actin stress fibers within the cell body as well as cortical actin are visible in those parts of the cells directly contacting the glass surface . The observed migration towards the glass surface gave rise to the investigation of this behavior. Migration in response to mechanical si gnals is termed durotaxis . Cells cultivated in collagen hydrogels or collagen hydrogels supported by cellulose sheets over a period of time were microscopicall y investigated to determine the distribution of cells . Cell distribution in unsupported collagen hydrogels was clearly in favor of hydrogel regions in close proximity to the glass surface. By applying supporting material in form of cellulose sheets, the cell culture was freely floating in the culture medium, resulting in an even distribution throughout the entire thickness of the cell culture system . As 3D cell culture systems make it more challenging to perform high quality imaging due to the inherent scattering and loss of intensity with increasing optical penetration, a post imaging processing tool set was evaluated and benchmarked in order to counteract these image corrupting effects and improve the image quality. This in turn also improves the compatibility of cellulose sheets with the commonly used tool set in life sciences: fluorescence microscopy. Special emphasis was put on the identification of a serviceable and performance -linked deconvolution setup . A GPU based CUDA Deconvolution plugin showed the best time performance but ultimately failed to produce the same quality level of image restoration as the three tested CPU based deconvolution applications. Among these three, the commercial HyugensPro software showed the best results in terms of increasing the contrast . The Iterative Deconvolution 3D plugin comes close to producing comparable results to the HuygensPro software, however, the time consumption for this application is up to 10 times larger. Finally, the plugin Deconvolution Lab showed reasonably satisfying results in terms of image restoration quality, while performing deconvolution slightly faster than HuygensPro. Finally, cellulose sheets are used for the cultivation of cells in 3D as an example of for paper as a versatile platform for the development of functional devices . Therefore a method is required that delivers spatially resolved, quantitative, sensitive, and, most importantly, also dynamic measurements . Optical microscopy has long been recognized as a method to characterize the heterogeneous and complex structure of paper. With fluorescence detection, the functionality has even been extended to provide chemical selectivity, e . g. to determine the distribution of secondary modifications like coatings and fillers throughout a sheet of paper. Here it is shown that quantitative widefield and confocal fluorescence microscopy are versatile methods to meet this set of demands. Confocal microscopy was used to achieve a detailed view of the interface between a hyd rophobic and rhodamine labeled polymer and a FITC labeled dextran solution. Furthermore, confocal microscopy revealed that the spatial propagation of the FITC labeled dextran solution occurs along the surface of the cellulose fibers, instead of the inter -fibers space. Widefield fluorescent microscopy was subsequently used for dynamic investigations of this spatial propagation

Shear velocity model for the Kyrgyz Tien Shan from joint inversion of receiver function and surface wave data

The Tien Shan is the largest active intracontinental orogenic belt on Earth. To better understand the processes causing mountains to form at great distances from a plate boundary, we analyse passive source seismic data collected on 40 broad band stations of the MANAS project (2005-2007) and 12 stations of the permanent KRNET seismic network to determine variations in crustal thickness and shear wavespeed across the range. We jointly invert P- and S-wave receiver functions with surface wave observations from both earthquakes and ambient noise to reduce the ambiguity inherent in the images obtained from the techniques applied individually. Inclusion of ambient noise data improves constraints on the upper crust by allowing dispersion measurements to be made at shorter periods. Joint inversion can also reduce the ambiguity in interpretation by revealing the extent to which various features in the receiver functions are amplified or eliminated by interference from multiples. The resulting wavespeed model shows a variation in crustal thickness across the range. We find that crustal velocities extend to ∼ 75 km beneath the Kokshaal Range, which we attribute to underthrusting of the Tarim Basin beneath the southern Tien Shan. This result supports the plate model of intracontinental convergence. Crustal thickness elsewhere beneath the range is about 50 km, including beneath the Naryn Valley in the central Tien Shan where previous studies reported a shallow Moho. This difference apparently is the result of wavespeed variations in the upper crust that were not previously taken into account. Finally, a high velocity lid appears in the upper mantle of the Central and Northern part of the Tien Shan, which we interpret as a remnant of material that may have delaminated elsewhere under the range.km, including beneath the Naryn Valley in the central Tien Shan where previous studies reported a shallow Moho. This difference apparently is the result of wavespeed variations in the upper crust that were not previously taken into account. Finally, a high velocity lid appears in the upper mantle of the Central and Northern part of the Tien Shan, which we interpret as a remnant of material that may have delaminated elsewhere under the range.This is the final published version. It's also available from Oxford Journals at http://gji.oxfordjournals.org/content/199/1/480.full

