Search Results
Statistical Analysis of Scanning Fluorescence Correlation Spectroscopy Data Differentiates Free from Hindered Diffusion
2018-7-20, Schneider, Falk, Waithe, Dominic, Lagerholm, B. Christoffer, Shrestha, Dilip, Sezgin, Erdinc, Eggeling, Christian, Fritzsche, Marco
Cells rely on versatile diffusion dynamics in their plasma membrane. Quantification of this often heterogeneous diffusion is essential to the understanding of cell regulation and function. Yet such measurements remain a major challenge in cell biology, usually due to low sampling throughput, a necessity for dedicated equipment, sophisticated fluorescent label strategies, and limited sensitivity. Here, we introduce a robust, broadly applicable statistical analysis pipeline for large scanning fluorescence correlation spectroscopy data sets, which uncovers the nanoscale heterogeneity of the plasma membrane in living cells by differentiating free from hindered diffusion modes of fluorescent lipid and protein analogues.
Spironaphthoxazine switchable dyes for biological imaging
2018, Xiong, Yaoyao, Vargas Jentzsch, Andreas, Osterrieth, Johannes W. M., Sezgin, Erdinc, Sazanovich, Igor V., Reglinski, Katharina, Galiani, Silvia, Parker, Anthony W., Eggeling, Christian, Anderson, Harry L.
Recent developments in super-resolution microscopy have significantly expanded the requirements for switchable dyes, leading to demand for specially designed molecular switches. We report the synthesis and characterization of a spironaphthoxazine photochromic switch (a derivative of palatinate purple) displaying high photoconversion (85-95%) under readily accessible 405 nm light, broad absorption in the visible, and excellent fatigue resistance. The indole substituent on this spironaphthoxazine is twisted out of conjugation with the naphthalene unit, yet it is crucial for activation with visible light. The open colored merocyanine form of the spironaphthoxazine reverts to the closed form with a lifetime of 4.7 s in dichloromethane at 20 °C; this thermal reversion is even faster in more polar solvents. The photochemical quantum yields for ring-opening and ring-closing are approximately 8% and 1%, respectively, in dichloromethane. The ring-opening and ring-closing reactions have been characterized by time-resolved infrared and transient absorption spectroscopies. Ring opening occurs rapidly (τ = 2.1 ns) and efficiently (∼90%) from the singlet excited state to form an intermediate (assigned as a cisoid merocyanine), which returns to the closed ground state (τ = 4.5 ns) in competition with relaxation to the transoid open form (τ = 40 ns). Photochemical ring closing is a faster and simpler process: the excited state proceeds to the closed spirooxazine with a time constant of 0.28 ns. This photochromic switch can be used in conjunction with commercial fluorescent dyes to create a small-molecule switchable fluorescent dyad that shows high contrast and good fatigue resistance in living cells. These properties make the dyads suitable for application in RESOLFT microscopy.
Nanoscale Spatiotemporal Diffusion Modes Measured by Simultaneous Confocal and Stimulated Emission Depletion Nanoscopy Imaging
2018-6-12, Schneider, Falk, Waithe, Dominic, Galiani, Silvia, Bernardino de la Serna, Jorge, Sezgin, Erdinc, Eggeling, Christian
The diffusion dynamics in the cellular plasma membrane provide crucial insights into molecular interactions, organization, and bioactivity. Beam-scanning fluorescence correlation spectroscopy combined with super-resolution stimulated emission depletion nanoscopy (scanning STED–FCS) measures such dynamics with high spatial and temporal resolution. It reveals nanoscale diffusion characteristics by measuring the molecular diffusion in conventional confocal mode and super-resolved STED mode sequentially for each pixel along the scanned line. However, to directly link the spatial and the temporal information, a method that simultaneously measures the diffusion in confocal and STED modes is needed. Here, to overcome this problem, we establish an advanced STED–FCS measurement method, line interleaved excitation scanning STED–FCS (LIESS–FCS), that discloses the molecular diffusion modes at different spatial positions with a single measurement. It relies on fast beam-scanning along a line with alternating laser illumination that yields, for each pixel, the apparent diffusion coefficients for two different observation spot sizes (conventional confocal and super-resolved STED). We demonstrate the potential of the LIESS–FCS approach with simulations and experiments on lipid diffusion in model and live cell plasma membranes. We also apply LIESS–FCS to investigate the spatiotemporal organization of glycosylphosphatidylinositol-anchored proteins in the plasma membrane of live cells, which, interestingly, show multiple diffusion modes at different spatial positions.
