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Live cell imaging

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
Brennan S Dirk, Logan R Van Nynatten, Jimmy D Dikeakos
Viruses must continuously evolve to hijack the host cell machinery in order to successfully replicate and orchestrate key interactions that support their persistence. The type-1 human immunodeficiency virus (HIV-1) is a prime example of viral persistence within the host, having plagued the human population for decades. In recent years, advances in cellular imaging and molecular biology have aided the elucidation of key steps mediating the HIV-1 lifecycle and viral pathogenesis. Super-resolution imaging techniques such as stimulated emission depletion (STED) and photoactivation and localization microscopy (PALM) have been instrumental in studying viral assembly and release through both cell-cell transmission and cell-free viral transmission...
October 19, 2016: Viruses
Xiaofeng Wu, Lihong Li, Wen Shi, Qiuyu Gong, Huimin Ma
Fluorescence imaging of tyrosinase (a cancer biomarker) in living organisms is of great importance for biological studies. However, selective detection of tyrosinase remains a great challenge because current fluorescent probes that contain the 4-hydroxyphenyl moiety show similar fluorescence responses to both tyrosinase and some reactive oxygen species (ROS), thereby suffering from ROS interference. Herein, a new tyrosinase-recognition 3-hydroxybenzyloxy moiety, which exhibits distinct fluorescence responses for tyrosinase and ROS, is proposed...
October 24, 2016: Angewandte Chemie
Michael R Dent, Ismael López-Duarte, Callum J Dickson, Phoom Chairatana, Harry L Anderson, Ian R Gould, Douglas Wylie, Aurimas Vyšniauskas, Nicholas J Brooks, Marina K Kuimova
Molecular rotors have emerged as versatile probes of microscopic viscosity in lipid bilayers, although it has proved difficult to find probes that stain both phases equally in phase-separated bilayers. Here, we investigate the use of a membrane-targeting viscosity-sensitive fluorophore based on a thiophene moiety with equal affinity for ordered and disordered lipid domains to probe ordering and viscosity within artificial lipid bilayers and live cell plasma membranes.
October 24, 2016: Chemical Communications: Chem Comm
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
Ashley Page, David Perry, Philip Young, Daniel A Mitchell, Bruno G Frenguelli, Patrick R Unwin
A vast range of interfacial systems exhibit charge heterogeneities on the nanoscale. These differences in local surface charge density are challenging to visualize, but recent work has shown the scanning ion conductance microscope (SICM) to be a very promising tool to spatially resolve and map surface charge and topography via a hopping potential sweep technique with a single nanopipette probe, with harmonic modulation of a bias applied between quasi-reference counter electrodes in the nanopipette and bulk solution, coupled with lock-in detection...
October 24, 2016: Analytical Chemistry
Zhan Zhou, Yuhui Zheng, Cheng Zhang Cheng, Jiajia Wen, Qianming Wang
Here we developed the first case of pyropheophorbide-a-loaded PEGylated-hybrid carbon nanohorns (CNH-Pyro) to study tumor targeting therapy. During incubation with living cells, CNH-Pyro exhibited very intense red emissions. The intracellular imaging results were carried out by flow cytometry based on four different kinds of cell lines (including three adherent cell lines and one suspension cell line). Compared with free pyropheophorbide-a, CNH-Pyro demonstrated enhanced photodynamic tumor ablation efficiency during in vitro experiments due to improved biocompatibility of the hybrid nanomaterial and the photothermal therapy effect derived from carbon-network structure...
January 1, 2017: Materials Science & Engineering. C, Materials for Biological Applications
Shoujie Liu, Hejun Li, Yangyang Su, Qian Guo, Leilei Zhang
Carbon nanotubes (CNTs) possess excellent mechanical properties for their role playing in reinforcement as imparting strength to brittle hydroxyapatite (HA) bioceramic coating. However, there are few reports relating to the in-situ grown carbon nanotubes reinforced hydroxyapatite (CNTs-HA) coating. Here we demonstrate the potential application in reinforcing biomaterials by an attempt to use in-situ grown of CNTs strengthen HA coating, using a combined method composited of injection chemical vapor deposition (ICVD) and pulsed electrodeposition...
January 1, 2017: Materials Science & Engineering. C, Materials for Biological Applications
Quan-Xing Mao, Lu Han, Yang Shu, Xu-Wei Chen, Jian-Hua Wang
In the practice of in vivo imaging with carbon nanodots (CNDs) as probe, the volume of CNDs solution introduced into living body should be kept at minimum, and a higher concentration is needed to ensure sufficient quantity of the probe for obtaining bright image. Therefore, the improvement on biocompatibility of the CNDs is among the most important and critical issues. We report herein the improvement on the biocompatibility of CNDs with modification by ionic liquid. Amide group functionalization of carbon nanodots is first conducted through microwave irradiation, followed by coupling the ionic liquid 1-carboxymethyl-3-methyl imidazolium bromide on the surface of the Amide-CNDs via covalent conjunction to produce the modified carbon nanodots (IL-CNDs)...
December 1, 2016: Talanta
Haiyang Liu, Bibo Zhang, Chunyan Tan, Feng Liu, Jiakun Cao, Ying Tan, Yuyang Jiang
A simple Schiff base (BMSA) prepared from salicylaldehyde and 2-(1H-benzo[d]imidazol-2-yl)aniline was evaluated as an efficient fluorescent chemosensor for the selective recognition of Al(3+)and Cu(2+) over other common metal ions. This sensor could detect Al3(+) in CH3OH/PBS with distinct emission red-shift (the detection limit 0.31μM)and Cu(2+)in CH3OH/Tris-HCL (the detection limit 0.54μM) with obvious fluorescence quenching. The obtained BMSA-Al(3+) and BMSA-Cu(2+) complexes could act as cascade sensors for detecting F(-) and S(2-), respectively...
