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Waja Wegner, Alexander C Mott, Seth G N Grant, Heinz Steffens, Katrin I Willig
The post-synaptic density (PSD) is an electron dense region consisting of ~1000 proteins, found at the postsynaptic membrane of excitatory synapses, which varies in size depending upon synaptic strength. PSD95 is an abundant scaffolding protein in the PSD and assembles a family of supercomplexes comprised of neurotransmitter receptors, ion channels, as well as signalling and structural proteins. We use superresolution STED (STimulated Emission Depletion) nanoscopy to determine the size and shape of PSD95 in the anaesthetised mouse visual cortex...
January 9, 2018: Scientific Reports
Michael Abadier, Akula Bala Pramod, Sara McArdle, Alex Marki, Zhichao Fan, Edgar Gutierrez, Alex Groisman, Klaus Ley
The adaptive immune response involves T cell differentiation and migration to sites of inflammation. T cell trafficking is initiated by rolling on inflamed endothelium. Tethers and slings, discovered in neutrophils, facilitate cell rolling at high shear stress. Here, we demonstrate that the ability to form tethers and slings during rolling is highly inducible in T helper 1 (Th1), Th17, and regulatory T (Treg) cells but less in Th2 cells. In vivo, endogenous Treg cells rolled stably in cremaster venules at physiological shear stress...
December 26, 2017: Cell Reports
Roderick P Tas, Anaël Chazeau, Bas M C Cloin, Maaike L A Lambers, Casper C Hoogenraad, Lukas C Kapitein
Microtubules are essential for polarized transport in neurons, but how their organization guides motor proteins to axons or dendrites is unclear. Because different motors recognize distinct microtubule properties, we used optical nanoscopy to examine the relationship between microtubule orientations, stability, and modifications. Nanometric tracking of motors to super-resolve microtubules and determine their polarity revealed that in dendrites, stable and acetylated microtubules are mostly oriented minus-end out, while dynamic and tyrosinated microtubules are oriented oppositely...
November 28, 2017: Neuron
Federico M Barabas, Luciano A Masullo, Martín D Bordenave, Sebastián A Giusti, Nicolás Unsain, Damián Refojo, Alfredo Cáceres, Fernando D Stefani
Fluorescence nanoscopy imaging permits the observation of periodic supramolecular protein structures in their natural environment, as well as the unveiling of previously unknown protein periodic structures. Deciphering the biological functions of such protein nanostructures requires systematic and quantitative analysis of large number of images under different experimental conditions and specific stimuli. Here we present a method and an open source software for the automated quantification of protein periodic structures in super-resolved images...
November 22, 2017: Scientific Reports
Xuzhu Zhang, Evangelos Sisamakis, Krzysztof Sozanski, Robert Holyst
Equilibrium and rate constants are key descriptors of complex-formation processes in a variety of chemical and biological reactions. However, these parameters are difficult to quantify, especially in the locally confined, heterogeneous, and dynamically changing living matter. Herein, we address this challenge by combining stimulated emission depletion (STED) nanoscopy with fluorescence correlation spectroscopy (FCS). STED reduces the length-scale of observation to tens of nanometres (2D)/attoliters (3D) and the time-scale-to microseconds, with direct, gradual control...
November 13, 2017: Journal of Physical Chemistry Letters
Jan Philip Kraack
Ultrafast, multi-dimensional infrared (IR) spectroscopy has been advanced in recent years to a versatile analytical tool with a broad range of applications to elucidate molecular structure on ultrafast timescales, and it can be used for samples in a many different environments. Following a short and general introduction on the benefits of 2D IR spectroscopy, the first part of this chapter contains a brief discussion on basic descriptions and conceptual considerations of 2D IR spectroscopy. Outstanding classical applications of 2D IR are used afterwards to highlight the strengths and basic applicability of the method...
October 25, 2017: Topics in Current Chemistry (Journal)
Sofia D'Abrantes, Sarah Gratton, Pamela Reynolds, Verena Kriechbaumer, Joseph McKenna, Stephen Barnard, Dave Clarke, Stanley W Botchway
Genomic deoxyribonucleic acid (DNA) is continuously being damaged by endogenous processes such as metabolism or by exogenous events such as radiation. The specific phosphorylation of histone H2AX on serine residue 139, described as γ-H2AX, is an excellent indicator or marker of DNA double-strand breaks (DSBs). The yield of γ-H2AX (foci) is shown to have some correlation with the dose of radiation or other DSB-causing agents. However, there is some discrepancy in the DNA DSB foci yield among imaging and other methods such as gel electrophoresis...
