Nanoscopy of bacterial cells immobilized by holographic optical tweezers

Diekmann R, Wolfson D, Spahn C, Heilemann M, Schüttpelz M, Huser T. Nanoscopy of bacterial cells immobilized by holographic optical tweezers. Nature Communications. 2016;7(1): 13711

Medien der Kooperation

Die digital-vernetzten Medien erfordern neue Analysen, Theorien und Geschichten. Sie verändern unseren Blick auf die Geschichte von Infrastrukturen, Öffentlichkeiten und Medienpraktiken. Was wären Ansätze für eine Medientheorie, die praktischen „skills“ des Mediengebrauchs,seiner soziotechnischen Materialität und den bürokratischen wie epistemischen Qualitäten der Medien gerecht wird? Die vorliegende Ausgabe 1/2015 der Navigationen widmet sich Medien als kooperativ bewerkstelligten Kooperationsbedingungen. Sie erbringen, so die These, konstitutive Vermittlungsleistungen zwischen der Organisation von Arbeit, Praktiken des Infrastrukturierens und der Genese von Öffentlichkeiten in wechselseitiger Interaktion

Characterization of an industry-grade CMOS camera well suited for single molecule localization microscopy – high performance super-resolution at low cost

Diekmann R, Till K, Müller M, Simonis M, Schüttpelz M, Huser T. Characterization of an industry-grade CMOS camera well suited for single molecule localization microscopy – high performance super-resolution at low cost. Scientific Reports. 2017;7(1): 14425.Many commercial as well as custom-built fluorescence microscopes use scientific-grade cameras that represent a substantial share of the instrument’s cost. This holds particularly true for super-resolution localization microscopy where high demands are placed especially on the detector with respect to sensitivity, noise, and also image acquisition speed. Here, we present and carefully characterize an industry-grade CMOS camera as a cost-efficient alternative to commonly used scientific cameras. Direct experimental comparison of these two detector types shows widely similar performance for imaging by single molecule localization microscopy (SMLM). Furthermore, high image acquisition speeds are demonstrated for the CMOS detector by ultra-fast SMLM imaging

Publisher Correction: Breaking the speed limit with multimode fast scanning of DNA by Endonuclease V.

Ahmadi A, Rosnes I, Blicher P, et al. Publisher Correction: Breaking the speed limit with multimode fast scanning of DNA by Endonuclease V. Nature communications. 2019;10(1): 1991.The original version of this Article was updated shortly after publication to add a link to the Peer Review file, which was inadvertently omitted. The Peer Review file is available to download as a Supplementary File from the HTML version of the Article

Fluoreszenzspektroskopische Methoden zur Charakterisierung von Protein-Wechselwirkungen

Schüttpelz M. Fluoreszenzspektroskopische Methoden zur Charakterisierung von Protein-Wechselwirkungen. Bielefeld (Germany): Bielefeld University; 2006.In der vorliegenden Arbeit werden neue Verfahren zum Nachweis und zur Quantifizierung von Protein-Wechselwirkungen vorgestellt und an biologischen Modellsystemen überprüft. Dazu wird ein sensitives, ausschließlich auf der nativen Fluoreszenz von Proteinen basierendes Nachweisverfahren entwickelt. Protein-Wechselwirkungen werden dabei nur durch eine Änderung der intrinsischen Fluoreszenzlebensdauer nachgewiesen. Dieses Verfahren wird anschließend eingesetzt, um die Bindung eines p53-Modellantikörpers an ein kurzes p53-Peptidepitop nachzuweisen und zu quantifizieren. p53-Autoantikörper sind unabhängige und hochspezifische Tumormarker mit einem hohen Potenzial zur Diagnose und Früherkennung von Krebserkrankungen, wodurch sie in der modernen Krebsforschung vermehrt an Bedeutung gewonnen haben. In einem zweiten Teil beschäftigt sich diese Arbeit mit dem Nachweis und der Aufklärung von Protein-RNA-Wechselwirkungen am Beispiel des Proteins AtGRP7 und seiner kodierenden RNA in der Pflanze Arabidopsis thaliana, die einen so genannten zirkadianen Slave-Oszillator darstellen. Mittels schneller Fluoreszenzkorrelationsspektroskopie (FCS) wird zum einen die spezifische Bindung des Proteins an seine RNA untersucht und auf wenige Nukleotide eingegrenzt, zum anderen werden auch die Konformationen bzw. Konformationsänderungen auf Grund der Protein-RNA-Wechselwirkung an Hand synthetischer Hairpin-Strukturen und nativer Oligonukleotide analysiert, um auf den molekularbiologischen Bindungsmechanismus zu schließen. Für diese Messungen werden unterschiedliche Farbstoff-markierte kurze DNA- und RNA-Oligonukleotide eingesetzt, die Teile der bekannten Bindesequenz des Proteins an seine RNA sind

