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Single molecule imaging

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https://www.readbyqxmd.com/read/27929472/three-dimensional-super-resolution-microscopy-of-f-actin-filaments-by-interferometric-photoactivated-localization-microscopy-ipalm
#1
Yilin Wang, Pakorn Kanchanawong
Fluorescence microscopy enables direct visualization of specific biomolecules within cells. However, for conventional fluorescence microscopy, the spatial resolution is restricted by diffraction to ~ 200 nm within the image plane and > 500 nm along the optical axis. As a result, fluorescence microscopy has long been severely limited in the observation of ultrastructural features within cells. The recent development of super resolution microscopy methods has overcome this limitation. In particular, the advent of photoswitchable fluorophores enables localization-based super resolution microscopy, which provides resolving power approaching the molecular-length scale...
December 1, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/27929459/perturbations-of-circulating-mirnas-in-irritable-bowel-syndrome-detected-using-a-multiplexed-high-throughput-gene-expression-platform
#2
Nicolaas H Fourie, Ralph M Peace, Sarah K Abey, LeeAnne B Sherwin, John W Wiley, Wendy A Henderson
The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in a single reaction. The miRNA assay counts transcripts by directly imaging and digitally counting miRNA molecules that are labeled with color-coded fluorescent barcoded probe sets (a reporter probe and a capture probe). Barcodes are hybridized directly to mature miRNAs that have been elongated by ligating a unique oligonucleotide tag (miRtag) to the 3' end...
November 30, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/27929085/multi-dimensional-super-resolution-imaging-enables-surface-hydrophobicity-mapping
#3
Marie N Bongiovanni, Julien Godet, Mathew H Horrocks, Laura Tosatto, Alexander R Carr, David C Wirthensohn, Rohan T Ranasinghe, Ji-Eun Lee, Aleks Ponjavic, Joelle V Fritz, Christopher M Dobson, David Klenerman, Steven F Lee
Super-resolution microscopy allows biological systems to be studied at the nanoscale, but has been restricted to providing only positional information. Here, we show that it is possible to perform multi-dimensional super-resolution imaging to determine both the position and the environmental properties of single-molecule fluorescent emitters. The method presented here exploits the solvatochromic and fluorogenic properties of nile red to extract both the emission spectrum and the position of each dye molecule simultaneously enabling mapping of the hydrophobicity of biological structures...
December 8, 2016: Nature Communications
https://www.readbyqxmd.com/read/27927704/nogo-a-regulates-vascular-network-architecture-in-the-postnatal-brain
#4
Thomas Wälchli, Alexandra Ulmann-Schuler, Christoph Hintermüller, Eric Meyer, Marco Stampanoni, Peter Carmeliet, Maximilian Y Emmert, Oliver Bozinov, Luca Regli, Martin E Schwab, Johannes Vogel, Simon P Hoerstrup
Recently, we discovered a new role for the well-known axonal growth inhibitory molecule Nogo-A as a negative regulator of angiogenesis in the developing central nervous system. However, how Nogo-A affected the three-dimensional (3D) central nervous system (CNS) vascular network architecture remained unknown. Here, using vascular corrosion casting, hierarchical, synchrotron radiation μCT-based network imaging and computer-aided network analysis, we found that genetic ablation of Nogo-A significantly increased the three-dimensional vascular volume fraction in the postnatal day 10 (P10) mouse brain...
November 10, 2016: Journal of Cerebral Blood Flow and Metabolism
https://www.readbyqxmd.com/read/27920086/a-role-for-mitotic-bookmarking-of-sox2-in-pluripotency-and-differentiation
#5
Cédric Deluz, Elias T Friman, Daniel Strebinger, Alexander Benke, Mahé Raccaud, Andrea Callegari, Marion Leleu, Suliana Manley, David M Suter
Mitotic bookmarking transcription factors remain bound to chromosomes during mitosis and were proposed to regulate phenotypic maintenance of stem and progenitor cells at the mitosis-to-G1 (M-G1) transition. However, mitotic bookmarking remains largely unexplored in most stem cell types, and its functional relevance for cell fate decisions remains unclear. Here we screened for mitotic chromosome binding within the pluripotency network of embryonic stem (ES) cells and show that SOX2 and OCT4 remain bound to mitotic chromatin through their respective DNA-binding domains...