Silicon Powder-Based Wafers for Low-Cost Photovoltaics: Laser Treatments and Nanowire Etching
2018, Jia, G., Plentz, J., Gawlik, A., Azar, A.S., Stokkan, G., Syvertsen, M., Carvalho, P.A., Dellith, J., Dellith, A., Andrä, G., Ulyashin, A.
In this study, laser-treated polycrystalline Si (pc-Si) wafers, fabricated by wire sawing of hot-pressed ingots sintered from Si powder, have been investigated. As-cut wafers and those with high-quality thin Si layers deposited on top of them by e-beam have been subjected to laser irradiation to clarify typical trends of structural modifications caused by laser treatments. Moreover, possibility to use laser-treated Si powder-based substrates for fabrication of advanced Si structures has been analysed. It is established that (i) Si powder-based wafers with thicknesses 180 μm can be fully (from the front to back side) or partly (subsurface region) remelted by a diode laser and grain sizes in laser-treated regions can be increased; (ii) a high-quality top layer can be fabricated by crystallization of an additional a-Si layer deposited by e-beam evaporation on top of the pc-Si; and (iii) silicon nanowires can be formed by metal-assisted wet chemical etching (MAWCE) of polished Si powder-based wafers and as-cut wafers irradiated with medium laser power, while a surface texturing on the as-cut pc-Si wafers occur, and no nanowires can form in the region subject to a liquid phase crystallization (LPC) caused by high-power laser treatments.
Dumbbell gold nanoparticle dimer antennas with advanced optical properties
2018, Herrmann, Janning F., Höppener, Christiane
Plasmonic nanoantennas have found broad applications in the fields of photovoltaics, electroluminescence, non-linear optics and for plasmon enhanced spectroscopy and microscopy. Of particular interest are fundamental limitations beyond the dipolar approximation limit. We introduce asymmetric gold nanoparticle antennas (AuNPs) with improved optical near-field properties based on the formation of sub-nanometer size gaps, which are suitable for studying matter with high-resolution and single molecule sensitivity. These dumbbell antennas are characterized in regard to their far-field and near-field properties and are compared to similar dimer and trimer antennas with larger gap sizes. The tailoring of the gap size down to sub-nanometer length scales is based on the integration of rigid macrocyclic cucurbituril molecules. Stable dimer antennas are formed with an improved ratio of the electromagnetic field enhancement and confinement. This ratio, taken as a measure of the performance of an antenna, can even exceed that exhibited by trimer AuNP antennas composed of comparable building blocks with larger gap sizes. Fluctuations in the far-field and near-field properties are observed, which are likely caused by distinct deviations of the gap geometry arising from the faceted structure of the applied colloidal AuNPs.
Light-responsive paper strips as CO-releasing material with a colourimetric response
2017, Reddy G., Upendar, Liu, Jingjing, Hoffmann, Patrick, Steinmetzer, Johannes, Görls, Helmar, Kupfer, Stephan, Askes, Sven H. C., Neugebauer, Ute, Gräfe, Stefanie, Schiller, Alexander
Carbon monoxide (CO) is known for its multifaceted role in human physiology, and molecules that release CO in a controlled way have been proposed as therapeutic drugs. In this work, a light-responsive CO-releasing molecule (CORM-Dabsyl) showed a strong colourimetric response upon photochemical CO-release, owing to the tight conjugation of a Mn(i) tricarbonyl centre to a dabsyl chromophoric ligand (L). Whereas the complex was very stable in the dark in nitrogen-purged aqueous media, CO-release was effectively triggered using 405 nm irradiation. CORM-Dabsyl, L and the inactive product iCORM-Dabsyl have been investigated by DFT and TD-DFT calculations. Only mild toxicity of CORM-Dabsyl was observed against LX-2 and HepaRG® human cell lines (IC50 ∼ 30 μM). Finally, to develop a CO storage and release material that is readily applicable to therapeutic situations, CORM-Dabsyl was loaded on low-cost and easily disposable paper strips, from which the light triggered CO-release was conveniently visible with the naked eye.
Mitochondria Targeted Protein-Ruthenium Photosensitizer for Efficient Photodynamic Applications
2017, Chakrabortty, Sabyasachi, Agrawalla, Bikram Keshari, Stumper, Anne, Vegi, Naidu M., Fischer, Stephan, Reichardt, Christian, Kögler, Michael, Dietzek, Benjamin, Feuring-Buske, Michaela, Buske, Christian, Rau, Sven, Weil, Tanja
Organelle-targeted photosensitization represents a promising approach in photodynamic therapy where the design of the active photosensitizer (PS) is very crucial. In this work, we developed a macromolecular PS with multiple copies of mitochondria-targeting groups and ruthenium complexes that displays highest phototoxicity toward several cancerous cell lines. In particular, enhanced anticancer activity was demonstrated in acute myeloid leukemia cell lines, where significant impairment of proliferation and clonogenicity occurs. Finally, attractive two-photon absorbing properties further underlined the great significance of this PS for mitochondria targeted PDT applications in deep tissue cancer therapy.
