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Fluorescent imaging

Kevin M Dean, Philippe Roudot, Erik S Welf, Theresa Pohlkamp, Gerard Garrelts, Joachim Herz, Reto Fiolka
In fluorescence microscopy, the serial acquisition of 2D images to form a 3D volume limits the maximum imaging speed. This is particularly evident when imaging adherent cells in a light-sheet fluorescence microscopy format, as their elongated morphologies require ~200 image planes per image volume. Here, by illuminating the specimen with three light-sheets, each independently detected, we present a light-efficient, crosstalk free, and volumetrically parallelized 3D microscopy technique that is optimized for high-speed (up to 14 Hz) subcellular (300 nm lateral, 600 nm axial resolution) imaging of adherent cells...
February 20, 2017: Optica
Katterine Salazar, Fernando Martínez, Margarita Pérez-Martín, Manuel Cifuentes, Laura Trigueros, Luciano Ferrada, Francisca Espinoza, Natalia Saldivia, Romina Bertinat, Katherine Forman, María José Oviedo, Antonio J López-Gambero, Christian Bonansco, Ernesto R Bongarzone, Francisco Nualart
Ascorbic acid (AA), the reduced form of vitamin C, acts as a neuroprotector by eliminating free radicals in the brain. Sodium/vitamin C co-transporter isoform 2 (SVCT2) mediates uptake of AA by neurons. It has been reported that SVCT2 mRNA is induced in astrocytes under ischemic damage, suggesting that its expression is enhanced in pathological conditions. However, it remains to be established if SVCT expression is altered in the presence of reactive astrogliosis generated by different brain pathologies. In the present work, we demonstrate that SVCT2 expression is increased in astrocytes present at sites of neuroinflammation induced by intracerebroventricular injection of a GFP-adenovirus or the microbial enzyme, neuraminidase...
September 23, 2017: Molecular Neurobiology
Zhao Ma, Xiaotian Wu, Christopher J Krueger, Antony K Chen
With numerous advancements in novel biochemical techniques, our knowledge of the role of RNAs in the regulation of cellular physiology and pathology has grown significantly over the past several decades. Nevertheless, detailed information regarding RNA processing, trafficking, and localization in living cells has been lacking due to technical limitations in imaging single RNA transcripts in living cells with high spatial and temporal resolution. In this review, we discuss techniques that have shown great promise for single RNA imaging, followed by highlights in our recent work in the development of molecular beacons (MBs), a class of nanoscale oligonucleotide-probes, for detecting individual RNA transcripts in living cells...
September 20, 2017: Genomics, Proteomics & Bioinformatics
Xiaoyun Wang, Yaowen Lai, Xiaojian Zhang, Jie Zhao
OBJECTIVES: Vibroacousitic disease (VAD) is caused by excessive exposure to low-frequency but high-intensity noise. The integrity of the brain blood barrier (BBB) is essential for the brain. The study aimed to investigate the effect of noise exposure on the BBB. METHODS: Healthy male Bama swine were exposed to 50, 70, 100, and 120Hz, 140dB noise for 30min. After exposure, CT brain imaging and ex vivo fluorescent imaging of parenchymal EB leakage were performed (each group consisted of N=3 swine)...
September 20, 2017: Neuroscience Letters
M A Ahmed, Z M Abou-Gamra, H A A Medien, M A Hamza
As known, porphyrins have central role in photosynthesis, biological oxidation and reduction and oxygen transport beside to their intensive color which qualify them to be good photosensitizers. Herein, tetra (4-carboxyphenyl) porphyrin (TCPP) was prepared by a simple one-pot synthesis to use as a visible antenna for TiO2 nanoparticles that were prepared via a simple template-free sol-gel method. Various loading percentages of TCPP (0.05-1%) were incorporated on the surface of TiO2 as photosensitizer for photocatalytic degradation of Rhodamine B (Rh B) dye as a primary cationic pollutant model...
September 18, 2017: Journal of Photochemistry and Photobiology. B, Biology
Na Mi Lee, Soo Ahn Chae, Hong Jun Lee
Neonatal hypoxic-ischemic brain injuries cause serious neurological sequelae, yet there is currently no effective treatment for them. We hypothesized that neurotrophic factors released into the medium by stem cells could supply hypoxia-damaged organotypic hippocampal slice cultures with regenerative abilities. We prepared organotypic slice cultures of the hippocampus of 7-day-old Sprague-Dawley rats based on the modified Stoppini method; slices were cultured for 14 days in vitro using either Gahwiler's medium (G-medium) or stem cell-conditioned medium (S-medium) as culture medium...