December 5, 2016: Genes & Development
https://www.readbyqxmd.com/read/27919618/electric-field-induced-suppression-of-pten-drives-epithelial-to-mesenchymal-transition-via-mtorc1-activation
#6
Tiantian Yan, Xupin Jiang, Xiaowei Guo, Wen Chen, Di Tang, Junhui Zhang, Xingyue Zhang, Dongxia Zhang, Qiong Zhang, Jiezhi Jia, Yuesheng Huang
BACKGROUND: Naturally occurring electric fields (EFs) are an intrinsic property of wounds. Endogenous EFs in skin wounds play critical roles in the dynamic and well-ordered biological process of wound healing. The epithelial-to-mesenchymal transition (EMT) allows keratinocytes to transition from sedentary cells to motile cells, facilitating wound healing. However, EMT-related studies have been performed without considering endogenous EFs. Thus, the relationship between electrical signals and the EMT remain elusive...
November 18, 2016: Journal of Dermatological Science
https://www.readbyqxmd.com/read/27911841/high-performance-multiplexed-fluorescence-in-situ-hybridization-in-culture-and-tissue-with-matrix-imprinting-and-clearing
#7
Jeffrey R Moffitt, Junjie Hao, Dhananjay Bambah-Mukku, Tian Lu, Catherine Dulac, Xiaowei Zhuang
Highly multiplexed single-molecule FISH has emerged as a promising approach to spatially resolved single-cell transcriptomics because of its ability to directly image and profile numerous RNA species in their native cellular context. However, background-from off-target binding of FISH probes and cellular autofluorescence-can become limiting in a number of important applications, such as increasing the degree of multiplexing, imaging shorter RNAs, and imaging tissue samples. Here, we developed a sample clearing approach for FISH measurements...
November 22, 2016: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/27903895/protein-dynamics-of-human-rpa-and-rad51-on-ssdna-during-assembly-and-disassembly-of-the-rad51-filament
#8
Chu Jian Ma, Bryan Gibb, YoungHo Kwon, Patrick Sung, Eric C Greene
Homologous recombination (HR) is a crucial pathway for double-stranded DNA break (DSB) repair. During the early stages of HR, the newly generated DSB ends are processed to yield long single-stranded DNA (ssDNA) overhangs, which are quickly bound by replication protein A (RPA). RPA is then replaced by the DNA recombinase Rad51, which forms extended helical filaments on the ssDNA. The resulting nucleoprotein filament, known as the presynaptic complex, is responsible for pairing the ssDNA with homologous double-stranded DNA (dsDNA), which serves as the template to guide DSB repair...
November 29, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27893965/single-cell-and-single-molecule-analysis-of-gene-expression-regulation
#9
Maria Vera, Jeetayu Biswas, Adrien Senecal, Robert H Singer, Hye Yoon Park
Recent advancements in single-cell and single-molecule imaging technologies have resolved biological processes in time and space that are fundamental to understanding the regulation of gene expression. Observations of single-molecule events in their cellular context have revealed highly dynamic aspects of transcriptional and post-transcriptional control in eukaryotic cells. This approach can relate transcription with mRNA abundance and lifetimes. Another key aspect of single-cell analysis is the cell-to-cell variability among populations of cells...
November 23, 2016: Annual Review of Genetics
https://www.readbyqxmd.com/read/27889453/simultaneous-real-time-imaging-of-leading-and-lagging-strand-synthesis-reveals-the-coordination-dynamics-of-single-replisomes
#10
Karl E Duderstadt, Hylkje J Geertsema, Sarah A Stratmann, Christiaan M Punter, Arkadiusz W Kulczyk, Charles C Richardson, Antoine M van Oijen
The molecular machinery responsible for DNA replication, the replisome, must efficiently coordinate DNA unwinding with priming and synthesis to complete duplication of both strands. Due to the anti-parallel nature of DNA, the leading strand is copied continuously, while the lagging strand is produced by repeated cycles of priming, DNA looping, and Okazaki-fragment synthesis. Here, we report a multidimensional single-molecule approach to visualize this coordination in the bacteriophage T7 replisome by simultaneously monitoring the kinetics of loop growth and leading-strand synthesis...
