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Single molecule imaging

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https://www.readbyqxmd.com/read/29148550/two-dimensional-2d-self-assembly-of-oligo-phenylene-ethynylene-molecules-and-their-triangular-platinum-ii-diimine-complexes-studied-using-stm
#1
Siqi Zhang, Yanfang Geng, Yuanpeng Fan, Wubiao Duan, Ke Deng, Dahui Zhao, Qingdao Zeng
In this investigation, the two-dimensional (2D) self-assembly nanostructures of a series of cyclic oligo(phenylene-ethynylene) (OPE) molecules (L1, L2-6 and L2-12) at the 1-phenyloctane/highly oriented pyrolytic graphite (HOPG) interface were thoroughly studied using scanning tunneling microscopy (STM). Comparative STM studies with their triangular Pt(ii) diimine complexes (C1, C2-6 and C2-12) were also carried out. Based on careful measurements on single molecule level STM images and density functional theory (DFT) calculations, the formation mechanisms of the nanoarrays formed were revealed...
November 17, 2017: Physical Chemistry Chemical Physics: PCCP
https://www.readbyqxmd.com/read/29147545/protein-labeling-for-live-cell-fluorescence-microscopy-with-a-highly-photostable-renewable-signal
#2
Nina G Bozhanova, Mikhail S Baranov, Natalia V Klementieva, Karen S Sarkisyan, Alexey S Gavrikov, Ilia V Yampolsky, Elena V Zagaynova, Sergey A Lukyanov, Konstantin A Lukyanov, Alexander S Mishin
We present protein-PAINT - the implementation of the general principles of PAINT (Point Accumulation for Imaging in Nanoscale Topography) for live-cell protein labeling. Our method employs the specific binding of cell-permeable fluorogenic dyes to genetically encoded protein tags. We engineered three mutants of the bacterial lipocalin Blc that possess different affinities to a fluorogenic dye and exhibit a strong increase in fluorescence intensity upon binding. This allows for rapid labeling and washout of intracellular targets on a time scale from seconds to a few minutes...
October 1, 2017: Chemical Science
https://www.readbyqxmd.com/read/29147508/chiral-nanoprobes-for-targeting-and-long-term-imaging-of-the-golgi-apparatus
#3
Rong Sheng Li, Peng Fei Gao, Hong Zhi Zhang, Lin Ling Zheng, Chun Mei Li, Jian Wang, Yuan Fang Li, Feng Liu, Na Li, Cheng Zhi Huang
The Golgi apparatus is an essential subcellular organelle. Targeting and monitoring the Golgi change at the single-cell level over a long time scale are critical but are challenges that have not yet been tackled. Inspired by the precise Golgi positioning ability of galactosyltransferase and protein kinase D, due to their cysteine residues, we developed a method for long-term Golgi imaging. Fluorescent molecules, carbon quantum dots (CQDs) and silica nanoparticles could target the Golgi when they are modified with l-cysteine...
October 1, 2017: Chemical Science
https://www.readbyqxmd.com/read/29144410/evaluation-of-different-single-walled-carbon-nanotube-surface-coatings-for-single-particle-tracking-applications-in-biological-environments
#4
Zhenghong Gao, Noémie Danné, Antoine Guillaume Godin, Brahim Lounis, Laurent Cognet
Fluorescence imaging of biological systems down to the single-molecule level has generated many advances in cellular biology. For applications within intact tissue, single-walled carbon nanotubes (SWCNTs) are emerging as distinctive single-molecule nanoprobes, due to their near-infrared photoluminescence properties. For this, SWCNT surfaces must be coated using adequate molecular moieties. Yet, the choice of the suspension agent is critical since it influences both the chemical and emission properties of the SWCNTs within their environment...
November 16, 2017: Nanomaterials
https://www.readbyqxmd.com/read/29144055/confocal-raman-spectroscopic-imaging-for-in-vitro-monitoring-of-active-ingredient-penetration-and-distribution-in-reconstructed-human-epidermis-model
#5
Lynda Miloudi, Franck Bonnier, Ali Tfayli, Florent Yvergnaux, Hugh J Byrne, Igor Chourpa, Emilie Munnier
Topically applied Active Cosmetic Ingredients (ACI) or Active Pharmaceutical Ingredients (API) efficacy is directly related to their efficiency of penetration in the skin. In vitro Reconstructed Human Epidermis (RHE) surrogate models offer in vivo like skin samples for transdermal studies. Using Delipidol®, an ACI currently used in the cosmetics industry, the capabilities to deliver accurate distribution maps and penetration profiles of this molecule by means of Confocal Raman spectroscopic Imaging have been demonstrated...
