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https://www.readbyqxmd.com/read/28924236/in-vivo-mouse-and-live-cell-sted-microscopy-of-neuronal-actin-plasticity-using-far-red-emitting-fluorescent-proteins
#1
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
https://www.readbyqxmd.com/read/28924139/srphi-ratiometric-ph-biosensors-for-super-resolution-microscopy
#2
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
https://www.readbyqxmd.com/read/28900102/photobleaching-in-sted-nanoscopy-and-its-dependence-on-the-photon-flux-applied-for-reversible-silencing-of-the-fluorophore
#3
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
https://www.readbyqxmd.com/read/28898567/multicolor-photo-crosslinkable-aiegens-toward-compact-nanodots-for-subcellular-imaging-and-sted-nanoscopy
#4
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
https://www.readbyqxmd.com/read/28887513/coherent-diffractive-imaging-of-single-helium-nanodroplets-with-a-high-harmonic-generation-source
#5
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
https://www.readbyqxmd.com/read/28875992/fluorescence-nanoscopy-in-cell-biology
#6
REVIEW
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...
September 6, 2017: Nature Reviews. Molecular Cell Biology
https://www.readbyqxmd.com/read/28845665/cell-permeant-large-stokes-shift-dyes-for-transfection-free-multicolor-nanoscopy
#7
Alexey N Butkevich, Gražvydas Lukinavičius, Elisa D'Este, Stefan W Hell
We designed cell-permeant red-emitting fluorescent dye labels with >140 nm Stokes shifts based on 9-iminoanthrone, 9-imino-10-silaxanthone, and 9-imino-10-germaxanthone fluorophores. The corresponding probes selectively targeting mitochondria, lysosomes, and F-actin demonstrate low toxicity and enable stimulated emission depletion (STED) nanoscopy in neurons, human fibroblasts, U2OS, and HeLa cells. In combination with known small Stokes shift dyes, our probes allow live-cell three-color STED nanoscopy of endogenous targets on popular setups with 775 nm STED wavelength...
August 30, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28842890/sequential-super-resolution-imaging-of-bacterial-regulatory-proteins-the-nucleoid-and-the-cell-membrane-in-single-fixed-e-coli-cells
#8
Christoph Spahn, Mathilda Glaesmann, Yunfeng Gao, Yong Hwee Foo, Marko Lampe, Linda J Kenney, Mike Heilemann
Despite their small size and the lack of compartmentalization, bacteria exhibit a striking degree of cellular organization, both in time and space. During the last decade, a group of new microscopy techniques emerged, termed super-resolution microscopy or nanoscopy, which facilitate visualizing the organization of proteins in bacteria at the nanoscale. Single-molecule localization microscopy (SMLM) is especially well suited to reveal a wide range of new information regarding protein organization, interaction, and dynamics in single bacterial cells...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28792749/hide-probes-a-new-toolkit-for-visualizing-organelle-dynamics-longer-and-at-super-resolution
#9
Alexander D Thompson, Joerg Bewersdorf, Derek Toomre, Alanna Schepartz
Living cells are complex and dynamic assemblies that carefully sequester and orchestrate multiple diverse processes that enable growth, division, regulation, movement, and communication. Membrane-bound organelles such as the endoplasmic reticulum, mitochondria, plasma membrane, and others are integral to these processes, and their functions demand dynamic reorganization in both space and time. Visualizing these dynamics in live cells over long time periods demands probes that label discrete organelles specifically, at high density, and withstand long-term irradiation...
August 9, 2017: Biochemistry
https://www.readbyqxmd.com/read/28717186/overstepping-the-upper-refractive-index-limit-to-form-ultra-narrow-photonic-nanojets
#10
Guoqiang Gu, Jun Song, Hongda Liang, Mengjie Zhao, Yue Chen, Junle Qu
In general, photonic nanojets (PNJs) occur only when the refractive index (Ri) difference between the microparticle and background media is less than 2. The minimum full width at half-maximum (FWHM) of the PNJ is ~130 nm (approximately one-third of the illumination wavelength λ = 400 nm) formed within the evanescent field region. This paper proposes and studies a method to overstep the Ri upper bound and generate ultra-narrow PNJs. Finite element method based numerical investigations and ray-optics theoretical analyses have realized ultra-narrow PNJs with FWHM as small as 114...
