Artículos de revistas: "In-Situ microscopie de fluorescence"

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Pernthaler, Jakob, Annelie Pernthaler y Rudolf Amann. "Automated Enumeration of Groups of Marine Picoplankton after Fluorescence In Situ Hybridization". Applied and Environmental Microbiology 69, n.º 5 (mayo de 2003): 2631–37. http://dx.doi.org/10.1128/aem.69.5.2631-2637.2003.

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ABSTRACT We describe here an automated system for the counting of multiple samples of double-stained microbial cells on sections of membrane filters. The application integrates an epifluorescence microscope equipped with motorized z-axis drive, shutters, and filter wheels with a scanning stage, a digital camera, and image analysis software. The relative abundances of specific microbial taxa are quantified in samples of marine picoplankton, as detected by fluorescence in situ hybridization (FISH) and catalyzed reporter deposition. Pairs of microscopic images are automatically acquired from numerous positions at two wavelengths, and microbial cells with both general DNA and FISH staining are counted after object edge detection and signal-to-background ratio thresholding. Microscopic fields that are inappropriate for cell counting are automatically excluded prior to measurements. Two nested walk paths guide the device across a series of triangular preparations until a user-defined number of total cells has been analyzed per sample. A backup autofocusing routine at incident light allows automated refocusing between individual samples and can reestablish the focal plane after fatal focusing errors at epifluorescence illumination. The system was calibrated to produce relative abundances of FISH-stained cells in North Sea samples that were comparable to results obtained by manual evaluation. Up to 28 preparations could be analyzed within 4 h without operator interference. The device was subsequently applied for the counting of different microbial populations in incubation series of North Sea waters. Automated digital microscopy greatly facilitates the processing of numerous FISH-stained samples and might thus open new perspectives for bacterioplankton population ecology.

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Leger, I., M. Robert-Nicoud y G. Brugal. "Combination of DNA in situ hybridization and immunocytochemical detection of nucleolar proteins: a contribution to the functional mapping of the human genome by fluorescence microscopy." Journal of Histochemistry & Cytochemistry 42, n.º 2 (febrero de 1994): 149–54. http://dx.doi.org/10.1177/42.2.8288860.

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The recent application of DNA cloning and non-radioactive in situ hybridization techniques has strengthened the hypothesis of an ordered chromatin structure in interphase nuclei. The arrangement of specific chromosomal regions is not random and is strongly suspected to vary with functional activity. The combination of in situ hybridization and immunocytochemistry, allowing simultaneous detection of nucleic acid sequences and specific antigens in the same nucleus, has already made significant contributions to the study of gene expression, to simultaneous karyotyping and phenotyping of tumor cells, and to in situ analysis of viral infections. This report emphasizes the considerable interest of such combined techniques for functional in situ mapping of the genome at the individual cell level. We propose a method that combines fluorescence immunocytochemical detection of nucleolar proteins and fluorescence in situ hybridization of centromeric and telomeric probes specific for chromosome 1 in two cultured human cell lines. The preparative constraints for a broad application of this procedure are defined so that the cell preparations can be further analyzed by fluorescence microscopic imaging techniques and confocal laser scan microscopy. The two selected sequences of the human chromosome 1 can be localized in the nucleus with respect to nucleolar proteins in a one-step fluorescence microscopic observation.

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Lu, Fang, Tingting Zhou, Yan Liu, Liying Song, Bin Zhang y Yuyan Li. "Application of Fluorescence In Situ Hybridization Assisted by Fluorescence Microscope in Detection of Her2 Gene in Breast Cancer Patients". Contrast Media & Molecular Imaging 2022 (11 de agosto de 2022): 1–6. http://dx.doi.org/10.1155/2022/3087681.