November 16, 2017: Journal of Biophotonics
https://www.readbyqxmd.com/read/29141863/eight-color-multiplex-immunohistochemistry-for-simultaneous-detection-of-multiple-immune-checkpoint-molecules-within-the-tumor-microenvironment
#6
Mark A J Gorris, Altuna Halilovic, Katrin Rabold, Anne van Duffelen, Iresha N Wickramasinghe, Dagmar Verweij, Inge M N Wortel, Johannes C Textor, I Jolanda M de Vries, Carl G Figdor
Therapies targeting immune checkpoint molecules CTLA-4 and PD-1/PD-L1 have advanced the field of cancer immunotherapy. New mAbs targeting different immune checkpoint molecules, such as TIM3, CD27, and OX40, are being developed and tested in clinical trials. To make educated decisions and design new combination treatment strategies, it is vital to learn more about coexpression of both inhibitory and stimulatory immune checkpoints on individual cells within the tumor microenvironment. Recent advances in multiple immunolabeling and multispectral imaging have enabled simultaneous analysis of more than three markers within a single formalin-fixed paraffin-embedded tissue section, with accurate cell discrimination and spatial information...
November 15, 2017: Journal of Immunology: Official Journal of the American Association of Immunologists
https://www.readbyqxmd.com/read/29141223/gradients-of-rac1-nanoclusters-support-spatial-patterns-of-rac1-signaling
#7
Amanda Remorino, Simon De Beco, Fanny Cayrac, Fahima Di Federico, Gaetan Cornilleau, Alexis Gautreau, Maria Carla Parrini, Jean-Baptiste Masson, Maxime Dahan, Mathieu Coppey
Rac1 is a small RhoGTPase switch that orchestrates actin branching in space and time and protrusion/retraction cycles of the lamellipodia at the cell front during mesenchymal migration. Biosensor imaging has revealed a graded concentration of active GTP-loaded Rac1 in protruding regions of the cell. Here, using single-molecule imaging and super-resolution microscopy, we show an additional supramolecular organization of Rac1. We find that Rac1 partitions and is immobilized into nanoclusters of 50-100 molecules each...
November 14, 2017: Cell Reports
https://www.readbyqxmd.com/read/29138775/microsecond-resolved-single-molecule-fret-time-series-measurements-based-on-the-line-confocal-optical-system-combined-with-hybrid-photodetectors
#8
Hiroyuki Oikawa, Takumi Takahashi, Supawich Kamonprasertsuk, Satoshi Takahashi
Single-molecule (sm) fluorescence time series measurements based on the line confocal optical system are a powerful strategy for the investigation of the structure, dynamics, and heterogeneity of biological macromolecules. This method enables the detection of more than several thousands of fluorescence photons per millisecond from single fluorophores, implying that the potential time resolution for measurements of the fluorescence resonance energy transfer (FRET) efficiency is 10 μs. However, the necessity of using imaging photodetectors in the method limits the time resolution in the FRET efficiency measurements to approximately 100 μs...
November 15, 2017: Physical Chemistry Chemical Physics: PCCP
https://www.readbyqxmd.com/read/29136076/enhancing-the-photostability-of-poly-phenylene-ethynylene-for-single-particle-studies
#9
C F Calver, B A Lago, K S Schanze, G Cosa
Single molecule fluorescence (SMF) studies on conjugated polymers yield enhanced information on exciton dynamics and on the interplay between polymer conformation/morphology and photophysical behavior. SMF studies, however, demand good signal stability, excellent photostability, and high photon yields (a measure of both photostability and brightness) and thus the development of strategies to help conjugated polymers (CPs) meet these requirements is a topic of great interest. Here, we evaluate the effect of a number of triplet quencher additives on the photostability of a 49-mer long poly(phenylene-ethynylene) conjugated polymer bearing carboxylate side groups (PPE-CO2-49) that is deposited onto 100 nm diameter SiO2 nanoparticles (NPs)...