July 17, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28696661/multicolor-super-resolution-fluorescence-microscopy-with-blue-and-carmine-small-photoblinking-polymer-dots
#11
Xuanze Chen, Zhihe Liu, Rongqin Li, Chunyan Shan, Zhiping Zeng, Boxin Xue, Weihong Yuan, Chi Mo, Peng Xi, Changfeng Wu, Yujie Sun
Advances in the development of small photoblinking semiconducting polymer dots (Pdots) have attracted great interest for use in super-resolution microscopy. However, multicolor super-resolution imaging using conventional small photoblinking Pdots remains a challenge due to their limited color choice, broad emission spectrum, and heavy spectrum crosstalk. Here, we introduce two types of small photoblinking Pdots with different colors and relatively narrow emission spectra: blue PFO Pdots and carmine PFTBT5 Pdots for blinking-based statistical nanoscopy...
August 22, 2017: ACS Nano
https://www.readbyqxmd.com/read/28686583/interfacing-3d-magnetic-twisting-cytometry-with-confocal-fluorescence-microscopy-to-image-force-responses-in-living-cells
#12
Yuejin Zhang, Fuxiang Wei, Yeh-Chuin Poh, Qiong Jia, Junjian Chen, Junwei Chen, Junyu Luo, Wenting Yao, Wenwen Zhou, Wei Huang, Fang Yang, Yao Zhang, Ning Wang
Cells and tissues can undergo a variety of biological and structural changes in response to mechanical forces. Only a few existing techniques are available for quantification of structural changes at high resolution in response to forces applied along different directions. 3D-magnetic twisting cytometry (3D-MTC) is a technique for applying local mechanical stresses to living cells. Here we describe a protocol for interfacing 3D-MTC with confocal fluorescence microscopy. In 3D-MTC, ferromagnetic beads are bound to the cell surface via surface receptors, followed by their magnetization in any desired direction...
July 2017: Nature Protocols
https://www.readbyqxmd.com/read/28679029/long-term-live-cell-sted-nanoscopy-of-primary-and-cultured-cells-with-the-plasma-membrane-hide-probe-dii-sir
#13
Alexander D Thompson, Mitchell H Omar, Felix Rivera-Molina, Zhiqun Xi, Anthony J Koleske, Derek K Toomre, Alanna Schepartz
Super-resolution imaging of live cells over extended time periods with high temporal resolution requires high-density labeling and extraordinary fluorophore photostability. Herein, we achieve this goal by combining the attributes of the high-density plasma membrane probe DiI-TCO and the photostable STED dye SiR-Tz. These components undergo rapid tetrazine ligation within the plasma membrane to generate the HIDE probe DiI-SiR. Using DiI-SiR, we visualized filopodia dynamics in HeLa cells over 25 min at 0.5 s temporal resolution, and visualized dynamic contact-mediated repulsion events in primary mouse hippocampal neurons over 9 min at 2 s temporal resolution...
August 21, 2017: Angewandte Chemie
https://www.readbyqxmd.com/read/28671662/long-time-lapse-nanoscopy-with-spontaneously-blinking-membrane-probes
#14
Hideo Takakura, Yongdeng Zhang, Roman S Erdmann, Alexander D Thompson, Yu Lin, Brian McNellis, Felix Rivera-Molina, Shin-Nosuke Uno, Mako Kamiya, Yasuteru Urano, James E Rothman, Joerg Bewersdorf, Alanna Schepartz, Derek Toomre
Imaging cellular structures and organelles in living cells by long time-lapse super-resolution microscopy is challenging, as it requires dense labeling, bright and highly photostable dyes, and non-toxic conditions. We introduce a set of high-density, environment-sensitive (HIDE) membrane probes, based on the membrane-permeable silicon-rhodamine dye HMSiR, that assemble in situ and enable long time-lapse, live-cell nanoscopy of discrete cellular structures and organelles with high spatiotemporal resolution. HIDE-enabled nanoscopy movies span tens of minutes, whereas movies obtained with labeled proteins span tens of seconds...
August 2017: Nature Biotechnology
https://www.readbyqxmd.com/read/28663931/development-of-bimolecular-fluorescence-complementation-using-rsegfp2-for-detection-and-super-resolution-imaging-of-protein-protein-interactions-in-live-cells
#15
Sheng Wang, Miao Ding, Xuanze Chen, Lei Chang, Yujie Sun
Direct visualization of protein-protein interactions (PPIs) at high spatial and temporal resolution in live cells is crucial for understanding the intricate and dynamic behaviors of signaling protein complexes. Recently, bimolecular fluorescence complementation (BiFC) assays have been combined with super-resolution imaging techniques including PALM and SOFI to visualize PPIs at the nanometer spatial resolution. RESOLFT nanoscopy has been proven as a powerful live-cell super-resolution imaging technique. With regard to the detection and visualization of PPIs in live cells with high temporal and spatial resolution, here we developed a BiFC assay using split rsEGFP2, a highly photostable and reversibly photoswitchable fluorescent protein previously developed for RESOLFT nanoscopy...
