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live cell single molecule imaging

Adam J M Wollman, Helen Miller, Simon Foster, Mark C Leake
Staphylococcus aureus is an important pathogen, giving rise to antimicrobial resistance in cell strains such as Methicillin Resistant S. aureus (MRSA). Here we report an image analysis framework for automated detection and image segmentation of cells in S. aureus cell clusters, and explicit identification of their cell division planes. We use a new combination of several existing analytical tools of image analysis to detect cellular and subcellular morphological features relevant to cell division from millisecond time scale sampled images of live pathogens at a detection precision of single molecules...
October 17, 2016: Physical Biology
Delong Zhang, Chen Li, Chi Zhang, Mikhail N Slipchenko, Gregory Eakins, Ji-Xin Cheng
Chemical contrast has long been sought for label-free visualization of biomolecules and materials in complex living systems. Although infrared spectroscopic imaging has come a long way in this direction, it is thus far only applicable to dried tissues because of the strong infrared absorption by water. It also suffers from low spatial resolution due to long wavelengths and lacks optical sectioning capabilities. We overcome these limitations through sensing vibrational absorption-induced photothermal effect by a visible laser beam...
September 2016: Science Advances
Shalin B Mehta, Molly McQuilken, Patrick J La Riviere, Patricia Occhipinti, Amitabh Verma, Rudolf Oldenbourg, Amy S Gladfelter, Tomomi Tani
Regulation of order, such as orientation and conformation, drives the function of most molecular assemblies in living cells but remains difficult to measure accurately through space and time. We built an instantaneous fluorescence polarization microscope, which simultaneously images position and orientation of fluorophores in living cells with single-molecule sensitivity and a time resolution of 100 ms. We developed image acquisition and analysis methods to track single particles that interact with higher-order assemblies of molecules...
September 27, 2016: Proceedings of the National Academy of Sciences of the United States of America
Johannes P Schneider, Marek Basler
To understand basic principles of living organisms one has to know many different properties of all cellular components, their mutual interactions but also their amounts and spatial organization. Live-cell imaging is one possible approach to obtain such data. To get multiple snapshots of a cellular process, the imaging approach has to be gentle enough to not disrupt basic functions of the cell but also have high temporal and spatial resolution to detect and describe the changes. Light microscopy has become a method of choice and since its early development over 300 years ago revolutionized our understanding of living organisms...
November 5, 2016: Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences
Diana Di Paolo, Oshri Afanzar, Judith P Armitage, Richard M Berry
For the past two decades, the use of genetically fused fluorescent proteins (FPs) has greatly contributed to the study of chemotactic signalling in Escherichia coli including the activation of the response regulator protein CheY and its interaction with the flagellar motor. However, this approach suffers from a number of limitations, both biological and biophysical: for example, not all fusions are fully functional when fused to a bulky FP, which can have a similar molecular weight to its fused counterpart; they may interfere with the native interactions of the protein and the chromophores of FPs have low brightness and photostability and fast photobleaching rates...
November 5, 2016: Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences
B T Cunningham, M Zhang, Y Zhuo, L Kwon, C Race
Photonic crystal surfaces that are designed to function as wavelength-selective optical resonators have become a widely adopted platform for label-free biosensing, and for enhancement of the output of photon-emitting tags used throughout life science research and in vitro diagnostics. While some applications, such as analysis of drug-protein interactions, require extremely high resolution and the ability to accurately correct for measurement artifacts, others require sensitivity that is high enough for detection of disease biomarkers in serum with concentrations less than 1 pg/ml...
May 15, 2016: IEEE Sensors Journal
Nan Li, Yong Yang, Kangmin He, Fayun Zhang, Libo Zhao, Wei Zhou, Jinghe Yuan, Wei Liang, Xiaohong Fang
Smad3 is an intracellular protein that plays a key role in propagating transforming growth factor β (TGF-β) signals from cell membrane to nucleus. However whether the transient process of Smad3 activation occurs on cell membrane and how it is regulated remains elusive. Using advanced live-cell single-molecule fluorescence microscopy to image and track fluorescent protein-labeled Smad3, we observed and quantified, for the first time, the dynamics of individual Smad3 molecules docking to and activation on the cell membrane...
2016: Scientific Reports
Anne Plochowietz, Ian Farrell, Zeev Smilansky, Barry S Cooperman, Achillefs N Kapanidis
Transfer RNA (tRNA) links messenger RNA nucleotide sequence with amino acid sequence during protein synthesis. Despite the importance of tRNA for translation, its subcellular distribution and diffusion properties in live cells are poorly understood. Here, we provide the first direct report on tRNA diffusion localization in live bacteria. We internalized tRNA labeled with organic fluorophores into live bacteria, applied single-molecule fluorescence imaging with single-particle tracking and localized and tracked single tRNA molecules over seconds...