November 15, 2016: Molecular Cell
https://www.readbyqxmd.com/read/27886170/tuning-charge-and-correlation-effects-for-a-single-molecule-on-a-graphene-device
#11
Sebastian Wickenburg, Jiong Lu, Johannes Lischner, Hsin-Zon Tsai, Arash A Omrani, Alexander Riss, Christoph Karrasch, Aaron Bradley, Han Sae Jung, Ramin Khajeh, Dillon Wong, Kenji Watanabe, Takashi Taniguchi, Alex Zettl, A H Castro Neto, Steven G Louie, Michael F Crommie
The ability to understand and control the electronic properties of individual molecules in a device environment is crucial for developing future technologies at the nanometre scale and below. Achieving this, however, requires the creation of three-terminal devices that allow single molecules to be both gated and imaged at the atomic scale. We have accomplished this by integrating a graphene field effect transistor with a scanning tunnelling microscope, thus allowing gate-controlled charging and spectroscopic interrogation of individual tetrafluoro-tetracyanoquinodimethane molecules...
November 25, 2016: Nature Communications
https://www.readbyqxmd.com/read/27883889/inferring-cell-state-transition-dynamics-from-lineage-trees-and-endpoint-single-cell-measurements
#12
Sahand Hormoz, Zakary S Singer, James M Linton, Yaron E Antebi, Boris I Shraiman, Michael B Elowitz
As they proliferate, living cells undergo transitions between specific molecularly and developmentally distinct states. Despite the functional centrality of these transitions in multicellular organisms, it has remained challenging to determine which transitions occur and at what rates without perturbations and cell engineering. Here, we introduce kin correlation analysis (KCA) and show that quantitative cell-state transition dynamics can be inferred, without direct observation, from the clustering of cell states on pedigrees (lineage trees)...
November 23, 2016: Cell Systems
https://www.readbyqxmd.com/read/27877104/sweet-taste-receptor-serves-to-activate-glucose-and-leptin-responsive-neurons-in-the-hypothalamic-arcuate-nucleus-and-participates-in-glucose-responsiveness
#13
Daisuke Kohno, Miho Koike, Yuzo Ninomiya, Itaru Kojima, Tadahiro Kitamura, Toshihiko Yada
The hypothalamic feeding center plays an important role in energy homeostasis. In the feeding center, whole-body energy signals including hormones and nutrients are sensed, processed, and integrated. As a result, food intake and energy expenditure are regulated. Two types of glucose-sensing neurons exist in the hypothalamic arcuate nucleus (ARC): glucose-excited neurons and glucose-inhibited neurons. While some molecules are known to be related to glucose sensing in the hypothalamus, the mechanisms underlying glucose sensing in the hypothalamus are not fully understood...
2016: Frontiers in Neuroscience
https://www.readbyqxmd.com/read/27876869/discerning-the-location-and-nature-of-coke-deposition-from-surface-to-bulk-of-spent-zeolite-catalysts
#14
Arun Devaraj, Murugesan Vijayakumar, Jie Bao, Mond F Guo, Miroslaw A Derewinski, Zhijie Xu, Michel J Gray, Sebastian Prodinger, Karthikeyan K Ramasamy
The formation of carbonaceous deposits (coke) in zeolite pores during catalysis leads to temporary deactivation of catalyst, necessitating regeneration steps, affecting throughput, and resulting in partial permanent loss of catalytic efficiency. Yet, even to date, the coke molecule distribution is quite challenging to study with high spatial resolution from surface to bulk of the catalyst particles at a single particle level. To address this challenge we investigated the coke molecules in HZSM-5 catalyst after ethanol conversion treatment by a combination of C K-edge X-ray absorption spectroscopy (XAS), (13)C Cross polarization-magic angle spinning nuclear magnetic resonance (CP-MAS NMR) spectroscopy, and atom probe tomography (APT)...
November 23, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27876857/smart-aggregation-imaging-for-single-molecule-localisation-with-spad-cameras
#15
Istvan Gyongy, Amy Davies, Neale A W Dutton, Rory R Duncan, Colin Rickman, Robert K Henderson, Paul A Dalgarno
Single molecule localisation microscopy (SMLM) has become an essential part of the super-resolution toolbox for probing cellular structure and function. The rapid evolution of these techniques has outstripped detector development and faster, more sensitive cameras are required to further improve localisation certainty. Single-photon avalanche photodiode (SPAD) array cameras offer single-photon sensitivity, very high frame rates and zero readout noise, making them a potentially ideal detector for ultra-fast imaging and SMLM experiments...
