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Here, we use cryo soft X-ray tomography (cryo-SXT), which delivers 3D ultrastructural volumes of intact cells without chemical fixation or staining, to gain insight about nanoparticle uptake for nanomedicine. We initially used dendritic polyglycerol sulfate (dPGS) with potential diagnostic and therapeutic applications in inflammation. Although dPGS-coated gold nanoparticle (dPGS-AuNP) uptake followed a conventional endocytic/degradative pathway in human lung epithelial cell lines (A549), with cryo-SXT, we detected ∼5% of dPGS-AuNPs in the cytoplasm, a level undetectable by confocal light microscopy. We also observed ∼5% of dPGS-AuNPs in a rarely identified subcellular site, namely, lipid droplets, which are important for cellular energy metabolism. Finally, we also found substantial changes in the quantity of cytoplasmic organelles upon dPGS-AuNP uptake over the 1–6 h incubation period; the number of small vesicles and mitochondria significantly increased, and the number of multivesicular bodies and the number and volume of lipid droplets significantly decreased. Although nearly all organelle numbers at 6 h were still significantly different from controls, most appeared to be returning to normal levels. To test for generality, we also examined cells after uptake of gold nanoparticles coated with a different agent, polyethylenimine (PEI), used for nucleic acid delivery. PEI nanoparticles did not enter lipid droplets, but they induced similar, albeit less pronounced, changes in the quantity of cytoplasmic organelles. We confirmed these changes in organelle quantities for both nanoparticle coatings by confocal fluorescence microscopy. We suggest this cytoplasmic remodeling could reflect a more common cellular response to coated gold nanoparticle uptake.
Unter Berücksichtigung praktischer und wissenschaftlich-technischer Erkenntnisse der letzten 15 Jahre - insbesondere auch auf der Basis verschiedener BAM-Forschungsergebnisse und Zulassungsverfahren - erfolgte eine Überarbeitung der Grundlagen, Bauprinzipien, Ausführungs- und Einbaukriterien sowie der Zulassungs- und Gütesicherungsmaßnahmen für insgesamt 4 Bauarten von Fugenfüllsystemen für hochbeanspruchte Verkehrsflächen. In diesem - für den Bundesfernstraßenbereich verbindlichen bautechnischen Regelwerk - finden insbesondere auch die Ergebnisse mehrerer wissenschaftlicher Vorhaben der BAM bezüglich labortechnischer Untersuchungen und Felduntersuchungen zum Funktionsverhalten und zur Dauerhaftigkeit erste baurechtliche Konsequenzen. Auf der Basis von Untersuchungen u.a. im Brückenbau wurden Regelungen für die Fugenausbildung in Asphalt und bei Befestigungskombinationen erarbeitet und umgesetzt. Damit konnten umfassende Regelungen für die maßgebenden Fahrbahnbeläge im Bundesfernstraßenbereich der Straßenbauverwaltung und ausführenden Industrieunternehmungen zur Verfügung gestellt werden. Die Neufassung dieser Regelungen dient darüber hinaus dem Ziel, den von der BAM vertretenen Wechsel hin zu einer performance-bezogenen Bewertungsmethodik, vorzubereiten.
Nach mehr als 15-jähriger Anwendung und mit der zwischenzeitlichen Einführung europäisch harmonisierter Normen für Fugenmaterialien war eine Anpassung Qualitäts- und Gütesicherungsanforderungen für Fugenfüllungen im Bundesfernstraßenbereich erforderlich. Hierbei ging es insbesondere darum, Maßnahmen und Verfahren zu entwickeln, um das nationale Qualitäts- und Gütesicherungsniveau weiter zu entwickeln und weiterhin sicher stellen zu können. Nach den Untersuchungserfahrungen im Labor und im Feld ging darum, neuartige Material- und Stoffkombinationen qualitätssicher dem Bauprozess zur Verfügung stellen zu können. Erforderliche Anforderungskriterien und zulässige Toleranzen der Lieferqualität wurden neu gefasst. Es wurde ein Verfahren entwickelt, europäische Bauprodukte qualitätsgesichert in den Bauprozess im Bundesfernstraßenbericht zu überführen.