September 12, 2016: Nucleic Acids Research
Haogang Cai, Shalom J Wind
Single-molecule fluorescence techniques provide a critical tool for probing biomolecular and cellular interactions with unprecedented resolution and precision. Unfortunately, many of these techniques are hindered by a common problem, namely, the nonspecific adsorption of target biomolecules. This issue is mostly addressed by passivating the glass surfaces with a poly(ethylene glycol) (PEG) brush. This is effective only at low concentrations of the probe molecule because there are defects inherent to polymer brushes formed on glass coverslips due to the presence of surface impurities...
October 4, 2016: Langmuir: the ACS Journal of Surfaces and Colloids
Herlinde De Keersmaecker, Eduard Fron, Susana Rocha, Takako Kogure, Atsushi Miyawaki, Johan Hofkens, Hideaki Mizuno
Photoswitchable fluorescent proteins are capable of changing their spectral properties upon light irradiation, thus allowing one to follow a chosen subpopulation of molecules in a biological system. Recently, we revealed a photoinduced absorption band shift of LSSmOrange, which was originally engineered to have a large energy gap between excitation and emission bands. Here, we evaluated the performance of LSSmOrange as a fluorescent tracer in living cells. The absorption maximum of LSSmOrange in HeLa cells shifted from 437 nm to 553 nm upon illumination with a 405-, 445-, 458-, or 488-nm laser on a laser-scanning microscope, whereas the emission band remained same (∼570 nm)...
September 6, 2016: Biophysical Journal
Stephen A Lee, Aleks Ponjavic, Chanrith Siv, Steven F Lee, Julie S Biteen
In recent years, single-molecule fluorescence imaging has been reconciling a fundamental mismatch between optical microscopy and subcellular biophysics. However, the next step in nanoscale imaging in living cells can be accessed only by optical excitation confinement geometries. Here, we review three methods of confinement that can enable nanoscale imaging in living cells: excitation confinement by laser illumination with beam shaping; physical confinement by micron-scale geometries in bacterial cells; and nanoscale confinement by nanophotonics...
September 27, 2016: ACS Nano
Xavier Pichon, Amandine Bastide, Adham Safieddine, Racha Chouaib, Aubin Samacoits, Eugenia Basyuk, Marion Peter, Florian Mueller, Edouard Bertrand
Translation is an essential step in gene expression. In this study, we used an improved SunTag system to label nascent proteins and image translation of single messenger ribonucleoproteins (mRNPs) in human cells. Using a dedicated reporter RNA, we observe that translation of single mRNPs stochastically turns on and off while they diffuse through the cytoplasm. We further measure a ribosome density of 1.3 per kilobase and an elongation rate of 13-18 amino acids per second. Tagging the endogenous POLR2A gene revealed similar elongation rates and ribosomal densities and that nearly all messenger RNAs (mRNAs) are engaged in translation...
September 12, 2016: Journal of Cell Biology
Minghai Chen, Sanying Liu, Wei Li, Zhiping Zhang, Xiaowei Zhang, Xian-En Zhang, Zongqiang Cui
Many cellular processes are governed by molecular machineries that involve multiple protein interactions. However, visualizing and identifying multiprotein complexes such as ternary complexes inside cells is always challenging, particularly in the subdiffraction cellular space. Here, we developed a three-fragment fluorescence complementation system (TFFC) based on the splitting of a photoactivatable fluorescent protein, mIrisFP, for the imaging of ternary complexes inside living cells. Using a combination of TFFC and photoactivated localization microscopy (PALM), namely, the TFFC-PALM technique, we are able to identify the multi-interaction of a ternary complex with nanometer-level spatial resolution and single-molecule sensitivity...
September 27, 2016: ACS Nano
Ayuko Sakane, Shin Yoshizawa, Masaomi Nishimura, Yuko Tsuchiya, Natsuki Matsushita, Kazuhisa Miyake, Kazuki Horikawa, Issei Imoto, Chiharu Mizuguchi, Hiroyuki Saito, Takato Ueno, Sachi Matsushita, Hisashi Haga, Shinji Deguchi, Kenji Mizuguchi, Hideo Yokota, Takuya Sasaki
In fundamental biological processes, cells often move in groups, a process termed collective cell migration. Collectively migrating cells are much better organized than a random assemblage of individual cells. Many molecules have been identified as factors involved in collective cell migration, and no one molecule is adequate to explain the whole picture. Here we show that JRAB/MICAL-L2, an effector protein of Rab13 GTPase, provides the "law and order" allowing myriad cells to behave as a single unit just by changing its conformation...
