Genetic analysis of the embryo
David K. Gardner, Ariel Weissman, Colin M. Howles, Zeev Shoham in Textbook of Assisted Reproductive Techniques, 2017
The PCR products are commonly separated by gel electropho- resis, and their migration depends chiefly on their size. The standard visualization techniques include radioactive label- ing, ethidium bromide, or silver staining. These techniques are rather insensitive, requiring a relatively large amount of DNA. Moreover, they cannot distinguish between products of a relatively similar size, nor provide an adequate esti- mate of quantity. Fluorescent PCR employs primers tagged with a fluorescent dye, which label the resulting amplicons, enabling detection by florescence-based DNA sequencers using a module such as GeneScan™ (Applied Biosystems- Thermo Fisher Scientific Inc., Waltham, MA, USA). A laser beam scans the acrylamide gel as the fluorescent products pass across the laser path by means of electrophoresis. The different fluorescent dyes absorb the light at a particular wavelength and emit fluorescence at a different wavelength. The emitted light passes through a filter, is digitally ampli- fied, and is analyzed by a computer. With this technique, it is possible to separate, detect, and analyze the fluorescent- labeled PCR products with sensitivity that is 1000-times greater than that achieved using conventional methods (28). This method also has a higher fragment-size resolution and is able to distinguish between products having a size differ- ence of even 1-2 bp. Thus, several primer sets can be multi- plexed even if their product sizes only vary slightly.
Crystallization of the Light-Harvesting Chlorophyll A/B Protein Complex from Chloroplast Membranes
Hartmut Michel in Crystallization of Membrane Proteins, 1991
Small crystals appeared occasionally within minutes, but more usually overnight, and continued to grow for several days or weeks to a maximum size of 0.5 to 0.7 mm (body diagonal) at temperatures ranging from 4° to room temperature. The largest specimens formed at 16 to 18°C, at which temperature they seemed to be stable for several months. Octahedral crystals were obtained using phosphate buffers ranging from pH 4.5 to pH 9.5 without any apparent effect on crystal shape, although crystals grown at pH 6 to pH 7 tended to be larger. They were very dark green or black and showed a faint, isotropic red fluorescence when examined in incandescent white light. Crystals frequently appeared triangular or hexagonal in outline (Figure 3), having shapes that derived from regular octahedra which indicated growth in the (1,1,1) direction. Fully developed octahedra were comparatively rare. Crystals had sharp edges and plane, well-developed faces. They were not birefringent.
Applications of Fluorescence Techniques to Automated Biological Analysis
R. Michael Gendreau in Spectroscopy in the Biomedical Sciences, 1986
One of the problems which has limited the acceptance of fluorescence techniques in the clinical laboratory has been the negative experiences many clinical chemists encountered with contamination in the early use of fluorescence techniques. The system described is able to eliminate such contamination problems in several ways, principally by using kinetic methods of analysis. Extensive method comparisons have been conducted against RIA methods to assess potential interferences. Figure 6 shows an example of a digoxin comparison between the Stratus® and an RIA system. This comparison is quite good. Additional comparisons have been conducted with other types of assays and none of the contamination problems experienced in the past have been apparent.
Universal existence of fluorescent carbon dots in beer and assessment of their potential toxicity
Published in Nanotoxicology, 2019
Haitao Wang, Shan Liu, Yukun Song, Bei-Wei Zhu, Mingqian Tan
Male BALB/c mice (18–22g) were purchased from Dalian Medical University. Approved protocols from the Institutional Animal Care and Use Committee at Dalian Polytechnic University were followed for all animal procedures. The mice were given 2 g kg−1 of CDs from Snow beer by a single oral administration to evaluate their biodistribution. The control group mice were orally administered a 0.9% NaCl solution. Mice were sacrificed at 0.5, 2, 6, 24 h after the CDs administration. The major organs were collected and imaged on a Multi-functional in vivo imaging system (Molecular Devices, San Jose, CA). The fluorescent intensity of organs under the excitation of 365 nm wavelength was recorded and shown as pseudocolor image. The fluorescence intensity was visualized by a different color. White-red indicates high fluorescence intensity, whereas blue-purple represents a weak intensity. Then these organ’s tissues were sectioned into slices for histological analysis. For acute oral toxicity investigate, mice were sacrificed on day 1 post-exposure. The control group mice were orally administrated a 0.9% NaCl and glucose-CDs of 2 g kg−1 body weight. The physiological and biochemical indexes were measured by using ADVIA 2400 Chemistry System (Siemens, Munich, Germany). Hematoxylin–eosin (H–E) staining of the major organs/tissue was carried out following the standard protocol.
Genetic analysis of KillerRed in C. elegans identifies a shared role of calcium genes in ROS-mediated neurodegeneration
Published in Journal of Neurogenetics, 2019
Lyndsay E. A. Young, Chelsea Shoben, Kyra Ricci, Daniel C. Williams
To test whether ROS production by KillerRed is affected by genetic background, we measured fluorescence of an ROS sensing molecular probe. Animals expressing KillerRed in the GABA neurons were treated with 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA), which is a membrane-permeant reporter that is converted to the fluorescent dichlorofluorescein (DCF) when oxidized by ROS (Labuschagne & Brenkman, 2013). Illumination of treated animals resulted in green fluorescence that overlapped with KillerRed fluorescence (Figure 5(A)). The intensity of fluorescence increased with longer illumination times. Notably, tissues that surround the GABA neurons, including other neuronal cell bodies along the ventral cord, did not have any detectable DCF fluorescence. This demonstrates that ROS production is dependent on both KillerRed and illumination and is consistent with the absence of neurodegeneration in illuminated animals that do not express KillerRed. We measured DCF fluorescence intensity in neuronal cell bodies of wild-type and mutant animals after illumination. The amount of DCF fluorescence was similar in all genetic backgrounds tested (Figure 5(B), bottom). Collectively, these data demonstrate that itr-1 or crt-1 does not affect KillerRed activity, and therefore indicate the suppression of neurodegeneration in these genetic backgrounds is not due to reduced ROS production.
Fluorescent melamine-formaldehyde/polyamine coatings for microcapsules enabling their tracking in composites
Published in Journal of Microencapsulation, 2022
Christian Neumann, Sophia Rosencrantz, Andreas Schmohl, Latnikova Alexandra
It is reasonable to assume that fluorescent Schiff base (Chio and Tappel 1969) structures are formed, since polycondensates made from aldehydes and primary amines are reported to absorb in the UV range (Veberg et al.2006). However, for acid catalysed polycondensation of MF resins, the formation of methylene bridges is much more favoured. Furthermore, Schiff base structures would also be formed for MF/EDA and MF/isophorone diamine, which does not exhibit any fluorescence. The different emission wavelengths (colours) suggest that it is not the binding between the MF prepolymer and the amine that is responsible for fluorescence but that a variety of fluorescent species are formed. Zhang et al. report the fluorescence of aliphatic amines after oxidation to oximes. It was also reported that an aggregation induced amplification of the emission intensity occurs. Based on the crystallographic data from the single crystal structure and theoretical calculations, they stated that the aggregation of oxime groups (C═N–OH) through intermolecular hydrogen bonding results in extended conjugation and a rigidified conformation, which lead to strong fluorescence (Zhang et al.2017).
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