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In order to study the important factors for evaluating the prognosis of breast cancer patients, a fluorescence microscopy-assisted fluorescence in situ hybridization technique was proposed. Compared with other detection techniques, fluorescence in situ hybridization (FISH) technology assisted by a fluorescence microscope has gradually gained favor in related fields due to its advantages of high detection specificity, high sensitivity, and strong experimental period. Combined with the basic overview of fluorescence microscopy and FISH technology, the advantages and application points of FISH technology assisted by fluorescence microscopy in the detection of the Her2 gene in breast cancer patients were studied and discussed. The results show that IHC can be used as the primary screening for HER2 gene status detection; IHC (2+) and IHC (3+) have false positives, which are related to chromosome 17 polysomy, so FISH should be done to confirm the diagnosis.

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Buchanan, Bailey C. y Jeong-Yeol Yoon. "Microscopic Imaging Methods for Organ-on-a-Chip Platforms". Micromachines 13, n.º 2 (19 de febrero de 2022): 328. http://dx.doi.org/10.3390/mi13020328.

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Microscopic imaging is essential and the most popular method for in situ monitoring and evaluating the outcome of various organ-on-a-chip (OOC) platforms, including the number and morphology of mammalian cells, gene expression, protein secretions, etc. This review presents an overview of how various imaging methods can be used to image organ-on-a-chip platforms, including transillumination imaging (including brightfield, phase-contrast, and holographic optofluidic imaging), fluorescence imaging (including confocal fluorescence and light-sheet fluorescence imaging), and smartphone-based imaging (including microscope attachment-based, quantitative phase, and lens-free imaging). While various microscopic imaging methods have been demonstrated for conventional microfluidic devices, a relatively small number of microscopic imaging methods have been demonstrated for OOC platforms. Some methods have rarely been used to image OOCs. Specific requirements for imaging OOCs will be discussed in comparison to the conventional microfluidic devices and future directions will be introduced in this review.

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Tatarov, Boyan, Detlef Müller, Matthias Tesche y Sung-Kyun Shin. "Lidar Innovations for Technologies and Environmental Sciences (LITES) – An Remote Sensing Infrastructure Facility: Setup and Measurements Examples". EPJ Web of Conferences 237 (2020): 07017. http://dx.doi.org/10.1051/epjconf/202023707017.

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At the University of Hertfordshire, we have been developing a new remote sensing facility (LITES) to explore the feasibility of using Raman and/or fluorescence backscattering for chemical aerosol profiling. This paper provides an overview of the instruments of the facility and measurement examples. LITES includes a ultra-high-energy Nd:YAG/OPO setup, spectroscopic equipment with high spectral resolution, several imaging and single detectors that allow for time-resolved (lidar) signal detection, a Raman/fluorescence microscope, and a suite of gas and aerosol chambers. We present examples of elastic, rotational and vibrational spectroscopic lidar signals, as well as in-situ microscopic spectrums of dust and bio-aerosol compounds.

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Leitch, A. R., W. Mosgoller, T. Schwarzacher, M. D. Bennett y J. S. Heslop-Harrison. "Genomic in situ hybridization to sectioned nuclei shows chromosome domains in grass hybrids". Journal of Cell Science 95, n.º 3 (1 de marzo de 1990): 335–41. http://dx.doi.org/10.1242/jcs.95.3.335.

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In situ hybridization using biotinylated total genomic DNA and avidin detection systems was adapted for examination of thin-sectioned plant material in the light and electron microscopes. Root tip material was preserved prior to sectioning, so that the in vivo disposition of the chromatin was maintained. Use of total genomic DNA from Secale africanum as a probe enabled the chromatin from the two parental genomes in the grass hybrid Hordeum chilense × S. africanum to be distinguished. The biotinylated probe preferentially labelled the chromosomes of S. africanum origin. DNA-DNA hybrids were visualized at the light-microscope level by Texas Red fluorescence and at the electron-microscope level by the enzymic precipitation of DAB (diaminobenzidine) or by colloidal gold particles. The use of thin sections allowed the location of probe hybridization to be established unequivocally in both metaphase and interphase nuclei. Analysis of interphase nuclei showed that chromatin originating from the two parental genomes did not intermix but occupied distinct domains.