October 15, 2016: Molecular Biology of the Cell
Thomas G Baboolal, Gregory I Mashanov, Tatiana A Nenasheva, Michelle Peckham, Justin E Molloy
Myosin 10 is an actin-based molecular motor that localizes to the tips of filopodia in mammalian cells. To understand how it is targeted to this distinct region of the cell we have used total internal reflection fluorescence microscopy (TIRFM) to study the movement of individual full length and truncated GFP-tagged molecules. Truncation mutants lacking the motor region failed to localize to filopodial tips but still bound transiently at the plasma membrane. Deletion of the single alpha helical (SAH) and anti-parallel coiled-coil forming regions, which lie between the motor and PH domains, reduced the instantaneous velocity of intrafilopodial movement but did not affect the number of substrate adherent filopodia...
August 26, 2016: Journal of Biological Chemistry
Mathew Stracy, Marcin Jaciuk, Stephan Uphoff, Achillefs N Kapanidis, Marcin Nowotny, David J Sherratt, Pawel Zawadzki
Nucleotide excision repair (NER) removes chemically diverse DNA lesions in all domains of life. In Escherichia coli, UvrA and UvrB initiate NER, although the mechanistic details of how this occurs in vivo remain to be established. Here, we use single-molecule fluorescence imaging to provide a comprehensive characterization of the lesion search, recognition and verification process in living cells. We show that NER initiation involves a two-step mechanism in which UvrA scans the genome and locates DNA damage independently of UvrB...
2016: Nature Communications
Masashi Ohmachi, Yoshie Fujiwara, Shuki Muramatsu, Koji Yamada, Osamu Iwata, Kengo Suzuki, Dan Ohtan Wang
Single-cell transfection is a powerful technique for delivering chemicals, drugs, or probes into arbitrary, specific single cells. This technique is especially important when the analysis of molecular function and cellular behavior in individual microscopic organisms such as protists requires the precise identification of the target cell, as fluorescence labeling of bulk populations makes tracking of individual motile protists virtually impossible. Herein, we have modified current single-cell electroporation techniques for delivering fluorescent markers into single Euglena gracilis, a motile photosynthetic microalga...
November 2016: Journal of Microbiological Methods
Britta Barlag, Oliver Beutel, Dennis Janning, Frederik Czarniak, Christian P Richter, Carina Kommnick, Vera Göser, Rainer Kurre, Florian Fabiani, Marc Erhardt, Jacob Piehler, Michael Hensel
The investigation of the subcellular localization, dynamics and interaction of proteins and protein complexes in prokaryotes is complicated by the small size of the cells. Super-resolution microscopy (SRM) comprise various new techniques that allow light microscopy with a resolution that can be up to ten-fold higher than conventional light microscopy. Application of SRM techniques to living prokaryotes demands the introduction of suitable fluorescent probes, usually by fusion of proteins of interest to fluorescent proteins with properties compatible to SRM...
2016: Scientific Reports
Jens C Schmidt, Arthur J Zaug, Thomas R Cech
Telomerase maintains genome integrity by adding repetitive DNA sequences to the chromosome ends in actively dividing cells, including 90% of all cancer cells. Recruitment of human telomerase to telomeres occurs during S-phase of the cell cycle, but the molecular mechanism of the process is only partially understood. Here, we use CRISPR genome editing and single-molecule imaging to track telomerase trafficking in nuclei of living human cells. We demonstrate that telomerase uses three-dimensional diffusion to search for telomeres, probing each telomere thousands of times each S-phase but only rarely forming a stable association...
August 25, 2016: Cell
Nils Gustafsson, Siân Culley, George Ashdown, Dylan M Owen, Pedro Matos Pereira, Ricardo Henriques
Despite significant progress, high-speed live-cell super-resolution studies remain limited to specialized optical setups, generally requiring intense phototoxic illumination. Here, we describe a new analytical approach, super-resolution radial fluctuations (SRRF), provided as a fast graphics processing unit-enabled ImageJ plugin. In the most challenging data sets for super-resolution, such as those obtained in low-illumination live-cell imaging with GFP, we show that SRRF is generally capable of achieving resolutions better than 150 nm...
2016: Nature Communications
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