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https://www.readbyqxmd.com/read/28534613/flashbody-a-next-generation-fluobody-with-fluorescence-intensity-enhanced-by-antigen-binding
#1
Devina Wongso, Jinhua Dong, Hiroshi Ueda, Tetsuya Kitaguchi
Fluorescent probes are valuable tools for visualizing the spatiotemporal dynamics of molecules in living cells. Here we developed a genetically-encoded antibody probe with antigen-dependent fluorescence intensity called "Flashbody". We first created a fusion of EGFP to the single chain variable region fragment (scFv) of antibody against seven amino acids of the bone Gla protein C-terminus (BGPC7) called BGP Fluobody, which successfully showed the intracellular localization of BGPC7-tagged protein. To generate BGP Flashbody, circularly permuted GFP was inserted in between two variable region fragments, and the linkers were optimized, resulting in fluorescence intensity increase of 300% upon binding with BGPC7 in a dose-dependent manner...
May 23, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28528628/millisecond-time-resolution-correlative-light-and-electron-microscopy-for-dynamic-cellular-processes
#2
Ludek Stepanek, Gaia Pigino
Molecular motors propel cellular components at velocities up to microns per second with nanometer precision. Imaging techniques combining high temporal and spatial resolution are therefore indispensable to understand the cellular mechanics at the molecular level. For example, intraflagellar transport (IFT) trains constantly shuttle ciliary components between the base and tip of the eukaryotic cilium. 3-D electron microscopy has revealed IFT train morphology and position, but was unable to correlate these features with the direction of train movement...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28523559/techniques-for-single-molecule-mrna-imaging-in-living-cells
#3
Kevin Czaplinski
Typical measurement of macromolecules in a biological sample typically averages the result over all the cells or molecules within the sample, and while these types of measurements provide very useful information, they completely miss heterogeneity among the components within the sample that could be a very important aspect of the sample's function. These techniques are also limited in their ability to examine intracellular spatial orientation of molecular activity, which is often a critical component to the regulation of biological processes, particularly in cells with unique spatial relationships, such as neurons...
2017: Advances in Experimental Medicine and Biology
https://www.readbyqxmd.com/read/28495969/fold-change-detection-and-scale-invariance-of-cell-cell-signaling-in-social-amoeba
#4
Keita Kamino, Yohei Kondo, Akihiko Nakajima, Mai Honda-Kitahara, Kunihiko Kaneko, Satoshi Sawai
Cell-cell signaling is subject to variability in the extracellular volume, cell number, and dilution that potentially increase uncertainty in the absolute concentrations of the extracellular signaling molecules. To direct cell aggregation, the social amoebae Dictyostelium discoideum collectively give rise to oscillations and waves of cyclic adenosine 3',5'-monophosphate (cAMP) under a wide range of cell density. To date, the systems-level mechanism underlying the robustness is unclear. By using quantitative live-cell imaging, here we show that the magnitude of the cAMP relay response of individual cells is determined by fold change in the extracellular cAMP concentrations...
May 11, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28494964/near-membrane-refractometry-using-supercritical-angle-fluorescence
#5
Maia Brunstein, Lopamudra Roy, Martin Oheim
Total internal reflection fluorescence (TIRF) microscopy and its variants are key technologies for visualizing the dynamics of single molecules or organelles in live cells. Yet truly quantitative TIRF remains problematic. One unknown hampering the interpretation of evanescent-wave excited fluorescence intensities is the undetermined cell refractive index (RI). Here, we use a combination of TIRF excitation and supercritical angle fluorescence emission detection to directly measure the average RI in the "footprint" region of the cell during image acquisition...
May 9, 2017: Biophysical Journal
https://www.readbyqxmd.com/read/28493880/identifying-stochastic-oscillations-in-single-cell-live-imaging-time-series-using-gaussian-processes
#6
Nick E Phillips, Cerys Manning, Nancy Papalopulu, Magnus Rattray
Multiple biological processes are driven by oscillatory gene expression at different time scales. Pulsatile dynamics are thought to be widespread, and single-cell live imaging of gene expression has lead to a surge of dynamic, possibly oscillatory, data for different gene networks. However, the regulation of gene expression at the level of an individual cell involves reactions between finite numbers of molecules, and this can result in inherent randomness in expression dynamics, which blurs the boundaries between aperiodic fluctuations and noisy oscillators...
May 11, 2017: PLoS Computational Biology
https://www.readbyqxmd.com/read/28488813/imaging-mass-spectrometry-for-metabolites-technical-progress-multimodal-imaging-and-biological-interactions
#7
REVIEW
Ying-Ning Ho, Lin-Jie Shu, Yu-Liang Yang
Imaging mass spectrometry (IMS) allows the study of the spatial distribution of small molecules in biological samples. IMS is able to identify and quantify chemicals in situ from whole tissue sections to single cells. Both vacuum mass spectrometry (MS) and ambient MS systems have advanced considerably over the last decade; however, some limitations are still hard to surmount. Sample pretreatment, matrix or solvent choices, and instrument improvement are the key factors that determine the successful application of IMS to different samples and analytes...
