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live cell single molecule imaging

Carlo Pellicciari
Histochemistry continues to be widely applied in biomedical research, being nowadays mostly addressed to detect and locate single molecules or molecular complexes inside cells and tissues, and to relate structural organization and function at the high resolution of the more advanced microscopical techniques. In the attempt to see whether histochemical novelties may be found in the recent literature, the articles published in the European Journal of Histochemistry in the period 2014-2016 have been reviewed. In the majority of the published papers, standardized methods have been preferred by scientists to make their results reliably comparable with the data in the literature, but  many papers (approximately one fourth of the published articles) described novel histochemical methods and procedures...
December 16, 2016: European Journal of Histochemistry: EJH
Adiki Raja Sekhar, Santhosh Kumar Sariki, R V Ramana Reddy, Alakesh Bisai, Pushpendra Kumar Sahu, Raghuvir S Tomar, Jeyaraman Sankar
The two first examples of zwitterionic BODIPYs have been synthesized via a simple SN-Ar methodology. The molecules exhibit excellent optical behavior, such as a large Stokes shift in solution and therefore a very intense emission, and can thus avoid self-quenching. The zwitterionic nature of the molecules was unambiguously elucidated using single crystal XRD studies. The electronic conjugation was investigated by NMR, DFT (NICS (0)) and XRD analysis. Due to their inherent ionic nature, their enhanced solubility in aqueous conditions was exploited for their utility in bio-imaging and cell viability studies...
January 5, 2017: Chemical Communications: Chem Comm
Stephanie Heinrich, Carina Patrizia Derrer, Azra Lari, Karsten Weis, Ben Montpetit
The transport of messenger RNAs (mRNAs) from the nucleus to cytoplasm is an essential step in the gene expression program of all eukaryotes. Recent technological advances in the areas of RNA-labeling, microscopy, and sequencing are leading to novel insights about mRNA biogenesis and export. This includes quantitative single molecule imaging (SMI) of RNA molecules in live cells, which is providing knowledge of the spatial and temporal dynamics of the export process. As this information becomes available, it leads to new questions, the reinterpretation of previous findings, and revised models of mRNA export...
January 4, 2017: BioEssays: News and Reviews in Molecular, Cellular and Developmental Biology
Adekunle T Bademosi, Elsa Lauwers, Pranesh Padmanabhan, Lorenzo Odierna, Ye Jin Chai, Andreas Papadopulos, Geoffrey J Goodhill, Patrik Verstreken, Bruno van Swinderen, Frédéric A Meunier
Syntaxin1A is organized in nanoclusters that are critical for the docking and priming of secretory vesicles from neurosecretory cells. Whether and how these nanoclusters are affected by neurotransmitter release in nerve terminals from a living organism is unknown. Here we imaged photoconvertible syntaxin1A-mEos2 in the motor nerve terminal of Drosophila larvae by single-particle tracking photoactivation localization microscopy. Opto- and thermo-genetic neuronal stimulation increased syntaxin1A-mEos2 mobility, and reduced the size and molecular density of nanoclusters, suggesting an activity-dependent release of syntaxin1A from the confinement of nanoclusters...
January 3, 2017: Nature Communications
Natalia V Klementieva, Anton I Pavlikov, Alexander A Moiseev, Nina G Bozhanova, Natalie M Mishina, Sergey A Lukyanov, Elena V Zagaynova, Konstantin A Lukyanov, Alexander S Mishin
Single-molecule localization microscopy relies on either controllable photoswitching of fluorescent probes or their robust blinking. We have found that blinking of monomeric red fluorescent proteins TagRFP, TagRFP-T, and FusionRed occurs at moderate illumination power and matches well with camera acquisition speed. It allows for super-resolution image reconstruction of densely labelled structures in live cells using various algorithms.
January 3, 2017: Chemical Communications: Chem Comm
Hiroyasu Hatakeyama, Yoshihito Nakahata, Hirokazu Yarimizu, Makoto Kanzaki
Quantum dots (QDs) are a powerful tool for quantitatively analyzing dynamic cellular processes by single-particle tracking. However, tracking of intracellular molecules with QDs is limited by their inability to penetrate the plasma membrane and bind to specific molecules of interest. Although several techniques for overcoming these problems have been proposed, they are either complicated or inconvenient. To address this issue, in this study, we developed a simple, convenient, and nontoxic method for labeling intracellular molecules in cells using HaloTag technology and electroporation...