December 1, 2016: Talanta
Ana Rita Araujo, Lendert Gelens, Rahuman S M Sheriff, Silvia D M Santos
Cell division is characterized by a sequence of events by which a cell gives rise to two daughter cells. Quantitative measurements of cell-cycle dynamics in single cells showed that despite variability in G1-, S-, and G2 phases, duration of mitosis is short and remarkably constant. Surprisingly, there is no correlation between cell-cycle length and mitotic duration, suggesting that mitosis is temporally insulated from variability in earlier cell-cycle phases. By combining live cell imaging and computational modeling, we showed that positive feedback is the molecular mechanism underlying the temporal insulation of mitosis...
October 20, 2016: Molecular Cell
Vimal Prabhu Pandiyan, Kedar Khare, Renu John
No abstract text is available yet for this article.
October 1, 2016: Journal of Biomedical Optics
Liangliang Chen, Yuancheng Peng, Juan Tian, Xiaohong Wang, Zhaosheng Kong, Tonglin Mao, Ming Yuan, Yunhai Li
How cell shape is controlled is a fundamental question in developmental biology, but the genetic and molecular mechanisms that determine cell shape are largely unknown. Arabidopsis trichomes have been used as a good model system to investigate cell shape at the single-cell level. Here we describe the trichome cell shape 1 (tcs1) mutants with the reduced trichome branch number in Arabidopsis. TCS1 encodes a coiled-coil domain-containing protein. Pharmacological analyses and observations of microtubule dynamics show that TCS1 influences the stability of microtubules...
October 2016: PLoS Genetics
Allegra T Aron, Morten O Loehr, Jana Bogena, Christopher J Chang
Iron is essential for sustaining life, as its ability to cycle between multiple oxidation states is critical for catalyzing chemical transformations in biological systems. However, without proper regulation, this same redox capacity can trigger oxidative stress events that contribute to aging along with diseases ranging from cancer to cardiovascular and neurodegenerative disorders. Despite its importance, methods for monitoring biological iron bound weakly to cellular ligands-the labile iron pool-to generate a response that preserves spatial and temporal information remain limited, owing to the potent fluorescence quenching ability of iron...
October 21, 2016: Journal of the American Chemical Society
Tao Peng, Howard C Hang
Over the past years, fluorescent proteins (e.g., green fluorescent proteins) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large sizes and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small-molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells...
October 21, 2016: Journal of the American Chemical Society
Hao Zhu, Jiangli Fan, Huiying Mu, Tao Zhu, Zhen Zhang, Jianjun Du, Xiaojun Peng
Polarity-sensitive fluorescent probes are powerful chemical tools for studying biomolecular structures and activities both in vitro and in vivo. However, the lack of "off-on" polarity-sensing probes has limited the accurate monitoring of biological processes that involve an increase in local hydrophilicity. Here, we design and synthesize a series of "off-on" polarity-sensitive fluorescent probes BP series consisting of the difluoroboron dippyomethene (BODIPY) fluorophore connected to a quaternary ammonium moiety via different carbon linkers...
October 21, 2016: Scientific Reports
Kwang-Youn Kim, Hyun-Jun Jang, Yong Ryul Yang, Kwang-Il Park, JeongKon Seo, Il-Woo Shin, Tae-Il Jeon, Soon-Cheol Ahn, Pann-Ghill Suh, Timothy F Osborne, Young-Kyo Seo
Dysregulated autophagy is associated with steatosis and non-alcoholic fatty liver disease (NAFLD), however the mechanisms connecting them remain poorly understand. Here, we show that co-administration of lovastatin and ezetimibe (L/E) significantly reverses hepatic triglyceride accumulation concomitant with an increase in SREBP-2 driven autophagy in mice fed a high-fat diet (HFD). We further show that the statin mediated increase in SREBP-2 directly activates expression of patatin-like phospholipase domain-containing enzyme 8 (PNPLA8) gene, and PNPLA8 associates with autophagosomes and is associated with a decrease in cellular triglyceride...
October 21, 2016: Scientific Reports
Sahishna Phaniraj, Zhe Gao, Digamber Rane, Blake R Peterson
The endoplasmic reticulum (ER) of eukaryotic cells plays critical roles in the processing of secreted and transmembrane proteins. Defects in these functions are associated with a wide range of pathologies. To image this organelle, cells are often treated with fluorescent ER-Tracker dyes. Although these compounds are selective, existing red fluorescent probes of the ER are costly glibenclamide derivatives that inhibit ER-associated sulphonylurea receptors. To provide simpler and more cost-effective red fluorescent probes of the ER, we synthesized amino analogues of the fluorophore resorufin...
December 2016: Dyes and Pigments: An International Journal
Teresa Delgado-Goni, Maria Falck Miniotis, Slawomir Wantuch, Harold G Parkes, Richard Marais, Paul Workman, Martin O Leach, Mounia Beloueche-Babari
Understanding the impact of BRAF signaling inhibition in human melanoma on key disease mechanisms is important for developing biomarkers of therapeutic response and combination strategies to improve long term disease control. This work investigates the downstream metabolic consequences of BRAF inhibition with vemurafenib, the molecular and biochemical processes that underpin them, their significance for antineoplastic activity and potential as non-invasive imaging response biomarkers.(1)H NMR spectroscopy showed that vemurafenib decreases the glycolytic activity of BRAF mutant (WM266...
October 7, 2016: Molecular Cancer Therapeutics
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