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https://www.readbyqxmd.com/read/28448050/single-molecule-analysis-of-laser-localized-psoralen-adducts
#1
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
#2
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
#3
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 19, 2017: Nature
https://www.readbyqxmd.com/read/28414097/single-molecule-and-super-resolution-imaging-of-transcription-in-living-bacteria
#4
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
#5
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
#6
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)...
April 6, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28389957/direct-imaging-of-intracellular-signaling-molecule-responsible-for-the-bacterial-chemotaxis
#7
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
#8
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
#9
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
#10
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
#11
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
https://www.readbyqxmd.com/read/28340410/development-of-fusogenic-glass-surfaces-that-impart-spatiotemporal-control-over-macrophage-fusion-direct-visualization-of-multinucleated-giant-cell-formation
#12
James J Faust, Wayne Christenson, Kyle Doudrick, Robert Ros, Tatiana P Ugarova
Implantation of synthetic material, including vascular grafts, pacemakers, etc. results in the foreign body reaction and the formation of multinucleated giant cells (MGCs) at the exterior surface of the implant. Despite the long-standing premise that fusion of mononucleated macrophages results in the formation of MGCs, to date, no published study has shown fusion in context with living specimens. This is due to the fact that optical-quality glass, which is required for the majority of live imaging techniques, does not promote macrophage fusion...
June 2017: Biomaterials
https://www.readbyqxmd.com/read/28337984/lipid-dependent-conformational-dynamics-underlie-the-functional-versatility-of-t-cell-receptor
#13
Xingdong Guo, Chengsong Yan, Hua Li, Wenmao Huang, Xiaoshan Shi, Min Huang, Yingfang Wang, Weiling Pan, Mingjun Cai, Lunyi Li, Wei Wu, Yibing Bai, Chi Zhang, Zhijun Liu, Xinyan Wang, Xiaohui F Zhang, Chun Tang, Hongda Wang, Wanli Liu, Bo Ouyang, Catherine C Wong, Yi Cao, Chenqi Xu
T-cell receptor-CD3 complex (TCR) is a versatile signaling machine that can initiate antigen-specific immune responses based on various biochemical changes of CD3 cytoplasmic domains, but the underlying structural basis remains elusive. Here we developed biophysical approaches to study the conformational dynamics of CD3ε cytoplasmic domain (CD3εCD). At the single-molecule level, we found that CD3εCD could have multiple conformational states with different openness of three functional motifs, i.e., ITAM, BRS and PRS...
April 2017: Cell Research
https://www.readbyqxmd.com/read/28324613/analysis-of-protein-kinetics-using-fluorescence-recovery-after-photobleaching-frap
#14
Nickolaos Nikiforos Giakoumakis, Maria Anna Rapsomaniki, Zoi Lygerou
Fluorescence recovery after photobleaching (FRAP) is a cutting-edge live-cell functional imaging technique that enables the exploration of protein dynamics in individual cells and thus permits the elucidation of protein mobility, function, and interactions at a single-cell level. During a typical FRAP experiment, fluorescent molecules in a defined region of interest within the cell are bleached by a short and powerful laser pulse, while the recovery of the fluorescence in the region is monitored over time by time-lapse microscopy...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324612/quantitative-image-analysis-of-single-molecule-mrna-dynamics-in-living-cells
#15
José Rino, Ana C de Jesus, Maria Carmo-Fonseca
Single mRNA molecules can be imaged in living cells by a method that consists in genetically inserting binding sites for a bacteriophage protein in the gene of interest. The resulting reporter transgene is then integrated in the genome of cells that express the phage protein fused to a fluorescent tag. Upon transcription, binding of the fluorescent protein to its target sequence makes the RNA visible. With this approach it is possible to track, in real time, the life cycle of a precursor mRNA at the site of transcription in the nucleus and transport of mature mRNA to the cytoplasm...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324608/two-color-total-internal-reflection-fluorescence-microscopy-of-exocytosis-in-endocrine-cells
#16
Adam J Trexler, Justin W Taraska
We describe a comprehensive method for imaging and analysis of local protein dynamics at single sites of exocytosis in living cultured endocrine cells. This method is well suited to quantitatively map the complex dynamics of individual molecules at single sites of vesicle fusion in live cells.
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324598/introduction-to-modern-methods-in-light-microscopy
#17
Joel Ryan, Abby R Gerhold, Vincent Boudreau, Lydia Smith, Paul S Maddox
For centuries, light microscopy has been a key method in biological research, from the early work of Robert Hooke describing biological organisms as cells, to the latest in live-cell and single-molecule systems. Here, we introduce some of the key concepts related to the development and implementation of modern microscopy techniques. We briefly discuss the basics of optics in the microscope, super-resolution imaging, quantitative image analysis, live-cell imaging, and provide an outlook on active research areas pertaining to light microscopy...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28315485/quantifying-transcription-factor-binding-dynamics-at-the-single-molecule-level-in-live-cells
#18
Diego M Presman, David A Ball, Ville Paakinaho, Jonathan B Grimm, Luke D Lavis, Tatiana S Karpova, Gordon L Hager
Progressive, technological achievements in the quantitative fluorescence microscopy field are allowing researches from many different areas to start unraveling the dynamic intricacies of biological processes inside living cells. From super-resolution microscopy techniques to tracking of individual proteins, fluorescence microscopy is changing our perspective on how the cell works. Fortunately, a growing number of research groups are exploring single-molecule studies in living cells. However, no clear consensus exists on several key aspects of the technique such as image acquisition conditions, or analysis of the obtained data...
March 14, 2017: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/28276025/single-molecule-tracking-and-localization-of-mitochondrial-protein-complexes-in-live-cells
#19
Timo Appelhans, Karin Busch
Mitochondria are the power plant of most non-green eukaryotic cells. An understanding of their function and regulation is only possible with the knowledge of the spatiotemporal dynamics of their proteins. Mitochondrial membrane proteins involved in diverse functions like protein import, cell respiration, metabolite transport, and mitochondrial morphology are mobile within membranes. Here, we provide a protocol for a superresolution fluorescence microscopy technique named tracking and localization microscopy (TALM) that allows for localization and diffusion analysis of single mitochondrial membrane proteins in situ in cell cultures...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28258033/rnp-transport-in-cell-biology-the-long-and-winding-road
#20
REVIEW
Carolina Eliscovich, Robert H Singer
Regulation of gene expression is key determinant to cell structure and function. RNA localization, where specific mRNAs are transported to subcellular regions and then translated, is highly conserved in eukaryotes ranging from yeast to extremely specialized and polarized cells such as neurons. Messenger RNA and associated proteins (mRNP) move from the site of transcription in the nucleus to their final destination in the cytoplasm both passively through diffusion and actively via directed transport. Dysfunction of RNA localization, transport and translation machinery can lead to pathology...
February 28, 2017: Current Opinion in Cell Biology
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