Linking Hydrogen (δ2H) Isotopes in Feathers and Precipitation: Sources of Variance and Consequences for Assignment to Isoscapes

Background: Tracking small migrant organisms worldwide has been hampered by technological and recovery limitations and sampling bias inherent in exogenous markers. Naturally occurring stable isotopes of H (d 2 H) in feathers provide an alternative intrinsic marker of animal origin due to the predictable spatial linkage to underlying hydrologically driven flow of H isotopes into foodwebs. This approach can assess the likelihood that a migrant animal originated from a given location(s) within a continent but requires a robust algorithm linking H isotopes in tissues of interest to an appropriate hydrological isotopic spatio-temporal pattern, such as weighted-annual rainfall. However, a number of factors contribute to or alter expected isotopic patterns in animals. We present results of an extensive investigation into taxonomic and environmental factors influencing feather d 2 H patterns across North America. Principal Findings: Stable isotope data were measured from 544 feathers from 40 species and 140 known locations. For d 2 H, the most parsimonious model explaining 83 % of the isotopic variance was found with amount-weighted growingseason precipitation d 2 H, foraging substrate and migratory strategy. Conclusions/Significance: This extensive H isotopic analysis of known-origin feathers of songbirds in North America and elsewhere reconfirmed the strong coupling between tissue d 2 H and global hydrologic d 2 H patterns, and accounting for variance associated with foraging substrate and migratory strategy, can be used in conservation and research for th

Microscopic characterization of functionalized paper as a platform for 3D cell cultures