October 20, 2017: Radiation Research
Qiuqiang Zhan, Haichun Liu, Baoju Wang, Qiusheng Wu, Rui Pu, Chao Zhou, Bingru Huang, Xingyun Peng, Hans Ågren, Sailing He
Stimulated emission depletion microscopy provides a powerful sub-diffraction imaging modality for life science studies. Conventionally, stimulated emission depletion requires a relatively high light intensity to obtain an adequate depletion efficiency through only light-matter interaction. Here we show efficient emission depletion for a class of lanthanide-doped upconversion nanoparticles with the assistance of interionic cross relaxation, which significantly lowers the laser intensity requirements of optical depletion...
October 20, 2017: Nature Communications
Stefan Mastel, Mark B Lundeberg, Pablo Alonso-González, Yuanda Gao, Kenji Watanabe, Takashi Taniguchi, James Hone, Frank H L Koppens, Alexey Y Nikitin, Rainer Hillenbrand
We developed THz-resonant scanning probe tips, yielding strongly enhanced and nanoscale confined THz near fields at their tip apex. The tips with length in the order of the THz wavelength (λ = 96.5 μm) were fabricated by focused ion beam (FIB) machining and attached to standard atomic force microscopy (AFM) cantilevers. Measurements of the near-field intensity at the very tip apex (25 nm radius) as a function of tip length, via graphene-based (thermoelectric) near-field detection, indicate their first and second order geometrical antenna resonances for tip length of 33 and 78 μm, respectively...
October 30, 2017: Nano Letters
Dongyu Li, Wei Qin, Bin Xu, Jun Qian, Ben Zhong Tang
Stimulated emission depletion (STED) nanoscopy is a typical super-resolution imaging technique that has become a powerful tool for visualizing intracellular structures on the nanometer scale. Aggregation-induced emission (AIE) luminogens are ideal fluorescent agents for bioimaging. Herein, long-term super-resolution fluorescence imaging of cancer cells, based on STED nanoscopy assisted by AIE nanoparticles (NPs) is realized. 2,3-Bis(4-(phenyl(4-(1,2,2-triphenylvinyl)phenyl)amino)phenyl) fumaronitrile (TTF), a typical AIE luminogen, is doped into colloidal mesoporous silica to form fluorescent NPs...
October 4, 2017: Advanced Materials
Ben E Urban, Lei Xiao, Biqin Dong, Siyu Chen, Yevgenia Kozorovitskiy, Hao F Zhang
Visualizing fine neuronal structures deep inside strong light-scattering brain tissue remains a challenge in neuroscience. Recent nanoscopy techniques have reached the necessary resolution, but often suffer from limited imaging depth, long imaging time, or high light fluence requirements. Here, we present two-photon Super-resolution Patterned Excitation Reconstruction (2P-SuPER) microscopy for three-dimensional imaging of dendritic spine dynamics at a maximum demonstrated imaging depth of 130 μm in living brain tissue with approximately 100 nm spatial resolution...
October 4, 2017: Journal of Biophotonics
Ashish Bhattarai, Alan G Joly, Wayne P Hess, Patrick Z El-Khoury
Tip-enhanced Raman scattering (TERS) can be used to image plasmon-enhanced local electric fields on the nanoscale. This is illustrated through ambient TERS measurements recorded using silver atomic force microscope tips coated with 4-mercaptobenzonitrile molecules and used to image step edges on an Au(111) surface. The observed two-dimensional TERS images uniquely map electric fields localized at Au(111) step edges following 671 nm excitation. We establish that our measurements are not only sensitive to spatial variations in the enhanced electric fields but also to their vector components...
October 5, 2017: Nano Letters
Thomas E Schaus, Sungwook Woo, Feng Xuan, Xi Chen, Peng Yin
Analysis of the spatial arrangement of molecular features enables the engineering of synthetic nanostructures and the understanding of natural ones. The ability to acquire a comprehensive set of pairwise proximities between components would satisfy an increasing interest in investigating individual macromolecules and their interactions, but current biochemical techniques detect only a single proximity partner per probe. Here, we present a biochemical DNA nanoscopy method that records nanostructure features in situ and in detail for later readout...
September 25, 2017: Nature Communications
Alexey N Butkevich, Haisen Ta, Michael Ratz, Stefan Stoldt, Stefan Jakobs, Vladimir N Belov, Stefan W Hell
A 810 nm STED nanoscopy setup and an appropriate combination of two fluorescent dyes (Si-rhodamine 680SiR and carbopyronine 610CP) have been developed for near-IR live-cell super-resolution imaging. Vimentin endogenously tagged using the CRISPR/Cas9 approach with the SNAP tag, together with a noncovalent tubulin label, provided reliable and cell-to-cell reproducible dual-color confocal and STED imaging of the cytoskeleton in living cells.