September 20, 2017: Brain Research
Chenghui Liu, Desheng Chen, Xiaobo Zhang, Liping Zhu, Zhengping Li
A new concept of single microbead (MB)-anchored fluorescent immunoassay (SMFIA) is proposed with greatly improved sensitivity. In the SMFIA, a single MB is manipulated as the reaction carrier so that the target-tethered fluorescent immunocomplexes will be highly concentrated on one MB. By monitoring the enriched fluorescence signal on the single MB through imaging, highly sensitive target quantification can be realized just by employing the most common sandwich immunoreactions without requirement of further signal amplification routes...
September 20, 2017: Chemistry, An Asian Journal
Yizhou Zhou, David Shaw, Cynthia Lam, Joni Tsukuda, Mandy Yim, Danming Tang, Salina Louie, Michael W Laird, Brad Snedecor, Shahram Misaghi
Establishing that a cell line was derived from a single cell progenitor and defined as clonally-derived for the production of clinical and commercial therapeutic protein drugs has been the subject of increased emphasis in cell line development (CLD). Several regulatory agencies have expressed that the prospective probability of clonality for CHO cell lines is assumed to follow the Poisson distribution based on the input cell count. The probability of obtaining monoclonal progenitors based on the Poisson distribution of all cells suggests that one round of limiting dilution may not be sufficient to assure the resulting cell lines are clonally-derived...
September 23, 2017: Biotechnology Progress
Tianyu Hu, Qiufeng Xu, Wei Lv, Qian Liu
Neuronal soma segmentation is essential for morphology quantification analysis. Rapid advances in light microscope imaging techniques have generated such massive amounts of data that time-consuming manual methods cannot meet requirements for high throughput. However, touching soma segmentation is still a challenge for automatic segmentation methods. In this paper, we propose a soma segmentation method that combines the Rayburst sampling algorithm and ellipsoid fitting. The improved Rayburst sampling algorithm is used to detect the soma surface; the ellipsoid fitting method then refines jagged sampled soma surface to generate smooth ellipsoidal shapes for efficient analysis...
September 22, 2017: Neuroinformatics
Małgorzata Grosbart, Dejan Ristić, Humberto Sánchez, Claire Wyman
Direct imaging is invaluable for understanding the mechanism of complex genome transactions where proteins work together to organize, transcribe, replicate and repair DNA. Scanning (or atomic) force microscopy is an ideal tool for this, providing 3D information on molecular structure at nm resolution from defined components. This is a convenient and practical addition to in vitro studies as readily obtainable amounts of purified proteins and DNA are required. The images reveal structural details on the size and location of DNA bound proteins as well as protein-induced arrangement of the DNA, which are directly correlated in the same complexes...
2018: Methods in Molecular Biology
Roderick P Tas, Trusanne G A A Bos, Lukas C Kapitein
The cytoskeleton is involved in many cellular processes. Over the last decade, super-resolution microscopy has become widely available to image cytoskeletal structures, such as microtubules and actin, with great detail. For example, Single-Molecule Localization Microscopy (SMLM) achieves resolutions of 5-50 nm through repetitive sparse labeling of samples, followed by Point-Spread-Function analysis of individual fluorophores. Whereas initially this approach depended on the controlled photoswitching of fluorophores targeted to the structure of interest, alternative techniques now depend on the transient binding of fluorescently labeled probes, such as the small polypeptide lifeAct that can transiently interact with polymerized actin...
2018: Methods in Molecular Biology
Jaap van Krugten, Erwin J G Peterman
Transportation of organelles and biomolecules is vital for many cellular processes. Single-molecule (SM) fluorescence microscopy can expose molecular aspects of the dynamics that remain unresolved in ensemble experiments. For example, trajectories of individual, moving biomolecules can reveal velocity and changes therein, including pauses. We use SM imaging to study the dynamics of motor proteins and their cargo in the cilia of living C. elegans. To this end, we employ standard fluorescent proteins, an epi-illuminated, wide-field fluorescence microscope and mostly open-source software...
2018: Methods in Molecular Biology
Aravindan Varadarajan, Felix Oswald, Yves J M Bollen
Single-molecule imaging in living cells can provide unique information about biological processes. Bacteria offer some particular challenges for single-molecule imaging due to their small size, only slightly larger than the diffraction limit of visible light. Here, we describe how reliable and reproducible single-molecule data can be obtained for a transmembrane protein in the Gram-negative bacterium Escherichia coli by using live-cell fluorescence microscopy. Fluorescent labeling of a protein by genetic fusion, cell culturing, sample preparation, imaging, and data analysis are discussed...
2018: Methods in Molecular Biology
Lingling Chen, Guiye Li, Yamin Li, Yingchao Li, Haiou Zhu, Li Tang, Paul French, James McGinty, Shuangchen Ruan
Optical clearing methods can facilitate deep optical imaging in biological tissue by reducing light scattering and this has enabled accurate three-dimensional signal visualization and quantification of complex biological structures. Unfortunately, existing optical clearing approaches present a compromise between maximizing clearing capability, the preservation of fluorescent protein emission and membrane integrity and the speed of sample processing - with the latter typically requiring weeks for cm scale tissue samples...