Diese Hinweisbroschüre erläutert allgemeine Planungsvoraussetzungen und Dimensionierungsregelungen, Baugrundsätze und Zulassungsbedingungen zur Anwendung innovativer Belagsdehnfugensysteme (Fahrbahnübergangskonstruktionen) in hochbeanspruchten Verkehrsflächen des Bundesfernstraßenbereiches. Alle planungsrelevanten, baustoffrelevanten, versuchstechnische, qualitätssichernden und baupraktischen Aspekte werden zur Unterstützung der Straßenbauverwaltungen, der Lieferfirmen und Ausführungsbetriebe erläutert. Die Veröffentlichung stellt eine technische Analyse aus Erfahrungssammlungen und Auswertung praktischer Erkenntnisse dar.
Multifunctional composite nanoprobes consisting of iron oxide nanoparticles linked to silver and gold nanoparticles, Ag–Magnetite and Au–Magnetite, respectively, were introduced by endocytic uptake into cultured fibroblast cells. The cells containing the non-toxic nanoprobes were shown to be displaceable in an external magnetic field and can be manipulated in microfluidic channels. The distribution of the composite nanostructures that are contained in the endosomal system is discussed on the basis of surfaceenhanced Raman scattering (SERS) mapping, quantitative laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) micromapping, and cryo soft X-ray tomography (cryo soft-XRT). Cryo soft-XRT of intact, vitrified cells reveals that the composite nanoprobes form intra-endosomal aggregates. The nanoprobes provide SERS signals from the biomolecular composition of their surface in the endosomal environment. The SERS data indicate the high stability of the nanoprobes and of their plasmonic properties in the harsh environment of endosomes and lysosomes. The spectra point at the molecular composition at the surface of the Ag–Magnetite and Au–Magnetite nanostructures that is very similar to that of other Composite structures, but different from the composition of pure silver and gold SERS nanoprobes used for intracellular investigations. As shown by the LA-ICP-MS data, the uptake efficiency of the magnetite composites is approximately two to three times higher than that of the pure gold and silver nanoparticles.
The molecular dynamics of the triphenylene-based discotic liquid crystal HAT6 is investigated by broadband dielectric spectroscopy, advanced dynamical calorimetry and neutron scattering. Differential scanning calorimetry in combination with X-ray scattering reveals that HAT6 has a plastic crystalline phase at low temperatures, a hexagonally ordered liquid crystalline phase at higher temperatures and undergoes a clearing transition at even higher temperatures. The dielectric spectra show several relaxation processes: a localized gamma-relaxation a lower temperature and a so called alpha-2-relaxation at higher temperatures. The relaxation rates of the alpha-2-relaxation have a complex temperature dependence and bear similarities to a dynamic glass transition. The relaxation rates estimated by hyper DSC, Fast Scanning calorimetry and AC Chip calorimetry have a different temperature dependence than the dielectric alpha-2-relaxation and follows the VFT-behavior characteristic for glassy dynamics. Therefore, this process is called alpha-1-relaxation. Its relaxation rates show a similarity with that of polyethylene. For this reason, the alpha-1-relaxation is assigned to the dynamic glass transition of the alkyl chains in the intercolumnar space. Moreover, this process is not observed by dielectric spectroscopy which supports its assignment. The alpha-2-relaxation was assigned to small scale translatorial and/or small angle fluctuations of the cores.
The neutron scattering data reveal two relaxation processes. The process observed at shorter relaxation times is assigned to the methyl group rotation. The second relaxation process at longer time scales agree in the temperature dependence of its relaxation rates with that of the dielectric gamma-relaxation.
Inconel 718 is a precipitation strengthened, nickel-based super alloy of interest for the Additive Manufacturing (AM) of low volume, complex parts to reduce production time and cost compared to conventional subtractive processes. The AM process involves repeated rapid melting, solidification and reheating, which exposes the material to non-equilibrium conditions that affect elemental segregation and the subsequent formation of solidification phases, either beneficial or detrimental. These variations are difficult to characterize due to the small length scale within the micron sized melt pool. To understand how the non-equilibrium conditions affect the initial solidification phases and their critical temperatures, a multi-length scale, multi modal approach has been taken to evaluate various methods for identifying the initial phases formed in the as-built Inconel 718 produced by laser-powder bed fusion (L-PBF) additive manufacturing (AM). Using a range of characterization tools from the bulk differential thermal analysis (DTA) and x-ray diffraction (XRD) to spatially resolved images using a variety of electron microscopy tools, a better understanding is obtained of how these minor phases can be properly identified regarding the amount and size, morphology and distribution. Using the most promising characterization techniques for investigation of the as-built specimens, those techniques were used to evaluate the specimens after various heat treatments. During the sequence of heat treatments, the initial as-built dendritic structures recrystallized into well-defined grains whose size was dependent on the temperature. Although the resulting strength was similar in all heat treated specimens, the elongation increased as the grain size was refined due to differences in the precipitated phase distribution and morphology.