November 14, 2017: Photochemical & Photobiological Sciences
https://www.readbyqxmd.com/read/29135007/nanoscale-imaging-and-force-probing-of-biomolecular-systems-using-atomic-force-microscopy-from-single-molecules-to-living-cells
#10
REVIEW
Mi Li, Dan Dang, Ning Xi, Yuechao Wang, Lianqing Liu
Due to the lack of adequate tools for observation, native molecular behaviors at the nanoscale have been poorly understood. The advent of atomic force microscopy (AFM) provides an exciting instrument for investigating physiological processes on individual living cells with molecular resolution, which attracts the attention of worldwide researchers. In the past few decades, AFM has been widely utilized to investigate molecular activities on diverse biological interfaces, and the performances and functions of AFM have also been continuously improved, greatly improving our understanding of the behaviors of single molecules in action and demonstrating the important role of AFM in addressing biological issues with unprecedented spatiotemporal resolution...
November 14, 2017: Nanoscale
https://www.readbyqxmd.com/read/29131610/real-time-imaging-of-single-molecule-enzyme-cascade-using-a-dna-origami-raft
#11
Lele Sun, Yanjing Gao, Yan Xu, Jie Chao, Huajie Liu, Lianhui Wang, Di Li, Chunhai Fan
The dynamics of enzymes are directly associated with their functions in various biological processes. Nevertheless, the ability to image motions of single enzymes in a highly parallel fashion remains a challenge. Here, we develop a DNA origami raft-based platform for in-situ real-time imaging of enzyme cascade at the single-molecule level. The motions of enzymes are rationally controlled via different tethering modes on a two-dimensional (2D) supported lipid bilayer (SLB). We construct an enzyme cascade by anchoring catalase on cholesterol-labeled double stranded (ds) DNA, and glucose oxidase on cholesterol-labeled origami rafts...
November 13, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/29131164/an-improved-ms2-system-for-accurate-reporting-of-the-mrna-life-cycle
#12
Evelina Tutucci, Maria Vera, Jeetayu Biswas, Jennifer Garcia, Roy Parker, Robert H Singer
The MS2-MCP system enables researchers to image multiple steps of the mRNA life cycle with high temporal and spatial resolution. However, for short-lived mRNAs, the tight binding of the MS2 coat protein (MCP) to the MS2 binding sites (MBS) protects the RNA from being efficiently degraded, and this confounds the study of mRNA regulation. Here, we describe a reporter system (MBSV6) with reduced affinity for the MCP, which allows mRNA degradation while preserving single-molecule detection determined by single-molecule FISH (smFISH) or live imaging...
November 13, 2017: Nature Methods
https://www.readbyqxmd.com/read/29130212/imaging-translation-dynamics-of-single-mrna-molecules-in-live-cells
#13
Suzan Ruijtenberg, Tim A Hoek, Xiaowei Yan, Marvin E Tanenbaum
mRNA translation is a key step in decoding the genetic information stored in DNA. Regulation of translation efficiency contributes to gene expression control and is therefore important for cell fate and function. Here, we describe a recently developed microscopy-based method that allows for visualization of translation of single mRNAs in live cells. The ability to measure translation dynamics of single mRNAs will enable a better understanding of spatiotemporal control of translation, and will provide unique insights into translational heterogeneity of different mRNA molecules in single cells...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29130211/detection-of-the-first-round-of-translation-the-trick-assay
#14
Franka Voigt, Jan Eglinger, Jeffrey A Chao
Quantitative fluorescence microscopy techniques are frequently applied to answer fundamental biological questions. Single-molecule RNA imaging methods have enabled the direct observation of the initial steps of the mRNA life cycle in living cells, however, the dynamic mechanisms that regulate mRNA translation are still poorly understood. We have developed an RNA biosensor that can assess the translational state of individual mRNA transcripts with spatiotemporal resolution in living cells. In this chapter, we describe how to perform a TRICK (translating RNA imaging by coat protein knock-off) experiment and specifically focus on a detailed description of our image processing and data analysis procedure...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29130208/real-time-fluorescence-imaging-of-single-molecule-endogenous-noncoding-rna-in-living-cells
#15
Hideaki Yoshimura, Takeaki Ozawa