Direct raman spectroscopic measurements of biological nitrogen fixation under natural conditions: An analytical approach for studying nitrogenase activity
2016, Jochum, Tobias, Fastnacht, Agnes, Trumbore, Susan E., Popp, Jürgen, Frosch, Torsten
Biological N2 fixation is a major input of bioavailable nitrogen, which represents the most frequent factor limiting the agricultural production throughout the world. Especially, the symbiotic association between legumes and Rhizobium bacteria can provide substantial amounts of nitrogen (N) and reduce the need for industrial fertilizers. Despite its importance in the global N cycle, rates of biological nitrogen fixation have proven difficult to quantify. In this work, we propose and demonstrate a simple analytical approach to measure biological N2 fixation rates directly without a proxy or isotopic labeling. We determined a mean N2 fixation rate of 78 ± 5 μmol N2 (g dry weight nodule)-1 h-1 of a Medicago sativa-Rhizobium consortium by continuously analyzing the amount of atmospheric N2 in static environmental chambers with Raman gas spectroscopy. By simultaneously analyzing the CO2 uptake and photosynthetic plant activity, we think that a minimum CO2 mixing ratio might be needed for natural N2 fixation and only used the time interval above this minimum CO2 mixing ratio for N2 fixation rate calculations. The proposed approach relies only on noninvasive measurements of the gas phase and, given its simplicity, indicates the potential to estimate biological nitrogen fixation of legume symbioses not only in laboratory experiments. The same methods can presumably also be used to detect N2 fluxes by denitrification from ecosystems to the atmosphere. (Figure Presented).
Nanoparticles Can Wrap Epithelial Cell Membranes and Relocate Them Across the Epithelial Cell Layer
2018-7-24, Urbančič, Iztok, Garvas, Maja, Kokot, Boštjan, Majaron, Hana, Umek, Polona, Cassidy, Hilary, Škarabot, Miha, Schneider, Falk, Galiani, Silvia, Arsov, Zoran, Koklic, Tilen, Matallanas, David, Čeh, Miran, Muševič, Igor, Eggeling, Christian, Štrancar, Janez
Although the link between the inhalation of nanoparticles and cardiovascular disease is well established, the causal pathway between nanoparticle exposure and increased activity of blood coagulation factors remains unexplained. To initiate coagulation tissue factor bearing epithelial cell membranes should be exposed to blood, on the other side of the less than a micrometre thin air-blood barrier. For the inhaled nanoparticles to promote coagulation, they need to bind lung epithelial-cell membrane parts and relocate them into the blood. To assess this hypothesis, we use advanced microscopy and spectroscopy techniques to show that the nanoparticles wrap themselves with epithelial-cell membranes, leading to the membrane’s disruption. The membrane-wrapped nanoparticles are then observed to freely diffuse across the damaged epithelial cell layer relocating epithelial cell membrane parts over the epithelial layer. Proteomic analysis of the protein content in the nanoparticles wraps/corona finally reveals the presence of the coagulation-initiating factors, supporting the proposed causal link between the inhalation of nanoparticles and cardiovascular disease.
An artificial photosynthetic system for photoaccumulation of two electrons on a fused dipyridophenazine (dppz)-pyridoquinolinone ligand
2018, Lefebvre, Jean-François, Schindler, Julian, Traber, Philipp, Zhang, Ying, Kupfer, Stephan, Gräfe, Stefanie, Baussanne, Isabelle, Demeunynck, Martine, Mouesca, Jean-Marie, Gambarelli, Serge, Artero, Vincent, Dietzek, Benjamin, Chavarot-Kerlidou, Murielle
Increasing the efficiency of molecular artificial photosynthetic systems is mandatory for the construction of functional devices for solar fuel production. Decoupling the light-induced charge separation steps from the catalytic process is a promising strategy, which can be achieved thanks to the introduction of suitable electron relay units performing charge accumulation. We report here on a novel ruthenium tris-diimine complex able to temporarily store two electrons on a fused dipyridophenazine-pyridoquinolinone π-extended ligand upon visible-light irradiation in the presence of a sacrificial electron donor. Full characterization of this compound and of its singly and doubly reduced derivatives thanks to resonance Raman, EPR and (TD)DFT studies allowed us to localize the two electron-storage sites and to relate charge photoaccumulation with proton-coupled electron transfer processes.