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live cell single molecule imaging

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
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
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
Elizabeth A Specht, Esther Braselmann, Amy E Palmer
Fluorescent tools have revolutionized our ability to probe biological dynamics, particularly at the cellular level. Fluorescent sensors have been developed on several platforms, utilizing either small molecule dyes or fluorescent proteins, to monitor proteins, RNA, DNA, small molecules, and even cellular properties, such as pH and membrane potential. We briefly summarize the impressive history of tool development for these various applications and then discuss the most recent noteworthy developments in more detail...
November 16, 2016: Annual Review of Physiology
Shenfei Zong, Xiaoyue Jiang, Zhuyuan Wang, Chen Chen, Ju Lu, Le Wang, Dan Zhu, Yiping Cui
Single molecule localization based super resolution imaging techniques (e.g. PALM/STORM) require that the employed optical nanoprobes possess signal fluctuations under certain excitations. Here, we present a Förster resonance energy transfer (FRET) based dual emission nanoprobe (denoted as FREDEN), which is suitable for PALM/STORM imaging. Basically, FREDEN is constructed by attaching Alexa Fluor 647 (A647) molecules onto semiconductor quantum dots (QDs). Dual emission is realized via FRET from QDs to A647...
December 7, 2016: Nanoscale
Chen Ji, Xuelin Lou
Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a determinant phosphoinositide of the plasma membrane (PM), and it is required to modulate ion channels, actin dynamics, exocytosis, endocytosis, intracellular signaling, and many other cellular processes. However, the spatial organization of PI(4,5)P2 in the PM is controversial, and its nanoscale distribution is poorly understood due to the technical limitations of research approaches...
October 15, 2016: Journal of Visualized Experiments: JoVE
Zhengjian Zhang, Brian P English, Jonathan B Grimm, Stephanie A Kazane, Wenxin Hu, Albert Tsai, Carla Inouye, Changjiang You, Jacob Piehler, Peter G Schultz, Luke D Lavis, Andrey Revyakin, Robert Tjian
Transcription of protein-encoding genes in eukaryotic cells requires the coordinated action of multiple general transcription factors (GTFs) and RNA polymerase II (Pol II). A "step-wise" preinitiation complex (PIC) assembly model has been suggested based on conventional ensemble biochemical measurements, in which protein factors bind stably to the promoter DNA sequentially to build a functional PIC. However, recent dynamic measurements in live cells suggest that transcription factors mostly interact with chromatin DNA rather transiently...
September 15, 2016: Genes & Development
Won-Ki Cho, Namrata Jayanth, Susan Mullen, Tzer Han Tan, Yoon J Jung, Ibrahim I Cissé
Live cell imaging of mammalian RNA polymerase II (Pol II) has previously relied on random insertions of exogenous, mutant Pol II coupled with the degradation of endogenous Pol II using a toxin, α-amanitin. Therefore, it has been unclear whether over-expression of labeled Pol II under an exogenous promoter may have played a role in reported Pol II dynamics in vivo. Here we label the endogenous Pol II in mouse embryonic fibroblast (MEF) cells using the CRISPR/Cas9 gene editing system. Using single-molecule based super-resolution imaging in the living cells, we captured endogenous Pol II clusters...
October 26, 2016: Scientific Reports
Jonathan B Grimm, Brian P English, Heejun Choi, Anand K Muthusamy, Brian P Mehl, Peng Dong, Timothy A Brown, Jennifer Lippincott-Schwartz, Zhe Liu, Timothée Lionnet, Luke D Lavis
Small-molecule fluorophores are important tools for advanced imaging experiments. We previously reported a general method to improve small, cell-permeable fluorophores which resulted in the azetidine-containing 'Janelia Fluor' (JF) dyes. Here, we refine and extend the utility of these dyes by synthesizing photoactivatable derivatives that are compatible with live-cell labeling strategies. Once activated, these derived compounds retain the superior brightness and photostability of the JF dyes, enabling improved single-particle tracking and facile localization microscopy experiments...
October 24, 2016: Nature Methods
Kalle Kipper, Ebba Gregorsson Lundius, Vladimir Curic, Ivana Nikic, Edward A Lemke, Manfred Wiessler, Johan Elf
Small synthetic fluorophores are in many ways superior to fluorescent proteins as labels for imaging. A major challenge is to use them for a protein-specific labeling in living cells. Here, we report on our use of noncanonical amino acids that are genetically encoded via the pyrrolysyl-tRNA/pyrrolysyl-RNA synthetase pair at artificially introduced TAG codons in a recoded E. coli strain. The strain is lacking endogenous TAG codons and the TAG-specific release factor RF1. The amino acids contain bioorthogonal groups that can be clicked to externally supplied dyes, thus enabling protein-specific labeling in live cells...
October 24, 2016: ACS Synthetic Biology
Timothy K Lee, Kevin Meng, Handuo Shi, Kerwyn Casey Huang
The peptidoglycan cell wall is an integral organelle critical for bacterial cell shape and stability. Proper cell wall construction requires the interaction of synthesis enzymes and the cytoskeleton, but it is unclear how the activities of individual proteins are coordinated to preserve the morphology and integrity of the cell wall during growth. To elucidate this coordination, we used single-molecule imaging to follow the behaviours of the two major peptidoglycan synthases in live, elongating Escherichia coli cells and after perturbation...
October 24, 2016: Nature Communications
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...
October 18, 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
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