May 10, 2017: Wiley Interdisciplinary Reviews. Systems Biology and Medicine
https://www.readbyqxmd.com/read/28484218/a-molecular-beacon-based-approach-for-live-cell-imaging-of-rna-transcripts-with-minimal-target-engineering-at-the-single-molecule-level
#8
Mingming Chen, Zhao Ma, Xiaotian Wu, Shiqi Mao, Yantao Yang, Jie Tan, Christopher J Krueger, Antony K Chen
Analysis of RNA dynamics and localization at the single-molecule level in living cells has been predominantly achieved by engineering target RNAs with large insertions of tandem repeat sequences that are bound by protein-based or oligonucleotide-based fluorescent probes. Thus, individual RNAs are tagged by multiple fluorescent probes, making them detectable by fluorescence microscopy. Since large insertions may affect RNA processes including trafficking and localization, here we present a strategy to visualize single RNA transcripts in living cells using molecular beacons (MBs) - fluorogenic oligonucleotide probes - with minimal target engineering...
May 8, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28481515/sequence-directed-covalent-protein-dna-linkages-in-a-single-step-using-huh-tags
#9
Klaus N Lovendahl, Amanda N Hayward, Wendy R Gordon
We present a robust strategy to covalently link proteins and DNA using HUH-endonuclease domains as fusion partners (HUH-tags). We show that HUH-tags react robustly with specific sequences of unmodified single-stranded DNA, and we have identified five tags that react orthogonally with distinct DNA sequences. We demonstrate the versatility of HUH-tags as fusion partners in Cas9-mediated gene editing and the construction of doubly DNA-tethered proteins for single-molecule studies. Finally we demonstrate application to cellular imaging in live and fixed cells...
May 16, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28448050/single-molecule-analysis-of-laser-localized-psoralen-adducts
#10
Jing Huang, Himabindu Gali, Julia Gichimu, Marina A Bellani, Durga Pokharel, Manikandan Paramasivam, Michael M Seidman
The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live cells. The DDR to helix distorting covalent DNA modifications, including interstrand DNA crosslinks (ICLs), is not as well defined. We have studied the DDR stimulated by ICLs, localized by laser photoactivation of immunotagged psoralens, in the nuclei of live cells. In order to address fundamental questions about adduct distribution and replication fork encounters, we combined laser localization with two other technologies...
April 20, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28426025/modular-low-light-microscope-for-imaging-cellular-bioluminescence-and-radioluminescence
#11
Tae Jin Kim, Silvan Türkcan, Guillem Pratx
Low-light microscopy methods are receiving increased attention as new applications have emerged. One such application is to allow longitudinal imaging of light-sensitive cells with no phototoxicity and no photobleaching of fluorescent biomarkers. Another application is for imaging signals that are inherently dim and undetectable using standard microscopy techniques, such as bioluminescence, chemiluminescence or radioluminescence. In this protocol, we provide instructions on how to build a modular low-light microscope (1-4 d) by coupling two microscope objective lenses, back to back from each other, using standard optomechanical components...
May 2017: Nature Protocols
https://www.readbyqxmd.com/read/28424513/super-multiplex-vibrational-imaging
#12
Lu Wei, Zhixing Chen, Lixue Shi, Rong Long, Andrew V Anzalone, Luyuan Zhang, Fanghao Hu, Rafael Yuste, Virginia W Cornish, Wei Min
The ability to visualize directly a large number of distinct molecular species inside cells is increasingly essential for understanding complex systems and processes. Even though existing methods have successfully been used to explore structure-function relationships in nervous systems, to profile RNA in situ, to reveal the heterogeneity of tumour microenvironments and to study dynamic macromolecular assembly, it remains challenging to image many species with high selectivity and sensitivity under biological conditions...
April 27, 2017: Nature
https://www.readbyqxmd.com/read/28414097/single-molecule-and-super-resolution-imaging-of-transcription-in-living-bacteria
#13
REVIEW
Mathew Stracy, Achillefs N Kapanidis
In vivo single-molecule and super-resolution techniques are transforming our ability to study transcription as it takes place in its native environment in living cells. This review will detail the methods for imaging single molecules in cells, and the data-analysis tools which can be used to extract quantitative information on the spatial organization, mobility, and kinetics of the transcription machinery from these experiments. Furthermore, we will highlight studies which have applied these techniques to shed new light on bacterial transcription...
April 13, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28404480/2d-and-3d-fish-of-expanded-repeat-rnas-in-human-lymphoblasts
#14
Martyna O Urbanek, Michał Michalak, Wlodzimierz J Krzyzosiak
The first methods for visualizing RNAs within cells were designed for simple imaging of specific transcripts in cells or tissues and since then significant technical advances have been made in this field. Today, high-resolution images can be obtained, enabling visualization of single transcript molecules, quantitative analyses of images, and precise localization of RNAs within cells as well as co-localization of transcripts with specific proteins or other molecules. In addition, tracking of RNA dynamics within single cell has become possible...
