Showing papers by "Stefan Seeger published in 2004"

Attoliter detection volumes by confocal total-internal-reflection fluorescence microscopy.

Abstract: We report a confocal total-internal-reflection fluorescence (TIRF) microscope that generates a detection volume for analyte molecules of less than 5 al 5×10-18 l at a water–glass interface. Compared with conventional confocal microscopy, this represents a reduction of almost 2 orders of magnitude, which is important in isolating individual molecules at high analyte concentrations, where many biologically relevant processes occur. Diffraction-limited supercritical focusing and fluorescence collection is accomplished by a parabolic mirror objective. The system delivers TIRF images with excellent spatial resolution and detects single molecules with a high signal-to-background ratio.

Deep-UV Laser-Based Fluorescence Lifetime Imaging Microscopy of Single Molecules

Abstract: Deep ultraviolet (deep-UV) laser-based fluorescence lifetime imaging microscopy of single dye molecules adsorbed on quartz glass surfaces is presented. The instrumental setup uses a mode-locked dio...

Highly (001)‐textured WS2−x films prepared by reactive radio frequency magnetron sputtering

Abstract: Highly (001)-oriented WS2−x films were grown onto oxidized silicon substrates by reactive magnetron sputtering from a metallic tungsten target in argon-hydrogen sulfide mixtures. The best films with respect to the van-der-Waals orientation, i.e. with the (001) planes parallel to the substrate surface, were grown by excitation of the plasma with radio frequency of 27.12 MHz. These films exhibit the largest grains and the lowest film strain. It is shown that this effect is not due to the lower deposition rate at this high excitation frequency. Instead it was found that the lower DC voltage at the sputtering target is advantageous for the film growth since the bombardment of the growing film by highly energetic particles is avoided by this type of plasma excitation. (© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

FRET studies of the interaction of dimeric cyanine dyes with DNA.

Abstract: Fluorescence Resonance Energy Transfer (FRET) is a powerful tool to determine distances between chromophores bound to macromolecules, since the efficiency of the energy transfer from an initially excited donor to an acceptor strongly depends on the distance between the two dye molecules The structure of the noncovalent complex of double-strand DNA (dsDNA) with thiazol orange dimers (TOTO) allows FRET analysis of two intercalated chromophores By intercalation of two different TOTO dyes we observe an energy transfer from TOTO-1 as donor and TOTO-3 as acceptor In this manner we are able to determine the mean distance between two proximate TOTO molecules bound to dsDNA Thus the maximum number of binding positions for this type of intercalation dyes in the dsDNA can be obtained Furthermore the dependency of the acceptor emission on the donor concentration is analysed The emission of TOTO-3 reaches a maximum when the acceptor-to-donor ratio is 1:10

Fast Detection of Single Nucleotide Polymorphisms (SNPs) by Primer Elongation with Monitoring of Supercritical-Angle Fluorescence

Abstract: We describe the rapid detection of single nucleotide polymorphisms (SNPs) by real-time observation of primer elongation. The enzymatic elongation of surface-bound primers is monitored by detecting the increase of surface-bound fluorescence caused by the incorporation of Cy5-labelled deoxycytidine 5′-triphosphate residues (Cy5-dCTPs) into the corresponding strand. In order to discriminate against the fluorescence from unbound Cy5-dCTPs, the detection volume was restricted to the surface by collecting supercritical-angle fluorescence. The efficiency of enzymatic double-stranded DNA synthesis is governed by the complementarity of the primer and template. An SNP in the sequence of the primer obstructs its elongation increasingly with decreased distance of the mismatch to the 3′ end of the primer. By real-time fluorescence detection during primer elongation, SNPs can be detected within a few minutes, which is significantly faster than in experiments where the fluorescence is measured after completion of the reaction. We demonstrate the efficiency of the method by detecting an SNP in the ErbB2 gene that is involved in causing a higher risk of breast cancer.

Real-time detection of polymerase activity using supercritical angle fluorescence.

Abstract: We investigated the incorporation efficiencies of different fluorescently labelled dNTPs with polymerases by complementary strand synthesis. For this reason single stranded DNA was immobilized on a coverslip and the increase of fluorescence due to the synthesis of the corresponding strand with tagged dNTPs was detected with a supercritical angle fluorescence biosensor in real-time. By comparison of the observed signal intensities it was possible to conclude that the system Cy5-dCTP—Klenow (exonuclease free) fragment gives the best incorporation yield of the investigated enzymes and dNTPs.

Langzeit-Scherfestigkeit von Geokunststoffen aus mehreren Komponenten 1

Abstract: 1. Einleitung Geokunststoffe sind oft aus mehreren Komponenten sandwich-artig aufgebaut. Man denke etwa an strukturierte Kunststoffdichtungsbahnen, bei denen in einem zweiten Fertigungsprozess nachtraglich Strukturpartikel auf die Oberflache aufgebracht werden [1], an Bentonitmatten, bei denen trockenes Bentonitpulver zwischen zwei Geotextilien eingepackt wird, die untereinander vernadelt oder vernaht werden [2], oder an Kunststoff-Dranmatten, die in der Regel aus einer Filterschicht, einem Drankern und einer Schutzschicht bestehen, die mit unterschiedlichen Methoden untereinander verbunden werden [3]. Werden solche Produkte in grosflachigen Abdichtungen auf mehr oder weniger steil geneigten und langen Boschungen eingebaut, so hangt die Standsicherheit des ganzen Abdichtungsbauwerks nicht nur von den Reibungskraften zwischen dem Geokunststoff und den benachbarten Komponenten der Abdichtung ab, sondern vor allem auch von der inneren Scherfestigkeit des Geokunststoffs. In welchem Ausmas dabei Scherkrafte mobilisiert werden, wird durch den Einbauzustand bestimmt. In manchen Fallen mag der Geokunststoff zwischen einem Erdstoff-Auflager und einer daruber liegenden Erdstoff-Deckschicht so eingezwangt sein, dass Aggregate in Auflager und Deckschicht sozusagen formschlussig ubereinander lagern und Scher- und Reibungskrafte gegenuber Kompression und Beigebeanspruchungen nur eine geringe Rolle spielen. In der Regel sind jedoch, gerade bei Dichtungsaufbauten aus mehreren Geokunststoffen, ebene und glatte Gleitflachen vorhanden und die Hangabtriebskrafte mussen in vollem Umfang durch innere Scherkrafte in den Abdichtungskomponenten und Reibungskrafte in den Grenzflachen zwischen den Komponenten ausgeglichen werden. Die innere Kurzzeit-Scherfestigkeit von Geokunststoffen wird in einem Scherversuch mit einem Kastenschergerat ermittelt [4]. Dazu wird bei sandwich-artigen Produkten deren Ober- und Unterseite auf dem jeweiligen Kasten fixiert und die Kasten mit konstanter Ge