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Peng Gao, G Ulrich Nienhaus
Low-resolution background in stimulated emission depletion (STED) nanoscopy can arise from incomplete depletion or re-excitation by the STED beam. We have recently introduced stimulated emission double depletion (STEDD), a technique to efficiently suppress this background. In STEDD, the conventional, doughnut-shaped STED pulse, which depletes excited fluorophores outside the center of the focal region, is followed by a second Gaussian STED pulse, which specifically depletes the central region. The background is removed by calculating a weighted difference of photon events collected before and after the second STED pulse...
February 15, 2017: Optics Letters
Fabian Göttfert, Tino Pleiner, Jörn Heine, Volker Westphal, Dirk Görlich, Steffen J Sahl, Stefan W Hell
Photobleaching remains a limiting factor in superresolution fluorescence microscopy. This is particularly true for stimulated emission depletion (STED) and reversible saturable/switchable optical fluorescence transitions (RESOLFT) microscopy, where adjacent fluorescent molecules are distinguished by sequentially turning them off (or on) using a pattern of light formed as a doughnut or a standing wave. In sample regions where the pattern intensity reaches or exceeds a certain threshold, the molecules are essentially off (or on), whereas in areas where the intensity is lower, that is, around the intensity minima, the molecules remain in the initial state...
February 13, 2017: Proceedings of the National Academy of Sciences of the United States of America
Jie Xiao, Taekjip Ha
No abstract text is available yet for this article.
February 10, 2017: Science
A Gozar, N E Litombe, Jennifer E Hoffman, I Božović
Helium ion beams (HIB) focused to subnanometer scales have emerged as powerful tools for high-resolution imaging as well as nanoscale lithography, ion milling, or deposition [ Ward et al. J. Vac. Sci. Technol. B 2006 , 24 , 2871 ; Hlawacek et al. J. Vac. Sci. Technol. B 2014 , 32 , 02081 ; Alkemade et al. Appl. Phys. A 2014 , 117 , 1727 ]. Quantifying irradiation effects is an essential step toward reliable device fabrication, but most of the depth profiling information is provided by computer simulations rather than the experiment [ Zeigler et al...
February 10, 2017: Nano Letters
Anna L Peters, Cornelis J F van Noorden
Cytochemistry is the discipline that is applied to visualize specific molecules in individual cells and has become an essential tool in life sciences. Immunocytochemistry was developed in the sixties of last century and is the most frequently used cytochemical application. However, metabolic mapping is the oldest cytochemical approach to localize activity of specific enzymes, but in the last decades of the previous century and the first decade of the present century it almost became obsolete. The popularity of this approach revived in the past few years...
2017: Methods in Molecular Biology
Judith Reindl, Stefanie Girst, Dietrich W M Walsh, Christoph Greubel, Benjamin Schwarz, Christian Siebenwirth, Guido A Drexler, Anna A Friedl, Günther Dollinger
The spatial distribution of DSB repair factors γH2AX, 53BP1 and Rad51 in ionizing radiation induced foci (IRIF) in HeLa cells using super resolution STED nanoscopy after low and high linear energy transfer (LET) irradiation was investigated. 53BP1 and γH2AX form IRIF with same mean size of (540 ± 40) nm after high LET irradiation while the size after low LET irradiation is significantly smaller. The IRIF of both repair factors show nanostructures with partial anti-correlation. These structures are related to domains formed within the chromatin territories marked by γH2AX while 53BP1 is mainly situated in the perichromatin region...
January 17, 2017: Scientific Reports
Alison R Dun, Gabriel J Lord, Rhodri S Wilson, Deirdre M Kavanagh, Katarzyna I Cialowicz, Shuzo Sugita, Seungmee Park, Lei Yang, Annya M Smyth, Andreas Papadopulos, Colin Rickman, Rory R Duncan
Eukaryotic plasma membrane organization theory has long been controversial, in part due to a dearth of suitably high-resolution techniques to probe molecular architecture in situ and integrate information from diverse data streams [1]. Notably, clustered patterning of membrane proteins is a commonly conserved feature across diverse protein families (reviewed in [2]), including the SNAREs [3], SM proteins [4, 5], ion channels [6, 7], and receptors (e.g., [8]). Much effort has gone into analyzing the behavior of secretory organelles [9-13], and understanding the relationship between the membrane and proximal organelles [4, 5, 12, 14] is an essential goal for cell biology as broad concepts or rules may be established...