June 1, 2017: Biomedical Optics Express
https://www.readbyqxmd.com/read/28661566/rational-engineering-of-photoconvertible-fluorescent-proteins-for-dual-color-fluorescence-nanoscopy-enabled-by-a-triplet-state-mechanism-of-primed-conversion
#16
Manuel Alexander Mohr, Andrei Yu Kobitski, Lluc Rullan Sabater, Karin Nienhaus, Christopher John Obara, Jennifer Lippincott-Schwartz, Gerd Ulrich Nienhaus, Periklis Pantazis
Green-to-red photoconvertible fluorescent proteins (pcFPs) are powerful tools for super-resolution localization microscopy and protein tagging. Recently, they have been found to undergo efficient photoconversion not only by the traditional 400-nm illumination but also by an alternative method termed primed conversion, employing dual wavelength illumination with blue and far-red/near-infrared light. Primed conversion has been reported only for Dendra2 and its mechanism has remained elusive. Here, we uncover the molecular mechanism of primed conversion by reporting the intermediate "primed" state to be a triplet dark state formed by intersystem crossing...
September 11, 2017: Angewandte Chemie
https://www.readbyqxmd.com/read/28657091/depleted-upconversion-luminescence-in-nayf4-yb-3-tm-3-nanoparticles-via-simultaneous-two-wavelength-excitation
#17
Hongxin Zhang, Tianqing Jia, Long Chen, Yuchan Zhang, Shian Zhang, Donghai Feng, Zhenrong Sun, Jianrong Qiu
We report optical depletion of upconversion luminescence (UCL) in NaYF4:Yb(3+),Tm(3+) nanoparticles excited simultaneously by 980 nm and 1550 nm lasers. The UCL intensity is greatly depleted, while the downshifted emission at 1210 nm is obviously enhanced. The disturbances of 1550 nm photons on energy transfer between Yb(3+) and Tm(3+), cross relaxation (CR), the thermal effect and the stimulated emission depletion (STED) process are qualitatively evaluated. Our investigations verify that the unexpected depletion phenomena are governed by the STED process...
July 21, 2017: Physical Chemistry Chemical Physics: PCCP
https://www.readbyqxmd.com/read/28536263/axodendritic-sorting-and-pathological-missorting-of-tau-are-isoform-specific-and-determined-by-axon-initial-segment-architecture
#18
Hans Zempel, Frank J A Dennissen, Yatender Kumar, Julia Luedtke, Jacek Biernat, Eva-Maria Mandelkow, Eckhard Mandelkow
Subcellular mislocalization of the microtubule-associated protein Tau is a hallmark of Alzheimer disease (AD) and other tauopathies. Six Tau isoforms, differentiated by the presence or absence of a second repeat or of N-terminal inserts, exist in the human CNS, but their physiological and pathological differences have long remained elusive. Here, we investigated the properties and distributions of human and rodent Tau isoforms in primary forebrain rodent neurons. We found that the Tau diffusion barrier (TDB), located within the axon initial segment (AIS), controls retrograde (axon-to-soma) and anterograde (soma-to-axon) traffic of Tau...
July 21, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28507079/distance-constraints-on-activation-of-trpv4-channels-by-akap150-bound-pkc%C3%AE-in-arterial-myocytes
#19
Sendoa Tajada, Claudia M Moreno, Samantha O'Dwyer, Sean Woods, Daisuke Sato, Manuel F Navedo, L Fernando Santana
TRPV4 (transient receptor potential vanilloid 4) channels are Ca(2+)-permeable channels that play a key role in regulating vascular tone. In arterial myocytes, opening of TRPV4 channels creates local increases in Ca(2+) influx, detectable optically as "TRPV4 sparklets." TRPV4 sparklet activity can be enhanced by the action of the vasoconstrictor angiotensin II (AngII). This modulation depends on the activation of subcellular signaling domains that comprise protein kinase C α (PKCα) bound to the anchoring protein AKAP150...
June 5, 2017: Journal of General Physiology
https://www.readbyqxmd.com/read/28495024/live-cell-nanoscopy-in-antiadhesion-therapy
#20
Joan A Geoghegan, Timothy J Foster, Pietro Speziale, Yves F Dufrêne
Live-cell nanoscopy has contributed significantly to assessing the inhibition of adhesion of uropathogenic Escherichia coli and Staphylococcus aureus by glycoconjugates and monoclonal antibodies, respectively, and of S. aureus surface attachment and cell-cell association by a synthetic peptide. This new technology shows promise for the development of antiadhesion therapies against bacterial pathogens.
May 8, 2017: Trends in Microbiology
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