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live cell single molecule imaging

Hong Huang, Fangyuan Dong, Yang Tian
Intracellular pH undertakes critical functions in the formation of a proton gradient and electrochemical potential that drives the adenosine triphosphate synthesis. It is also involved in various metabolic processes occurring in mitochondria, such as the generation of reactive oxygen species, calcium regulation, as well as the triggering of cell proliferation and apoptosis. Meanwhile, the aberrant accumulation of O2(•-) within mitochondria is frequently intertwined with mitochondrial dysfunction and disease development...
December 20, 2016: Analytical Chemistry
Navneet C Verma, Syamantak Khan, Chayan K Nandi
The advancement of high-resolution bioimaging has always been dependent on the discovery of bright and easily available fluorescent probes. Fluorescent carbon nanodots, an interesting class of relatively new nanomaterials, have emerged as a versatile alternative due to their superior optical properties, non-toxicity, cell penetrability and easy routes to synthesis. Although a plethora of reports is available on bioimaging using carbon dots, single-molecule-based super-resolution imaging is rare in the literature...
October 11, 2016: Methods and Applications in Fluorescence
Chanda Kumari, Dibyendu Sain, Ashish Kumar, Sushanta Debnath, Partha Saha, Swapan Dey
A new coumarin based turn on fluorescent sensor (R1) was reported for the detection of highly hazardous Cd(2+) with excellent selectivity and sensitivity without any interference of other metal ions. The single crystal X-ray structure analysis of the sensor showed the actual geometry of the molecule. For the first time, a Cd(2+) induced FRET mechanism was observed and explained accordingly. Instant naked eye detection of Cd(2+) through a sharp colour change signified the practical applicability of R1. The sensor of high quantum yield was applied in the intracellular detection of poisonous Cd(2+) in living HeLa S3 cells...
February 3, 2017: Dalton Transactions: An International Journal of Inorganic Chemistry
Lokesh Agrawal, Satyajit Sahu, Subrata Ghosh, Takashi Shiga, Daisuke Fujita, Anirban Bandyopadhyay
A substantial ion flow in a normally wet protein masks any other forms of signal transmission. We use hysteresis and linear conduction (both are artifacts) as a marker to precisely wet a protein, which restricts the ionic conduction (hysteresis disappears), and at the same time, it is not denatured (quantized conductance and Raman spectra are intact). Pure electric visualization of proteins at work by eliminating the screening of ions, electrons, would change the way we study biology. Here we discuss the technical challenges resolved for imaging a protein or live cell using nonlinear dielectric response (spatial distribution of conductance, capacitance and phase, GCP trio)...
January 31, 2017: Journal of Integrative Neuroscience
Jun Lao, Hua He, Xiaojuan Wang, Zhencai Wang, Yanzhuo Song, Bin Yang, Naseer Ullahkhan, Baosheng Ge, Fang Huang
The role of dimerization and oligomerization of G-protein-coupled receptors in their signal transduction is highly controversial. Delineating this issue can greatly facilitate rational drug design. With single-molecule imaging, we show that chemokine receptor CXCR4 exists mainly as a monomer in normal mammalian living cells and forms dimers and higher-order oligomers at a high expression level, such as in cancer cells. Chemotaxis tests demonstrate that the signal transduction activity of CXCR4 does not depend only on its expression level, indicating a close relation with the oligomeric status of CXCR4...
February 9, 2017: Journal of Physical Chemistry. B
Kristine O Vasquez, Jeffrey D Peterson
Hepatocellular and cholestatic forms of drug induced liver injury (DILI) are major reasons for late stage termination of small molecule drug discovery research projects. Biochemical serum markers are limited in their ability to sensitively and specifically detect both of these common DILI forms in preclinical models, and tissue-specific approaches to assessing this are labor intensive, requiring extensive animal dosing, tissue preparation, and pathology assessment. In vivo fluorescent imaging offers non-invasive detection of biological changes detected directly in the livers of living animals...
January 23, 2017: Journal of Pharmacology and Experimental Therapeutics
Guoliang Liu, Jingjing Li, Da-Qian Feng, Jun-Jie Zhu, Wei Wang
An activatable silver nanoclusters beacon (ASNCB) was synthesized through a facile one-pot approach and applied for multiplex DNAs, small molecule, and protein sensing. Multifunctional single-stranded DNA sequences are rationally designed and used for ASNCB in situ synthesis. Via target-responsive structure transformation of ASNCB, target recognition induced ASNCB conformational transition and lit up the fluorescent signal of silver nanoclusters. By further implementing two different color ASNCBs (520 and 600 nm), the parallel multiplexed analysis of two target genes (Influenza A virus genes H1N1 and H5N1) is achieved...
January 3, 2017: Analytical Chemistry
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, and increased photostability and has been shown to outperform fluorescence. However, to employ SERS tags for early cancer detection, it is important to understand their interaction with cells and determine their cytotoxicity...
January 6, 2017: 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
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