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Deerinck, Thomas J., Maryann E. Martone, Varda Lev-Ram, David P. L. Green, Roger Y. Tsien, David L. Spector, Sui Huang y Mark H. Ellisman. "3-Dimensional immunolabeling and in situ hybridization detection using fluorescence photooxidation and intermediate-voltage Electron Microscopy". Proceedings, annual meeting, Electron Microscopy Society of America 52 (1994): 164–65. http://dx.doi.org/10.1017/s0424820100168554.

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The confocal laser scanning microscope has become a powerful tool in the study of the 3-dimensional distribution of proteins and specific nucleic acid sequences in cells and tissues. This is also proving to be true for a new generation of high contrast intermediate voltage electron microscopes (IVEM). Until recently, the number of labeling techniques that could be employed to allow examination of the same sample with both confocal and IVEM was rather limited. One method that can be used to take full advantage of these two technologies is fluorescence photooxidation. Specimens are labeled by a fluorescent dye and viewed with confocal microscopy followed by fluorescence photooxidation of diaminobenzidine (DAB). In this technique, a fluorescent dye is used to photooxidize DAB into an osmiophilic reaction product that can be subsequently visualized with the electron microscope. The precise reaction mechanism by which the photooxidation occurs is not known but evidence suggests that the radiationless transfer of energy from the excited-state dye molecule undergoing the phenomenon of intersystem crossing leads to the formation of reactive oxygen species such as singlet oxygen. It is this reactive oxygen that is likely crucial in the photooxidation of DAB.

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Jovin, Thomas M., Michel Robert-Nicoud, Donna J. Arndt-Jovin y Thorsten Schormann. "3-D imaging of cells using a confocal laser scanning microscope and digital image processing". Proceedings, annual meeting, Electron Microscopy Society of America 46 (1988): 96–97. http://dx.doi.org/10.1017/s0424820100102560.

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Light microscopic techniques for visualizing biomolecules and biochemical processes in situ have become indispensable in studies concerning the structural organization of supramolecular assemblies in cells and of processes during the cell cycle, transformation, differentiation, and development. Confocal laser scanning microscopy offers a number of advantages for the in situ localization and quantitation of fluorescence labeled targets and probes: (i) rejection of interfering signals emanating from out-of-focus and adjacent structures, allowing the “optical sectioning” of the specimen and 3-D reconstruction without time consuming deconvolution; (ii) increased spatial resolution; (iii) electronic control of contrast and magnification; (iv) simultanous imaging of the specimen by optical phenomena based on incident, scattered, emitted, and transmitted light; and (v) simultanous use of different fluorescent probes and types of detectors.We currently use a confocal laser scanning microscope CLSM (Zeiss, Oberkochen) equipped with 3-laser excitation (u.v - visible) and confocal optics in the fluorescence mode, as well as a computer-controlled X-Y-Z scanning stage with 0.1 μ resolution.

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9

Shah, Jyotsna S., Olivia Mark, Eddie Caoili, Akhila Poruri, Richard I. Horowitz, Alan D. Ashbaugh y Ranjan Ramasamy. "A Fluorescence In Situ Hybridization (FISH) Test for Diagnosing Babesiosis". Diagnostics 10, n.º 6 (6 de junio de 2020): 377. http://dx.doi.org/10.3390/diagnostics10060377.

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Apicomplexan parasites of the genus Babesia cause babesiosis in humans and animals. The microscopic examination of stained blood smears, detection of serum antibodies by immunoassays, and PCR-based identification of parasite nucleic acid in blood are common laboratory methods for diagnosing babesiosis. The present study evaluated a commercially available Babesia genus-specific fluorescence in situ hybridization (FISH) test for detecting Babesia parasites in blood smears. The FISH test detected Babesia duncani and Babesia microti, two common species that cause human infections in the USA, and other Babesia species of human and veterinary importance in less than two hours. The Babesia genus-specific FISH test supplements other existing laboratory methods for diagnosing babesiosis and may be particularly useful in resource-limited laboratories.