Two-photon excited fluorescence detection at 420 nm for label-free detection of small aromatics and proteins in microchip electrophoresis

Schulze P, Schüttpelz M, Sauer M, Belder D. Two-photon excited fluorescence detection at 420 nm for label-free detection of small aromatics and proteins in microchip electrophoresis. LAB ON A CHIP. 2007;7(12):1841-1844.Two photon excited (TPE) fluorescence detection was applied to native fluorescence detection of aromatics in microchip electrophoresis (MCE). This technique was evaluated as an alternative to common one photon excitation in the deep UV spectral range. TPE enables fluorescence detection of unlabeled aromatic compounds, even in non-deep UV-transparent microfluidic chips. In this study, we demonstrate the proof of concept of native TPE fluorescence detection of small aromatics in commercial microfluidic glass chips. Label-free TPE fluorescence detection of native proteins and small aromatics in MCE was achieved within the micromolar concentration range, utilising 420 nm excitation light

UV fluorescence lifetime imaging microscopy: A label-free method for detection and quantification of protein interactions

Schüttpelz M, Müller C, Neuweiler H, Sauer M. UV fluorescence lifetime imaging microscopy: A label-free method for detection and quantification of protein interactions. ANALYTICAL CHEMISTRY. 2006;78(3):663-669.Due to the ability to detect multiple parameters simultaneously, protein microarrays have found widespread applications from basic biological research to diagnosis of diseases. Generally, readout of protein microarrays is performed by fluorescence detection using either dye-labeled detector antibodies or direct labeling of the target proteins. We developed a method for the label-free detection and quantification of proteins based on time-gated, wide-field, camera-based UV fluorescence lifetime imaging microscopy to gain lifetime information from each pixel of a sensitive CCD camera. The method relies on differences in the native fluorescence lifetime of proteins and takes advantage of binding-induced lifetime changes for the unequivocal detection and quantification of target proteins. Since fitting of the fluorescence decay for every pixel in an image using a classical exponential decay model is time-consuming and unstable at very low fluorescence intensities, we used a new, very robust and fast alternative method to generate UV fluorescence lifetime images by calculating the average lifetime of the decay for each pixel in the image stack using a model-free average decay time algorithm. To validate the method, we demonstrate the detection and quantification of p53 antibodies, a tumor marker in cancer diagnosis. Using tryptophan-containing capture peptides, we achieved a detection sensitivity for monoclonal antibodies down to the pico-molar concentration range. The obtained affinity constant, K-a,of (1.4 +/- 0.6) x 10(9) M-1, represents a typical value for antigen/antibody binding and is in agreement with values determined by traditional binding assays

RNA-Protein-Interaktion im Rampenlicht

Lummer M, Schüttpelz M, Sauer M, Staiger D. RNA-Protein-Interaktion im Rampenlicht. BIOforum. 2009;6:28-30

Sodium sulfite as a switching agent for single molecule based super-resolution optical microscopy

Engdahl AK, Grauberger O, Schüttpelz M, Huser T. Sodium sulfite as a switching agent for single molecule based super-resolution optical microscopy. bioRxiv. 2021.Photoinduced off-switching of organic fluorophores is routinely used in super-resolution microscopy to separate and localize single fluorescent molecules, but the method typically relies on the use of complex imaging buffers. The most common buffers use primary thiols to reversibly reduce excited fluorophores to a non-fluorescent dark state, but these thiols have a limited shelf life and additionally require high illumination intensities in order to efficiently switch the emission of fluorophores. Recently a high-index, thiol-containing imaging buffer emerged which used sodium sulfite as an oxygen scavenger, but the switching properties of sulfite was not reported on. Here, we show that sodium sulfite in common buffer solutions reacts with fluorescent dyes, such as Alexa Fluor 647 and Alexa Fluor 488 under low to medium intensity illumination to form a semi-stable dark state. The duration of this dark state can be tuned by adding glycerol to the buffer. This simplifies the realization of different super-resolution microscopy modalities such as direct Stochastic Reconstruction Microscopy (dSTORM) and Super-resolution Optical Fluctuation Microscopy (SOFI). We characterize sulfite as a switching agent and compare it to the two most common switching agents by imaging cytoskeleton structures such as microtubules and the actin cytoskeleton in human osteosarcoma cells