November 23, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27873472/perfect-timing-splicing-and-transcription-rates-in-living-cells
#16
REVIEW
Tara Alpert, Lydia Herzel, Karla M Neugebauer
An important step toward understanding gene regulation is the elucidation of the time necessary for the completion of individual steps. Measurement of reaction rates can reveal potential nodes for regulation. For example, measurements of in vivo transcription elongation rates reveal regulation by DNA sequence, gene architecture, and chromatin. Pre-mRNA splicing is regulated by transcription elongation rates and vice versa, yet the rates of RNA processing reactions remain largely elusive. Since the 1980s, numerous model systems and approaches have been used to determine the precise timing of splicing in vivo...
November 21, 2016: Wiley Interdisciplinary Reviews. RNA
https://www.readbyqxmd.com/read/27872870/nanoscale-organization-of-synaptic-adhesion-proteins-revealed-by-single-molecule-localization-microscopy
#17
Ingrid Chamma, Florian Levet, Jean-Baptiste Sibarita, Matthieu Sainlos, Olivier Thoumine
The advent of superresolution imaging has created a strong need for both optimized labeling strategies and analysis methods to probe the nanoscale organization of complex biological structures. We present a thorough description of the distribution of synaptic adhesion proteins at the nanoscopic scale, namely presynaptic neurexin-[Formula: see text] ([Formula: see text]), and its two postsynaptic binding partners neuroligin-1 (Nlg1) and leucine-rich-repeat transmembrane protein 2 (LRRTM2). We monitored these proteins in the membrane of neurons by direct stochastic optical reconstruction microscopy, after live surface labeling with Alexa647-conjugated monomeric streptavidin...
October 2016: Neurophotonics
https://www.readbyqxmd.com/read/27870840/single-nanotube-tracking-reveals-the-nanoscale-organization-of-the-extracellular-space-in-the-live-brain
#18
Antoine G Godin, Juan A Varela, Zhenghong Gao, Noémie Danné, Julien P Dupuis, Brahim Lounis, Laurent Groc, Laurent Cognet
The brain is a dynamic structure with the extracellular space (ECS) taking up almost a quarter of its volume. Signalling molecules, neurotransmitters and nutrients transit via the ECS, which constitutes a key microenvironment for cellular communication and the clearance of toxic metabolites. The spatial organization of the ECS varies during sleep, development and aging and is probably altered in neuropsychiatric and degenerative diseases, as inferred from electron microscopy and macroscopic biophysical investigations...
November 21, 2016: Nature Nanotechnology
https://www.readbyqxmd.com/read/27869821/synthetic-recording-and-in-situ-readout-of-lineage-information-in-single-cells
#19
Kirsten L Frieda, James M Linton, Sahand Hormoz, Joonhyuk Choi, Ke-Huan K Chow, Zakary S Singer, Mark W Budde, Michael B Elowitz, Long Cai
Reconstructing the lineage relationships and dynamic event histories of individual cells within their native spatial context is a long-standing challenge in biology. Many biological processes of interest occur in optically opaque or physically inaccessible contexts, necessitating approaches other than direct imaging. Here, we describe a new synthetic system that enables cells to record lineage information and event histories in the genome in a format that can be subsequently read out in single cells in situ...
November 21, 2016: Nature
https://www.readbyqxmd.com/read/27865781/imaging-transcriptional-regulation-of-eukaryotic-mrna-genes-advances-and-outlook
#20
REVIEW
Jie Yao
Regulation of eukaryotic transcription in vivo occurs at distinct stages. Previous research has identified many active or repressive transcription factors (TFs) and core transcription components and studied their functions in vitro and in vivo. Nonetheless, how individual TFs act in concert to regulate mRNA gene expression in a single cell remains poorly understood. Direct observation of TF assembly and disassembly and various biochemical reactions during transcription of a single-copy gene in vivo is the ideal approach to study this problem...
November 16, 2016: Journal of Molecular Biology
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