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https://www.readbyqxmd.com/read/28792749/hide-probes-a-new-toolkit-for-visualizing-organelle-dynamics-longer-and-at-super-resolution
#1
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
#2
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
#3
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 new 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...
July 11, 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
#4
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
#5
Alanna Schepartz, Alexander D Thompson, Mitchell H Omar, Felix Rivera-Molina, Zhiqun Xi, Anthony J Koleske, Derek K Toomre
Super-resolution imaging of live cells over extended time periods with high temporal resolution requires high-density labeling and extraordinary fluorophore photostability. Here 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 sec temporal resolution, and visualized dynamic contact-mediated repulsion events in primary mouse hippocampal neurons over 9 min at 2 sec temporal resolution...
July 5, 2017: Angewandte Chemie
https://www.readbyqxmd.com/read/28671662/long-time-lapse-nanoscopy-with-spontaneously-blinking-membrane-probes
#6
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
#7
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/a-triplet-state-mechanism-of-primed-conversion-enables-rational-engineering-of-photoconvertible-fluorescent-proteins-for-dual-color-fluorescence-nanoscopy
#8
Periklis Pantazis, Manuel Mohr, Andrei Kobitski, Karin Nienhaus, Christopher Obara, Jennifer Lippincott-Schwartz, Gerd Ulrich Nienhaus, Lluc Rulan Sabatter
Photoconvertible fluorescent proteins (pcFPs) are powerful tools for super-resolution localization microscopy and protein tagging. Recently, they have been found to undergo efficient photoconversion 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...
June 29, 2017: Angewandte Chemie
https://www.readbyqxmd.com/read/28657091/depleted-upconversion-luminescence-in-nayf4-yb-3-tm-3-nanoparticles-via-simultaneous-two-wavelength-excitation
#9
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
#10
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
#11
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
#12
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
https://www.readbyqxmd.com/read/28466025/review-of-combined-isotopic-and-optical-nanoscopy
#13
REVIEW
Katharina N Richter, Silvio O Rizzoli, Sebastian Jähne, Angela Vogts, Jelena Lovric
Investigating the detailed substructure of the cell is beyond the ability of conventional optical microscopy. Electron microscopy, therefore, has been the only option for such studies for several decades. The recent implementation of several super-resolution optical microscopy techniques has rendered the investigation of cellular substructure easier and more efficient. Nevertheless, optical microscopy only provides an image of the present structure of the cell, without any information on its long-temporal changes...
April 2017: Neurophotonics
https://www.readbyqxmd.com/read/28463260/investigation-of-axial-and-transverse-focal-spot-sizes-of-fresnel-zone-plates
#14
Tao Liu, Qiang Liu, Shuming Yang, Zhuangde Jiang, Tong Wang, Guofeng Zhang
An axial spot size formula is given for a standard binary Fresnel zone plate (FZP) illuminated with a linearly polarized beam. The axial spot size, characterized by the full width at half-maximum (FWHM), can be accurately expressed by the Jiang-Wilson formula, viz., d<sub>z</sub>=0.9λ<sub>0</sub>/[η-(η<sup>2</sup>-NA<sup>2</sup>)<sup>1/2</sup>], when the number of transparent annuli is no less than 3. λ<sub>0</sub> is the illumination light wavelength, η is the refractive index, and NA is the equivalent numerical aperture of a FZP...
May 1, 2017: Applied Optics
https://www.readbyqxmd.com/read/28457706/a-versatile-tool-for-live-cell-imaging-and-super-resolution-nanoscopy-studies-of-hiv-1-env-distribution-and-mobility
#15
Volkan Sakin, Janina Hanne, Jessica Dunder, Maria Anders-Össwein, Vibor Laketa, Ivana Nikić, Hans-Georg Kräusslich, Edward A Lemke, Barbara Müller
The envelope glycoproteins (Env) of HIV-1 mediate cell entry through fusion of the viral envelope with a target cell membrane. Intramembrane mobility and clustering of Env trimers at the viral budding site are essential for its function. Previous live-cell and super-resolution microscopy studies were limited by lack of a functional fluorescent Env derivative, requiring antibody labeling for detection. Introduction of a bio-orthogonal amino acid by genetic code expansion, combined with click chemistry, offers novel possibilities for site-specific, minimally invasive labeling...