Visualizing RNA in living cells is increasingly important to facilitate accumulation of knowledge about the relation between specific RNA dynamics and physiological events. Single-molecule fluorescence imaging of target RNAs is an excellent approach to analyzing intracellular RNA motion, but it requires special techniques for probe design and microscope setup. Herein, we present a principle and protocol of an RNA visualization probe based on an RNA binding protein of the Pumilio homology domain (PUM-HD). We also describe the setup and operation of a microscope, and introduce an application to visualize telomeric repeats-containing RNA with telomeres and a telomere-related protein: hnRNPA1...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29130202/optimizing-molecular-beacons-for-intracellular-analysis-of-rna
#16
Mingming Chen, Yantao Yang, Christopher J Krueger, Antony K Chen
Conventional molecular beacons (MBs) have been used extensively for imaging specific endogenous RNAs in living cells, but their tendency to generate false-positive signals as a result of nuclease degradation and/or nonspecific binding limits sensitive and accurate imaging of intracellular RNAs. In an attempt to overcome this limitation, MBs have been synthesized with various chemically modified oligonucleotide backbones to confer greater biostability. We have recently developed a new MB architecture composed of 2'-O-methyl RNA (2Me), a fully phosphorothioate (PS) modified loop domain and a phosphodiester stem (2Me/PSLOOP MB)...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29130201/quantifying-gene-expression-in-living-cells-with-ratiometric-bimolecular-beacons
#17
Yantao Yang, Mingming Chen, Christopher J Krueger, Andrew Tsourkas, Antony K Chen
Molecular beacons (MBs), a class of oligonucleotide-based probes, have enabled researchers to study various RNA molecules in their native live-cell contexts. However, it is also increasingly recognized that, when delivered into cells, MBs have the tendency to be sequestered into the nucleus where they may generate false positive signals. In an attempt to overcome this issue, MBs have been synthesized with chemically modified oligonucleotide backbones to confer greater biostability. Alternatively, strategies have been developed to minimize nuclear entry...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29130196/super-resolution-single-molecule-fish-at-the-drosophila-neuromuscular-junction
#18
Joshua S Titlow, Lu Yang, Richard M Parton, Ana Palanca, Ilan Davis
The lack of an effective, simple, and highly sensitive protocol for fluorescent in situ hybridization (FISH) at the Drosophila larval neuromuscular junction (NMJ) has hampered the study of mRNA biology. Here, we describe our modified single molecule FISH (smFISH) methods that work well in whole mount Drosophila NMJ preparations to quantify primary transcription and count individual cytoplasmic mRNA molecules in specimens while maintaining ultrastructural preservation. The smFISH method is suitable for high-throughput sample processing and 3D image acquisition using any conventional microscopy imaging modality and is compatible with the use of antibody colabeling and transgenic fluorescent protein tags in axons, glia, synapses, and muscle cells...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29130195/detection-and-automated-analysis-of-single-transcripts-at-subcellular-resolution-in-zebrafish-embryos
#19
L Carine Stapel, Coleman Broaddus, Nadine L Vastenhouw
Single molecule fluorescence in situ hybridization (smFISH) is a method to visualize single mRNA molecules. When combined with cellular and nuclear segmentation, transcripts can be assigned to different cellular compartments resulting in quantitative information on transcript levels at subcellular resolution. The use of smFISH in zebrafish has been limited by the lack of protocols and an automated image analysis pipeline for samples of multicellular organisms. Here we present a protocol for smFISH on zebrafish cryosections...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29130187/the-secret-life-of-rna-lessons-from-emerging-methodologies
#20
Caroline Medioni, Florence Besse
The last past decade has witnessed a revolution in our appreciation of transcriptome complexity and regulation. This remarkable expansion in our knowledge largely originates from the advent of high-throughput methodologies, and the consecutive discovery that up to 90% of eukaryotic genomes are transcribed, thus generating an unanticipated large range of noncoding RNAs (Hangauer et al., 15(4):112, 2014). Besides leading to the identification of new noncoding RNA species, transcriptome-wide studies have uncovered novel layers of posttranscriptional regulatory mechanisms controlling RNA processing, maturation or translation, and each contributing to the precise and dynamic regulation of gene expression...
2018: Methods in Molecular Biology
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