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Shah, Jyotsna S. y Ranjan Ramasamy. "Fluorescence In Situ Hybridization (FISH) Tests for Identifying Protozoan and Bacterial Pathogens in Infectious Diseases". Diagnostics 12, n.º 5 (21 de mayo de 2022): 1286. http://dx.doi.org/10.3390/diagnostics12051286.

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Diagnosing and treating many infectious diseases depends on correctly identifying the causative pathogen. Characterization of pathogen-specific nucleic acid sequences by PCR is the most sensitive and specific method available for this purpose, although it is restricted to laboratories that have the necessary infrastructure and finance. Microscopy, rapid immunochromatographic tests for antigens, and immunoassays for detecting pathogen-specific antibodies are alternative and useful diagnostic methods with different advantages and disadvantages. Detection of ribosomal RNA molecules in the cytoplasm of bacterial and protozoan pathogens by fluorescence in-situ hybridization (FISH) using sequence-specific fluorescently labelled DNA probes, is cheaper than PCR and requires minimal equipment and infrastructure. A LED light source attached to most laboratory light microscopes can be used in place of a fluorescence microscope with a UV lamp for FISH. A FISH test hybridization can be completed in 30 min at 37 °C and the whole test in less than two hours. FISH tests can therefore be rapidly performed in both well-equipped and poorly-resourced laboratories. Highly sensitive and specific FISH tests for identifying many bacterial and protozoan pathogens that cause disease in humans, livestock and pets are reviewed, with particular reference to parasites causing malaria and babesiosis, and mycobacteria responsible for tuberculosis.

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11

de Haas, R. R., N. P. Verwoerd, M. P. van der Corput, R. P. van Gijlswijk, H. Siitari y H. J. Tanke. "The use of peroxidase-mediated deposition of biotin-tyramide in combination with time-resolved fluorescence imaging of europium chelate label in immunohistochemistry and in situ hybridization." Journal of Histochemistry & Cytochemistry 44, n.º 10 (octubre de 1996): 1091–99. http://dx.doi.org/10.1177/44.10.8813073.

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The application of europium chelates as delayed fluorescent labels in FISH and immunocytochemistry is hampered by their relatively low quantum yield. To increase the intensity of the delayed fluorescence, we have used a recently introduced peroxidase-mediated amplification system. This system results in a large accumulation of biotin-tyramide, which is detected using streptavidin-europium chelate as label. Optimal staining conditions were evaluated for the immunocytochemical detection of vimentin in cryosections of rat liver, for DNA in situ hybridization (alphoid type probes and 40-KB cosmid probes), and for RNA in situ hybridization (detection of 28S ribosomal RNA, human elongation factor mRNA, and luciferase mRNA). Using a time-resolved fluorescence microscope, intense europium fluorescence was obtained in all these applications when the tyramide amplification system was applied. The signals were strong enough to be observed by eye using the microscope in the time-delayed mode. The routine application of this technique for localization and quantization of antigens or nucleic acid sequences in tissue exhibiting strong autofluorescence is discussed.

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12

TSUKAHARA, Satoshi. "In Situ Fluorescence Microscopic Measurements of Complexation Reactions at Liquid/Liquid Interface". International Journal of the Society of Materials Engineering for Resources 13, n.º 1 (2005): 1–8. http://dx.doi.org/10.5188/ijsmer.13.1.

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13

Kensche, A., S. Basche, W. H. Bowen, M. Hannig y C. Hannig. "Fluorescence microscopic visualization of non cellular components during initial bioadhesion in situ". Archives of Oral Biology 58, n.º 10 (octubre de 2013): 1271–81. http://dx.doi.org/10.1016/j.archoralbio.2013.07.006.

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14

Ferri, Gian-Luca, Jorma Isola, Peter Berger y Gabriele Giro. "Direct Eye Visualization of Cy5 Fluorescence for Immunocytochemistry and In Situ Hybridization". Journal of Histochemistry & Cytochemistry 48, n.º 3 (marzo de 2000): 437–44. http://dx.doi.org/10.1177/002215540004800314.