May 18, 2017: Cell Chemical Biology
https://www.readbyqxmd.com/read/28451345/protein-based-fluorescent-nanoparticles-for-super-resolution-sted-imaging-of-live-cells
#16
Li Shang, Peng Gao, Haixia Wang, Radian Popescu, Dagmar Gerthsen, Gerd Ulrich Nienhaus
Development of nanoparticles for super-resolution imaging (sriNPs) can greatly enrich the toolbox of robust optical probes for biological studies. Moreover, sriNPs enable us to monitor the behavior of engineered nanomaterials in complex biological environments with high spatial resolution, which is important for advancing our understanding of nano-bio interactions. Up to now, reports on sriNPs have been scarce. In this work, we report a facile strategy to prepare protein-based fluorescent NPs that can be utilized as probes in super-resolution microscopy...
March 1, 2017: Chemical Science
https://www.readbyqxmd.com/read/28445760/determining-the-spatial-relationship-of%C3%A2-membrane-bound-aquaporin-4-autoantibodies-by-sted-nanoscopy
#17
John N Soltys, Stephanie A Meyer, Hannah Schumann, Emily A Gibson, Diego Restrepo, Jeffrey L Bennett
Determining the spatial relationship of individual proteins in dense assemblies remains a challenge for superresolution nanoscopy. The organization of aquaporin-4 (AQP4) into large plasma membrane assemblies provides an opportunity to image membrane-bound AQP4 antibodies (AQP4-IgG) and evaluate changes in their spatial distribution due to alterations in AQP4 isoform expression and AQP4-IgG epitope specificity. Using stimulated emission depletion nanoscopy, we imaged secondary antibody labeling of monoclonal AQP4-IgGs with differing epitope specificity bound to isolated tetramers (M1-AQP4) and large orthogonal arrays of AQP4 (M23-AQP4)...
April 25, 2017: Biophysical Journal
https://www.readbyqxmd.com/read/28437075/fluorescent-photoswitchable-diarylethenes-for-biolabeling-and-single-molecule-localization-microscopies-with-optical-superresolution
#18
Benoît Roubinet, Michael Weber, Heydar Shojaei, Mark Bates, Mariano L Bossi, Vladimir N Belov, Masahiro Irie, Stefan W Hell
A modular assembly of water-soluble diarylethenes (DAEs), applicable as biomarkers for optical nanoscopy, is reported. Reversibly photoswitchable 1,2-bis(2-alkyl-6-phenyl-1-benzothiophene-1,1-dioxide-3-yl)perfluorocyclopentenes possessing a fluorescent "closed" form were decorated with one or two methoxy group(s) attached to the para-position(s) of phenyl ring(s) and two, four, or eight carboxylic acid groups. Antibody conjugates of these DAEs feature low aggregation, efficient photoswitching in aqueous buffers, specific staining of cellular structures, and photophysical properties (high emission efficiencies and low cycloreversion quantum yields) enabling their application in superresolution microscopy...
May 4, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28428254/a-novel-physiological-role-for-arf1-in-the-formation-of-bidirectional-tubules-from-the-golgi
#19
Francesca Bottanelli, Nicole Kilian, Andreas M Ernst, Felix Rivera-Molina, Lena K Schroeder, Emil B Kromann, Mark D Lessard, Roman S Erdmann, Alanna Schepartz, David Baddeley, Joerg Bewersdorf, Derek Toomre, James E Rothman
Capitalizing on CRISPR/Cas9 gene-editing techniques and super-resolution nanoscopy, we explore the role of the small GTPase ARF1 in mediating transport steps at the Golgi. Besides its well-established role in generating COPI vesicles, we find that ARF1 is also involved in the formation of long (∼3 µm), thin (∼110 nm diameter) tubular carriers. The anterograde and retrograde tubular carriers are both largely free of the classical Golgi coat proteins coatomer (COPI) and clathrin. Instead, they contain ARF1 along their entire length at a density estimated to be in the range of close packing...
June 15, 2017: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/28417977/multicolour-nanoscopy-of-fixed-and-living-cells-with-a-single-sted-beam-and-hyperspectral-detection
#20
Franziska R Winter, Maria Loidolt, Volker Westphal, Alexey N Butkevich, Carola Gregor, Steffen J Sahl, Stefan W Hell
The extension of fluorescence nanoscopy to larger numbers of molecular species concurrently visualized by distinct markers is of great importance for advanced biological applications. To date, up to four markers had been distinguished in STED experiments featuring comparatively elaborate imaging schemes and optical setups, and exploiting various properties of the fluorophores. Here we present a simple yet versatile STED design for multicolour imaging below the diffraction limit. A hyperspectral detection arrangement (hyperSTED) collects the fluorescence in four spectral channels, allowing the separation of four markers with only one excitation wavelength and a single STED beam...
April 18, 2017: Scientific Reports
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