A surface-labeled lyophilized lymphocyte (sLL) preparation has been developed using human peripheral blood mononuclear cells prelabeled with a fluorescein isothiocyanate conjugated anti-CD4 monoclonal antibody. The sLL preparation is intended to be used as a reference material for CD4+ cell counting including the development of higher order reference measurement procedures and has been evaluated in the pilot study CCQM-P102. This study was conducted across 16 laboratories from eight countries to assess the ability of participants to quantify the CD4+ cell count of this reference material and to document cross-laboratory variability plus associated measurement uncertainties. Twelve different flow cytometer platforms were evaluated using a standard protocol that included calibration beads used to obtain quantitative measurements of CD4+ T cell counts. There was good overall cross-platform and counting method agreement with a grand mean of the laboratory calculated means of (301.7 ± 4.9) µL-1 CD4+ cells. Excluding outliers, greater than 90% of participant data agreed within ±15%. A major contribution to variation of sLL CD4+ cell counts was tube to tube variation of the calibration beads, amounting to an uncertainty of 3.6%. Variation due to preparative steps equated to an uncertainty of 2.6%. There was no reduction in variability when data files were centrally reanalyzed. Remaining variation was attributed to instrument specific differences. CD4+ cell counts obtained in CCQM-P102 are in excellent agreement and show the robustness of both the measurements and the data analysis and hence the suitability of sLL as a reference material for interlaboratory comparisons and external quality assessment.
This report focuses on the characterization of CD4 expression level in terms of equivalent number of reference fluorophores (ERF). Twelve different flow cytometer platforms across sixteen laboratories were utilized in this study. As a first step the participants were asked to calibrate the fluorescein isothiocyanate (FITC) channel of each flow cytometer using commercially available calibration standard consisting of five populations of microspheres. Each population had an assigned value of equivalent fluorescein fluorophores (EFF denotes a special case of the generic term ERF with FITC as the reference fluorophore). The EFF values were assigned at the National Institute of Standards and Technology (NIST). A surface-labelled lyophilized cell preparation was provided by the National Institute of Biological Standards and Control (NIBSC), using human peripheral blood mononuclear cells (PBMC) pre-labeled with a FITC conjugated anti-CD4 monoclonal antibody. Three PBMC sample vials, provided to each participant, were used for the CD4 expression analysis. The PBMC are purported to have a fixed number of surface CD4 receptors. On the basis of the microsphere calibration, the EFF value of the PBMC samples was measured to characterize the population average CD4 expression level of the PBMC preparations. Both the results of data analysis performed by each participant and the results of centralized analysis of all participants' raw data are reported. Centralized analysis gave a mean EFF value of 22,300 and an uncertainty of 750, corresponding to 3.3% (level of confidence 68%) of the mean EFF value. The next step will entail the measurement of the ERF values of the lyophilized PBMC stained with labels for other fluorescence channels. The ultimate goal is to show that lyophilized PBMC is a suitable biological reference cell material for multicolor flow cytometry and that it can be used to present multicolor flow cytometry measurements in terms of ABC (antibodies bound per cell) units.
A non-invasive method to monitor the humoral immune response in mice after immunization is described. From fecal pellets of an individual mouse, a sufficient amount of active immunoglobulins or their fragments can be extracted to perform a regular examination of the status of the immune response by immunoassay. Hapten-specific antibodies from the feces of mice from three immunization trials showed very similar characteristics to those obtained from serum at a given date. Therefore, it can be suspected that some serum IgG enters the intestinal lumen and ends up in the feces, where they appear to be considerably stable. Hapten-specific IgAs were not found in the feces. Being able to analyze antibody titers in feces could be an interesting animal welfare refinement to standard practice that does not entail repeated blood sampling.