April 9, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28392350/choosing-the-right-fluorophore-for-single-molecule-fluorescence-studies-in-a-lipid-environment
#15
Zhenfu Zhang, Dan Yomo, Claudiu Gradinaru
Nonspecific interactions between lipids and fluorophores can alter the outcomes of single-molecule spectroscopy of membrane proteins in live cells, liposomes or lipid nanodiscs and of cytosolic proteins encapsulated in liposomes or tethered to supported lipid bilayers. To gain insight into these effects, we examined interactions between 9 dyes that are commonly used as labels for single-molecule fluorescence (SMF) and 6 standard lipids including cationic, zwitterionic and anionic types. The diffusion coefficients of dyes in the absence and presence of set amounts of lipid vesicles were measured by fluorescence correlation spectroscopy (FCS)...
July 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28389957/direct-imaging-of-intracellular-signaling-molecule-responsible-for-the-bacterial-chemotaxis
#16
Hajime Fukuoka
To elucidate the mechanisms by which cells respond to extracellular stimuli, the behavior of intracellular signaling proteins in a single cell should be directly examined, while simultaneously recording the cellular response. In Escherichia coli, an extracellular chemotactic stimulus is thought to induce a switch in the rotational direction of the flagellar motor, elicited by the binding and dissociation of the phosphorylated form of CheY (CheY-P) to and from the motor. We recently provided direct evidence for the binding of CheY-P to a functioning flagellar motor in live cells...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28383040/imaging-modes-of-atomic-force-microscopy-for-application-in-molecular-and-cell-biology
#17
REVIEW
Yves F Dufrêne, Toshio Ando, Ricardo Garcia, David Alsteens, David Martinez-Martin, Andreas Engel, Christoph Gerber, Daniel J Müller
Atomic force microscopy (AFM) is a powerful, multifunctional imaging platform that allows biological samples, from single molecules to living cells, to be visualized and manipulated. Soon after the instrument was invented, it was recognized that in order to maximize the opportunities of AFM imaging in biology, various technological developments would be required to address certain limitations of the method. This has led to the creation of a range of new imaging modes, which continue to push the capabilities of the technique today...
April 6, 2017: Nature Nanotechnology
https://www.readbyqxmd.com/read/28377799/electronic-tuning-of-self-healing-fluorophores-for-live-cell-and-single-molecule-imaging
#18
Qinsi Zheng, Steffen Jockusch, Zhou Zhou, Roger B Altman, Hong Zhao, Wesley Asher, Michael Holsey, Signe Mathiasen, Peter Geggier, Jonathan A Javitch, Scott C Blanchard
Bright, long-lasting organic fluorophores enable a broad range of imaging applications. "Self-healing" fluorophores, in which intra-molecularly linked protective agents quench photo-induced reactive species, exhibit both enhanced photostability and biological compatibility. However, the self-healing strategy has yet to achieve its predicted potential, particularly in the presence of ambient oxygen where live-cell imaging studies must often be performed. To identify key bottlenecks in this technology that can be used to guide further engineering developments, we synthesized a series of Cy5 derivatives linked to the protective agent cyclooctatetraene (COT) and examined the photophysical mechanisms curtailing their performance...
January 1, 2017: Chemical Science
https://www.readbyqxmd.com/read/28362417/application-of-genetically-encoded-fluorescent-nitric-oxide-no%C3%A2-probes-the-genops-for-real-time-imaging-of-no%C3%A2-signals-in-single-cells
#19
Emrah Eroglu, Rene Rost, Helmut Bischof, Sandra Blass, Anna Schreilechner, Benjamin Gottschalk, Maria R Depaoli, Christiane Klec, Suphachai Charoensin, Corina T Madreiter-Sokolowski, Jeta Ramadani, Markus Waldeck-Weiermair, Wolfgang F Graier, Roland Malli
Nitric Oxide (NO•) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO• in vivo and in vitro, the real-time monitoring of NO• at the single-cell level is very challenging. The physiological or pathological effects of NO• are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO• are highly desirable...
March 16, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28350161/multiparametric-atomic-force-microscopy-imaging-of-biomolecular-and-cellular-systems
#20
David Alsteens, Daniel J Müller, Yves F Dufrêne
There is a need in biochemical research for new tools that can image and manipulate biomolecular and cellular systems at the nanoscale. During the past decades, there has been tremendous progress in developing atomic force microscopy (AFM) techniques to analyze biosystems, down to the single-molecule level. Force-distance (FD) curve-based AFM in particular has enabled researchers to map and quantify biophysical properties and biomolecular interactions on a wide variety of specimens. Despite its great potential, this AFM method has long been limited by its low spatial and temporal resolutions...
March 28, 2017: Accounts of Chemical Research
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