September 22, 2017: Scientific Reports
Daryll Vanover, Daisy V Smith, Emmeline L Blanchard, Eric Alonas, Jonathan L Kirschman, Aaron W Lifland, Chiara Zurla, Philip J Santangelo
The human respiratory syncytial virus G protein plays an important role in the entry and assembly of filamentous virions. Here, we report the use of fluorescently labeled soybean agglutinin to selectively label the respiratory syncytial virus G protein in living cells without disrupting respiratory syncytial virus infectivity or filament formation and allowing for interrogations of respiratory syncytial virus virion assembly. Using this approach, we discovered that plasma membrane-bound respiratory syncytial virus G rapidly recycles from the membrane via clathrin-mediated endocytosis...
September 22, 2017: Nature Communications
Gabriela Kania, Magdalena Sternak, Agnieszka Jasztal, Stefan Chlopicki, Agnieszka Błażejczyk, Anna Nasulewicz-Goldeman, Joanna Wietrzyk, Krzysztof Jasiński, Tomasz Skórka, Szczepan Zapotoczny, Maria Nowakowska
Bioreactivity of superparamagnetic iron oxide nanoparticles (SPION) coated with thin layers of either cationic or anionic chitosan derivatives and serving as contrast agents in magnetic resonance imaging (MRI) was studied in vivo using BALB/c mouse model. Synthesized dual-modal fluorescing SPION were tracked in time using both fluorescent imaging and MRI. Although SPION started to be excreted by kidneys relatively shortly after administration they were uptaken by liver enhancing MRI contrast even up to 7 days...
September 19, 2017: Nanomedicine: Nanotechnology, Biology, and Medicine
Giampaolo Di Martino, Claudio Reato, Debora Verri, Federica Dell'Orto, Alessandro Buda
STUDY OBJECTIVE: To present our minimally invasive laparoscopic approach for sentinel lymph node (SLN) mapping with indocyanine green (ICG) fluorescence using with two florescence systems. DESIGN: step by step video description of the technique showing the most frequent typical and atypical location of SLN's (educational video). SETTING: Lymph node staging in apparent confined endometrial cancer. PATIENTS: women underwent sentinel node mapping in a minimally invasive setting...
September 19, 2017: Journal of Minimally Invasive Gynecology
Magda Białkowska, Irena Deperasińska, Artur Makarewicz, Bolesław Kozankiewicz
Highly terrylene doped single crystals of p-terphenyl, obtained by co-sublimation of both components, showed bright spots in the confocal fluorescence images. Polarization of the fluorescence excitation spectra, blinking and bleaching, and saturation behavior allowed us to attribute them to single molecules of terrylene anomalously embedded between two neighbor layers of the host crystal, in the (a,b) plane. Such an orientation of terrylene molecules results in much more efficient absorption and collection of the fluorescence photons than in the case of previously investigated molecules embedded in the substitution sites...
September 21, 2017: Journal of Chemical Physics
Melanie L Sutton-McDowall, Martin Gosnell, Ayad G Anwer, Melissa White, Malcolm Purdey, Andrew D Abell, Ewa M Goldys, Jeremy G Thompson
STUDY QUESTION: Can we separate embryos cultured under either 7% or 20% oxygen atmospheres by measuring their metabolic heterogeneity? SUMMARY ANSWER: Metabolic heterogeneity and changes in metabolic profiles in morula exposed to two different oxygen concentrations were not detectable using traditional fluorophore and two-channel autofluorescence but were detectable using hyperspectral microscopy. WHAT IS KNOWN ALREADY: Increased genetic and morphological blastomere heterogeneity is associated with compromised developmental competence of embryos and currently forms the basis for embryo scoring within the clinic...
October 1, 2017: Human Reproduction
Mingrong Cheng, Weiping Zhu, Qing Li, Dejian Dai, Yiming Hou
The present study investigated the synthesis of biotinylated chitosan (Bio-CS) from chitosan using a nanomaterial skeleton with biotin and the successful targeting of the formulation in liver cancer cells. Bio-CS was validated by fourier transformed infrared spectroscopy and hydrogen(-1) nuclear magnetic resonance spectroscopy. Bio-CS and plasmid DNA were used to construct Bio-CS/plasmid DNA nanoparticles according to the optimal molar ratio of 1:1 and the optimal pH-value of 5.5. Under these conditions, the parameters mean particle size, potential, encapsulation rate and drug loading, were 82...
August 29, 2017: Oncotarget
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