February 6, 2017: Current Biology: CB
Yves F Dufrêne
Studying the structure, properties, and interactions of microbial cells is key to understanding the functions of the microbiome. Recent advances in nanotechnology have offered new tools to probe microbes at the single-molecule and single-cell levels. In this issue of ACS Nano, Kumar et al. present an atomic force microscopy method that is capable of imaging the nanoscale organization of bacterial proteins in native, curved membranes. This study represents an important step forward in the development of nanoscopy techniques for analyzing biological systems with large curvature and vertical dimensions, such as membrane vesicles and bacterial cells...
January 12, 2017: ACS Nano
Mario Raab, Carolin Vietz, Fernando Daniel Stefani, Guillermo Pedro Acuna, Philip Tinnefeld
Over the last decade, two fields have dominated the attention of sub-diffraction photonics research: plasmonics and fluorescence nanoscopy. Nanoscopy based on single-molecule localization offers a practical way to explore plasmonic interactions with nanometre resolution. However, this seemingly straightforward technique may retrieve false positional information. Here, we make use of the DNA origami technique to both control a nanometric separation between emitters and a gold nanoparticle, and as a platform for super-resolution imaging based on single-molecule localization...
January 11, 2017: Nature Communications
Joseph J Kim, Neal K Bennett, Mitchel S Devita, Sanjay Chahar, Satish Viswanath, Eunjee A Lee, Giyoung Jung, Paul P Shao, Erin P Childers, Shichong Liu, Anthony Kulesa, Benjamin A Garcia, Matthew L Becker, Nathaniel S Hwang, Anant Madabhushi, Michael P Verzi, Prabhas V Moghe
While distinct stem cell phenotypes follow global changes in chromatin marks, single-cell chromatin technologies are unable to resolve or predict stem cell fates. We propose the first such use of optical high content nanoscopy of histone epigenetic marks (epi-marks) in stem cells to classify emergent cell states. By combining nanoscopy with epi-mark textural image informatics, we developed a novel approach, termed EDICTS (Epi-mark Descriptor Imaging of Cell Transitional States), to discern chromatin organizational changes, demarcate lineage gradations across a range of stem cell types and robustly track lineage restriction kinetics...
January 4, 2017: Scientific Reports
Gabriela Matela, Peng Gao, Gernot Guigas, Antonia F Eckert, Karin Nienhaus, G Ulrich Nienhaus
Here we present mGarnet2, a monomeric, far-red fluorescent marker protein derived from mRuby, with absorption and emission bands peaking at 598 and 671 nm, respectively. The protein shows excellent performance as a live-cell fusion marker for STED nanoscopy with 640 nm excitation and 780 nm depletion wavelengths.
January 3, 2017: Chemical Communications: Chem Comm
Federico M Barabas, Luciano A Masullo, Fernando D Stefani
Until recently, PC control and synchronization of scientific instruments was only possible through closed-source expensive frameworks like National Instruments' LabVIEW. Nowadays, efficient cost-free alternatives are available in the context of a continuously growing community of open-source software developers. Here, we report on Tormenta, a modular open-source software for the control of camera-based optical microscopes. Tormenta is built on Python, works on multiple operating systems, and includes some key features for fluorescence nanoscopy based on single molecule localization...
December 2016: Review of Scientific Instruments
Robin Diekmann, Deanna L Wolfson, Christoph Spahn, Mike Heilemann, Mark Schüttpelz, Thomas Huser
Imaging non-adherent cells by super-resolution far-field fluorescence microscopy is currently not possible because of their rapid movement while in suspension. Holographic optical tweezers (HOTs) enable the ability to freely control the number and position of optical traps, thus facilitating the unrestricted manipulation of cells in a volume around the focal plane. Here we show that immobilizing non-adherent cells by optical tweezers is sufficient to achieve optical resolution well below the diffraction limit using localization microscopy...
December 13, 2016: Nature Communications
Neranga Abeyasinghe, Santosh Kumar, Kai Sun, John F Mansfield, Rongchao Jin, Theodore Goodson
New approaches in molecular nanoscopy are greatly desired for interrogation of biological, organic, and inorganic objects with sizes below the diffraction limit. Our current work investigates emergent monolayer-protected gold quantum dots (nanoclusters, NCs) composed of 25 Au atoms by utilizing two-photon-excited fluorescence (TPEF) near-field scanning optical microscopy (NSOM) at single NC concentrations. Here, we demonstrate an approach to synthesize and isolate single NCs on solid glass substrates. Subsequent investigation of the NCs using TPEF NSOM reveals that, even when they are separated by distances of several tens of nanometers, we can excite and interrogate single NCs individually...