January 1, 2017: Molecular Biology of the Cell
Byung Hun Lee, Seong-Woo Bae, Jaeyoun Jay Shim, Sung Young Park, Hye Yoon Park
Local protein synthesis mediates precise spatio-temporal regulation of gene expression for neuronal functions such as long-term plasticity, axon guidance and regeneration. To reveal the underlying mechanisms of local translation, it is crucial to understand mRNA transport, localization and translation in live neurons. Among various techniques for mRNA analysis, fluorescence microscopy has been widely used as the most direct method to study localization of mRNA. Live-cell imaging of single RNA molecules is particularly advantageous to dissect the highly heterogeneous and dynamic nature of messenger ribonucleoprotein (mRNP) complexes in neurons...
December 2016: Molecules and Cells
Francisco Balzarotti, Yvan Eilers, Klaus C Gwosch, Arvid H Gynnå, Volker Westphal, Fernando D Stefani, Johan Elf, Stefan W Hell
We introduce MINFLUX, a concept for localizing photon emitters in space. By probing the emitter with a local intensity minimum of excitation light, MINFLUX minimizes the fluorescence photons needed for high localization precision. A 22-fold reduction of photon detections over that required in popular centroid-localization is demonstrated. In superresolution microscopy, MINFLUX attained ~1-nm precision, resolving molecules only 6 nm apart. Tracking single fluorescent proteins by MINFLUX increased the temporal resolution and the number of localizations per trace by 100-fold, as demonstrated with diffusing 30S ribosomal subunits in living Escherichia coli As conceptual limits have not been reached, we expect this localization modality to break new ground for observing the dynamics, distribution, and structure of macromolecules in living cells and beyond...
December 22, 2016: Science
Shuya Kasai, Shinji Kajimoto, Yuma Ito, Tomo Saito, Ken-Ichi Yasumoto, Makio Tokunaga, Kumiko Sakata-Sogawa, Hiroshi Fukumura, Kazuhiro Sogawa
Inhibitory PAS domain protein (IPAS) is a dual function protein acting as a transcriptional repressor and as a pro-apoptotic protein. Simultaneous dual-color single-molecule imaging of EGFP-IPAS coexpressed with Mit-TagRFP-T in living HeLa cells revealed that fraction of EGFP-IPAS was arrested in the nucleus and on mitochondria. Transiently expressed Cerulean-IPAS in HEK293T cells was present in nuclear speckles when coexpressed with Citrine-HIF-1α or Citrine-HLF. Fluorescence lifetime imaging microscopy (FLIM) analysis of Citrine-IPAS-Cerulean in living CHO-K1 cells clarified the presence of intramolecular FRET...
December 21, 2016: Journal of Biochemistry
Yi Liao, Yilai Li, Jeremy W Schroeder, Lyle A Simmons, Julie S Biteen
PolC is one of two essential replicative DNA polymerases found in the Gram-positive bacterium Bacillus subtilis. The B. subtilis replisome is eukaryotic-like in that it relies on a two DNA polymerase system for chromosomal replication. To quantitatively image how the replicative DNA polymerase PolC functions in B. subtilis, we applied photobleaching-assisted microscopy, three-dimensional superresolution imaging, and single-particle tracking to examine the in vivo behavior of PolC at single-molecule resolution...
December 20, 2016: Biophysical Journal
Manjari Bhamidipati, Laura Fabris
In recent years we and others have become interested in evaluating the use of surface-enhanced Raman scattering (SERS)-tags for early cancer detection and in designing new approaches to demonstrate the applicability of this spectroscopic technique in the clinic. SERS-based imaging, in particular, offers ultra sensitivity up to the single molecule, multiplexing capability, increased photostability, and has been shown to outperform fluorescence. However, in order to employ SERS tags for early cancer detection, it is important to understand their interaction with cells and determine their cytotoxicity...