To achieve an understanding and complete description of the functional properties of three dimensional (3D) cell culture systems, a large set of parameters is required, which clearly contrasts this cell cultivation approach from traditional two dimensional (2D), planar cultivation techniques . As an alternative to describe the characteristics of a 3D cell culture system by its physicochemical properties (e. g. stiffness, porosit y, level of crosslinking), the behavior of the cultivated cells can be used as a read-out parameter to characterize the 3D cultivation system . In this work, the cellular parameters membrane dynamics, actin fiber morphology and migration were used to investigate the differences between classic, planar and a collagen based three dimensional cell culture system Membrane dynamics – assessed by FRAP measurements of CAAX -mCherry – as well as actin formation – visualized by in vitro staining with LifeAct -tagRFP – showed distinct differences when investigated in planar or three dimensional systems. FRAP experiments with CAAX - mCherry showed, that even though the overall membrane composition does not appear to be different, mobility of the membrane is significantl y higher in three dimensional cell culture systems than in two dimensional . A view at the actin cytoskeleton revealed the already established difference: stress fibers and cortical actin are more pronounced in planar cell culture systems compared to cells c ultivated in three dimensional systems. Interestingly, cells originall y seeded in collagen hydrogels which migrated towards the glass surface show features in actin cytoskeleton formation resembling both culture conditions: both, actin stress fibers within the cell body as well as cortical actin are visible in those parts of the cells directly contacting the glass surface . The observed migration towards the glass surface gave rise to the investigation of this behavior. Migration in response to mechanical si gnals is termed durotaxis . Cells cultivated in collagen hydrogels or collagen hydrogels supported by cellulose sheets over a period of time were microscopicall y investigated to determine the distribution of cells . Cell distribution in unsupported collagen hydrogels was clearly in favor of hydrogel regions in close proximity to the glass surface. By applying supporting material in form of cellulose sheets, the cell culture was freely floating in the culture medium, resulting in an even distribution throughout the entire thickness of the cell culture system . As 3D cell culture systems make it more challenging to perform high quality imaging due to the inherent scattering and loss of intensity with increasing optical penetration, a post imaging processing tool set was evaluated and benchmarked in order to counteract these image corrupting effects and improve the image quality. This in turn also improves the compatibility of cellulose sheets with the commonly used tool set in life sciences: fluorescence microscopy. Special emphasis was put on the identification of a serviceable and performance -linked deconvolution setup . A GPU based CUDA Deconvolution plugin showed the best time performance but ultimately failed to produce the same quality level of image restoration as the three tested CPU based deconvolution applications. Among these three, the commercial HyugensPro software showed the best results in terms of increasing the contrast . The Iterative Deconvolution 3D plugin comes close to producing comparable results to the HuygensPro software, however, the time consumption for this application is up to 10 times larger. Finally, the plugin Deconvolution Lab showed reasonably satisfying results in terms of image restoration quality, while performing deconvolution slightly faster than HuygensPro. Finally, cellulose sheets are used for the cultivation of cells in 3D as an example of for paper as a versatile platform for the development of functional devices . Therefore a method is required that delivers spatially resolved, quantitative, sensitive, and, most importantly, also dynamic measurements . Optical microscopy has long been recognized as a method to characterize the heterogeneous and complex structure of paper. With fluorescence detection, the functionality has even been extended to provide chemical selectivity, e . g. to determine the distribution of secondary modifications like coatings and fillers throughout a sheet of paper. Here it is shown that quantitative widefield and confocal fluorescence microscopy are versatile methods to meet this set of demands. Confocal microscopy was used to achieve a detailed view of the interface between a hyd rophobic and rhodamine labeled polymer and a FITC labeled dextran solution. Furthermore, confocal microscopy revealed that the spatial propagation of the FITC labeled dextran solution occurs along the surface of the cellulose fibers, instead of the inter -fibers space. Widefield fluorescent microscopy was subsequently used for dynamic investigations of this spatial propagation

L'Auto-vélo : automobilisme, cyclisme, athlétisme, yachting, aérostation, escrime, hippisme / dir. Henri Desgranges

27 mars 19271927/03/27 (A28,N9598)

Cross-Linking Cellulosic Fibers with Photoreactive Polymers: Visualization with Confocal Raman and Fluorescence Microscopy

The properties of paper sheets can be tuned by adjusting the surface or bulk chemistry using functional polymers that are applied during (online) or after (offline) papermaking processes. In particular, polymers are widely used to enhance the mechanical strength of the wet state of paper sheets. However, the mechanical strength depends not only on the chemical nature of the polymeric additives but also on the distribution of the polymer on and in the lignocellulosic paper. Here, we analyze the photochemical attachment and distribution of hydrophilic polydimethylacrylamide-<i>co</i>-methacrylate-benzophenone P­(DMAA-<i>co</i>-MABP) copolymers with defined amounts of photoreactive benzophenone moieties in model paper sheets. Raman microscopy was used for the unambiguous identification of P­(DMAA-<i>co</i>-MABP) and cellulose specific bands and thus the copolymer distribution within the cellulose matrix. Two-dimensional Raman spectral maps at the intersections of overlapping cellulose fibers document that the macromolecules only partially surround the cellulose fibers, favor to attach to the fiber surface, and connect the cellulose fibers at crossings. Moreover, the copolymer appears to accumulate preferentially in holes, vacancies, and dips on the cellulose fiber surface. Correlative brightfield, Raman, and confocal laser scanning microscopy finally reveal a reticular three-dimensional distribution of the polymer and show that the polymer is predominately deposited in regions of high capillarity (i.e., in proximity to fine cellulose fibrils). These data provide deeper insights into the effects of paper functionalization with a copolymer and aid in understanding how these agents ultimately influence the local and overall properties of paper