December 21, 2016: Journal of the American Chemical Society
Marius Reinhart, M Cristina Cardoso
Scientific discoveries and technological advancements are inseparable but not always take place in a coherent chronological manner. In the next, we will provide a seemingly unconnected and serendipitous series of scientific facts that, in the whole, converged to unveil DNA and its duplication. We will not cover here the many and fundamental contributions from microbial genetics and in vitro biochemistry. Rather, in this journey, we will emphasize the interplay between microscopy development culminating on super resolution fluorescence microscopy (i...
December 9, 2016: Protoplasma
Xuanze Chen, Rongqin Li, Zhihe Liu, Kai Sun, Zezhou Sun, Danni Chen, Gaixia Xu, Peng Xi, Changfeng Wu, Yujie Sun
Two types of small photoblinking Pdots with high brightness, strong photostability, and favorable biocompatibility, are designed. Super-resolution optical fluctuation imaging is achieved using these Pdots. Imaging of subcellular structures demonstrates that these small photoblinking Pdots are outstanding probes for fast, long-term super-resolution fluorescence imaging.
February 2017: Advanced Materials
Emiliano Cortés, Paloma A Huidobro, Hugo G Sinclair, Stina Guldbrand, William J Peveler, Timothy Davies, Simona Parrinello, Frederik Görlitz, Chris Dunsby, Mark A A Neil, Yonatan Sivan, Ivan P Parkin, Paul M W French, Stefan A Maier
Plasmonic nanoparticles influence the absorption and emission processes of nearby emitters due to local enhancements of the illuminating radiation and the photonic density of states. Here, we use the plasmon resonance of metal nanoparticles in order to enhance the stimulated depletion of excited molecules for super-resolved nanoscopy. We demonstrate stimulated emission depletion (STED) nanoscopy with gold nanorods with a long axis of only 26 nm and a width of 8 nm. These particles provide an enhancement of up to 50% of the resolution compared to fluorescent-only probes without plasmonic components irradiated with the same depletion power...
November 22, 2016: ACS Nano
Jie Xiao, Yves F Dufrêne
Microbial cells have developed sophisticated multicomponent structures and machineries to govern basic cellular processes, such as chromosome segregation, gene expression, cell division, mechanosensing, cell adhesion and biofilm formation. Because of the small cell sizes, subcellular structures have long been difficult to visualize using diffraction-limited light microscopy. During the last three decades, optical and force nanoscopy techniques have been developed to probe intracellular and extracellular structures with unprecedented resolutions, enabling researchers to study their organization, dynamics and interactions in individual cells, at the single-molecule level, from the inside out, and all the way up to cell-cell interactions in microbial communities...
October 26, 2016: Nature Microbiology
Pablo Alonso-González, Alexey Y Nikitin, Yuanda Gao, Achim Woessner, Mark B Lundeberg, Alessandro Principi, Nicolò Forcellini, Wenjing Yan, Saül Vélez, Andreas J Huber, Kenji Watanabe, Takashi Taniguchi, Félix Casanova, Luis E Hueso, Marco Polini, James Hone, Frank H L Koppens, Rainer Hillenbrand
Terahertz (THz) fields are widely used for sensing, communication and quality control. In future applications, they could be efficiently confined, enhanced and manipulated well below the classical diffraction limit through the excitation of graphene plasmons (GPs). These possibilities emerge from the strongly reduced GP wavelength, λp, compared with the photon wavelength, λ0, which can be controlled by modulating the carrier density of graphene via electrical gating. Recently, GPs in a graphene/insulator/metal configuration have been predicted to exhibit a linear dispersion (thus called acoustic plasmons) and a further reduced wavelength, implying an improved field confinement, analogous to plasmons in two-dimensional electron gases (2DEGs) near conductive substrates...
October 24, 2016: Nature Nanotechnology
Karin Nienhaus, Gerd Ulrich Nienhaus
Photoactivatable fluorescent proteins (FPs) have become essential markers for nanoscopy on live specimens. In this issue of ACS Nano, Wang et al. present a reversibly photoswitching FP, GMars-Q, which they promote as an advanced marker for RESOLFT imaging because of its low residual intensity in the off state and low switching fatigue. Here, we explain the observed peculiar photobleaching behavior of GMars-Q by a mechanism that involves efficient shelving of proteins in dark states, resulting in low switching fatigue and low residual off intensity...
October 10, 2016: ACS Nano
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