December 19, 2016: Bioconjugate Chemistry
Enrico Monachino, Lisanne M Spenkelink, Antoine M van Oijen
Single-molecule manipulation and imaging techniques have become important elements of the biologist's toolkit to gain mechanistic insights into cellular processes. By removing ensemble averaging, single-molecule methods provide unique access to the dynamic behavior of biomolecules. Recently, the use of these approaches has expanded to the study of complex multiprotein systems and has enabled detailed characterization of the behavior of individual molecules inside living cells. In this review, we provide an overview of the various force- and fluorescence-based single-molecule methods with applications both in vitro and in vivo, highlighting these advances by describing their applications in studies on cytoskeletal motors and DNA replication...
January 2, 2017: Journal of Cell Biology
Kazushi Suzuki, Taichi Kimura, Hajime Shinoda, Guirong Bai, Matthew J Daniels, Yoshiyuki Arai, Masahiro Nakano, Takeharu Nagai
Luminescence imaging has gained attention as a promising bio-imaging modality in situations where fluorescence imaging cannot be applied. However, wider application to multicolour and dynamic imaging is limited by the lack of bright luminescent proteins with emissions across the visible spectrum. Here we report five new spectral variants of the bright luminescent protein, enhanced Nano-lantern (eNL), made by concatenation of the brightest luciferase, NanoLuc, with various colour hues of fluorescent proteins...
December 14, 2016: Nature Communications
Katalin Czöndör, Olivier Thoumine
One of the difficulties for studying the mechanisms of synaptogenesis stems from the spatial unpredictability of contact formation between neurons, and the involvement of many parallel adhesive pathways mediating axon/dendrite recognition. To circumvent these limitations, we describe here a method allowing the investigation of synaptic contacts at controlled locations with high precision and statistics. Specifically, primary neurons are cultured on micropatterned substrates comprising arrays of micron-scale dots coated with purified synaptogenic adhesion molecules...
2017: Methods in Molecular Biology
Yoriko Lill, Lorne D Jordan, Chuck R Smallwood, Salete M Newton, Markus A Lill, Phillip E Klebba, Ken Ritchie
The important process of nutrient uptake in Escherichia coli, in many cases, involves transit of the nutrient through a class of beta-barrel proteins in the outer membrane known as TonB-dependent transporters (TBDTs) and requires interaction with the inner membrane protein TonB. Here we have imaged the mobility of the ferric enterobactin transporter FepA and TonB by tracking them in the membranes of live E. coli with single-molecule resolution at time-scales ranging from milliseconds to seconds. We employed simple simulations to model/analyze the lateral diffusion in the membranes of E...
2016: PloS One
Kai Wen Teng, Yuji Ishitsuka, Pin Ren, Yeoan Youn, Xiang Deng, Pinghua Ge, Andrew S Belmont, Paul R Selvin
Site-specific fluorescent labeling of proteins inside live mammalian cells has been achieved by employing Streptolysin O, a bacterial enzyme which forms temporary pores in the membrane and allows delivery of virtually any fluorescent probes, ranging from labeled IgG's to small ligands, with high efficiency (>85% of cells). The whole process, including recovery, takes 30 min, and the cell is ready to be imaged immediately. A variety of cell viability tests were performed after treatment with SLO to ensure that the cells have intact membranes, are able to divide, respond normally to signaling molecules, and maintains healthy organelle morphology...
December 9, 2016: ELife
Krishna Agarwal, Radek Macháň
Single-molecule localization techniques are restricted by long acquisition and computational times, or the need of special fluorophores or biologically toxic photochemical environments. Here we propose a statistical super-resolution technique of wide-field fluorescence microscopy we call the multiple signal classification algorithm which has several advantages. It provides resolution down to at least 50 nm, requires fewer frames and lower excitation power and works even at high fluorophore concentrations...
December 9, 2016: Nature Communications
Lu Chen, Simin Fang, Xianjin Xiao, Bo Zheng, Meiping Zhao
Cell penetrating peptides (CPPs) are very useful tools for delivery of DNA molecules into living cells without damaging the cell membranes. However, covalent conjugation of DNAs to CPPs is technically difficult, and the reactions between DNA and target nucleases are also liable to be affected by the cationic CPP molecules. In this work, we demonstrate that the electrostatic interactions between CPPs and single-stranded DNA (ssDNA) were stronger than those between CPP and double-stranded DNA (dsDNA). Taking advantage of this property, we developed an ssDNA protected CPP-DNA fluorescent probe which allowed for noninvasive and efficient cellular uptake and rapid imaging of target nucleases in living cells...
December 6, 2016: Analytical Chemistry
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
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