October 2, 2017: ACS Chemical Biology
Waja Wegner, Peter Ilgen, Carola Gregor, Joris van Dort, Alexander C Mott, Heinz Steffens, Katrin I Willig
The study of proteins in dendritic processes within the living brain is mainly hampered by the diffraction limit of light. STED microscopy is so far the only far-field light microscopy technique to overcome the diffraction limit and resolve dendritic spine plasticity at superresolution (nanoscopy) in the living mouse. After having tested several far-red fluorescent proteins in cell culture we report here STED microscopy of the far-red fluorescent protein mNeptune2, which showed best results for our application to superresolve actin filaments at a resolution of ~80 nm, and to observe morphological changes of actin in the cortex of a living mouse...
September 18, 2017: Scientific Reports
Douglas S Richardson, Carola Gregor, Franziska R Winter, Nicolai T Urban, Steffen J Sahl, Katrin I Willig, Stefan W Hell
Fluorescence-based biosensors have become essential tools for modern biology, allowing real-time monitoring of biological processes within living cells. Intracellular fluorescent pH probes comprise one of the most widely used families of biosensors in microscopy. One key application of pH probes has been to monitor the acidification of vesicles during endocytosis, an essential function that aids in cargo sorting and degradation. Prior to the development of super-resolution fluorescence microscopy (nanoscopy), investigation of endosomal dynamics in live cells remained difficult as these structures lie at or below the ~250 nm diffraction limit of light microscopy...
September 18, 2017: Nature Communications
Joanna Oracz, Volker Westphal, Czesław Radzewicz, Steffen J Sahl, Stefan W Hell
In STED (stimulated emission depletion) nanoscopy, the resolution and signal are limited by the fluorophore de-excitation efficiency and photobleaching. Here, we investigated their dependence on the pulse duration and power of the applied STED light for the popular 750 nm wavelength. In experiments with red- and orange-emitting dyes, the pulse duration was varied from the sub-picosecond range up to continuous-wave conditions, with average powers up to 200 mW at 80 MHz repetition rate, i.e. peak powers up to 1 kW and pulse energies up to 2...
September 12, 2017: Scientific Reports
Xiaofeng Fang, Xuanze Chen, Rongqin Li, Zhihe Liu, Haobin Chen, Zezhou Sun, Bo Ju, Yifei Liu, Sean Xiao-An Zhang, Dan Ding, Yujie Sun, Changfeng Wu
Aggregation induced emission (AIE) has attracted considerable interest for the development of fluorescence probes. However, controlling the bioconjugation and cellular labeling of AIE dots is a challenging problem. Here, this study reports a general approach for preparing small and bioconjugated AIE dots for specific labeling of cellular targets. The strategy is based on the synthesis of oxetane-substituted AIEgens to generate compact and ultrastable AIE dots via photo-crosslinking. A small amount of polymer enriched with oxetane groups is cocondensed with most of the AIEgens to functionalize the nanodot surface for subsequent streptavidin bioconjugation...
September 12, 2017: Small
Daniela Rupp, Nils Monserud, Bruno Langbehn, Mario Sauppe, Julian Zimmermann, Yevheniy Ovcharenko, Thomas Möller, Fabio Frassetto, Luca Poletto, Andrea Trabattoni, Francesca Calegari, Mauro Nisoli, Katharina Sander, Christian Peltz, Marc J Vrakking, Thomas Fennel, Arnaud Rouzée
Coherent diffractive imaging of individual free nanoparticles has opened routes for the in situ analysis of their transient structural, optical, and electronic properties. So far, single-shot single-particle diffraction was assumed to be feasible only at extreme ultraviolet and X-ray free-electron lasers, restricting this research field to large-scale facilities. Here we demonstrate single-shot imaging of isolated helium nanodroplets using extreme ultraviolet pulses from a femtosecond-laser-driven high harmonic source...
September 8, 2017: Nature Communications
Steffen J Sahl, Stefan W Hell, Stefan Jakobs
Fluorescence nanoscopy uniquely combines minimally invasive optical access to the internal nanoscale structure and dynamics of cells and tissues with molecular detection specificity. While the basic physical principles of 'super-resolution' imaging were discovered in the 1990s, with initial experimental demonstrations following in 2000, the broad application of super-resolution imaging to address cell-biological questions has only more recently emerged. Nanoscopy approaches have begun to facilitate discoveries in cell biology and to add new knowledge...
November 2017: Nature Reviews. Molecular Cell Biology
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