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Cyanine 5.18 (or Cy5) is a fluorochrome emitting in the long-red/far-red range, usually regarded as unsuitable for direct observation by the human eye. We describe here the optimization of a direct visualization approach to Cy5 labeling, based on a standard fluorescence microscope with mercury light excitation and applicable to both immunocytochemistry and fluorescent in situ hybridization. Crucial factors were (a) an excitation path in the microscope not absorbing light in the orange-red range, up to 640 nm, (b) a 588-640-nm excitation filter range, distinctly below the excitation optimum for Cy5, (c) a 650-700-nm emission filter range, transmitting the low-wavelength portion of Cy5 emission, and (d) high-efficiency filter set components allowing a narrow gap between excitation and emission ranges without visible cross-talk of excitation light in the emission path.

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15

MATSUTA, MORIMASA, MAYUMI MATSUTA, KOHSUKE SASAKI, HIDEKI HARIU, IWAO NISHIYA, MAKOTO KATOU, ANNE KALLIONIEMI, HEINZ-ULRICH WEIER y JOE W. GRAY. "Application of Confocal Laser Scanning Microscope on Fluorescence in situ Hybridization (FISH)." Acta Histochemica et Cytochemica 26, n.º 5 (1993): 415–21. http://dx.doi.org/10.1267/ahc.26.415.

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16

Huang, Z., W. You, R. P. Haugland, V. B. Paragas, N. A. Olson y R. P. Haugland. "A novel fluorogenic substrate for detecting alkaline phosphatase activity in situ." Journal of Histochemistry & Cytochemistry 41, n.º 2 (febrero de 1993): 313–17. http://dx.doi.org/10.1177/41.2.8419466.

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We describe here the in situ detection of alkaline phosphatase (APase) activity with a new fluorogenic substrate, 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (CPPCQ). CPPCQ is very soluble and colorless. APase converts it into a rapidly precipitating product, whose strong fluorescence marks the sites of APase activity. The detected APase was either a probing enzyme anchored to epidermal growth factor (EGF) receptors of fixed human epidermoid carcinoma cell line (A431) by biotinylated EGF and streptavidin-APase conjugates or an endogenous marker existing in a fixed canine kidney cell line (MDCK). With CPPCQ staining, the EGF receptors and the endogenous APase were both visualized by fluorescence microscopy as contrasting, photostable, and well-resolved fluorescent stains. The EGF receptor staining was specific since it could be blocked by excessive unlabeled EGF. In contrast, fluorescein-labeled EGF failed to specifically stain the EGF receptors under the same fluorescent microscope. The endogenous APase staining with CPPCQ was sensitive to heating, levamisole and L-homoarginine, showing an APase tissue specificity of the liver/bone/kidney type. Therefore, CPPCQ appears to be a novel substrate dye for sensitive fluorescence APase histochemistry.

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17

Good, M. J., W. J. Hage, C. L. Mummery, S. W. De Laat y J. Boonstra. "Localization and quantification of epidermal growth factor receptors on single cells by confocal laser scanning microscopy." Journal of Histochemistry & Cytochemistry 40, n.º 9 (septiembre de 1992): 1353–61. http://dx.doi.org/10.1177/40.9.1506672.

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We have established a method for quantifying binding of fluorescence-labeled growth factors to their receptors on single cells in situ with the confocal laser scanning microscope (CLSM). Biotinylated epidermal growth factor (EGF) coupled to phycoerythrin-labeled anti-biotin was used to compare the levels of fluorescence on three different cell types for which the number of EGF factors was known from Scatchard analysis of [125I]-EGF binding. The results showed that as few as 10,000 receptors/cell were detectable above back-ground. This method will provide a rapid and quantifiable alternative to autoradiography for ligand binding to single cells in situ.

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Thimm, Torsten y Christoph C. Tebbe. "Protocol for Rapid Fluorescence In Situ Hybridization of Bacteria in Cryosections of Microarthropods". Applied and Environmental Microbiology 69, n.º 5 (mayo de 2003): 2875–78. http://dx.doi.org/10.1128/aem.69.5.2875-2878.2003.

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ABSTRACT A protocol was developed to detect bacteria inhabiting microarthropods by means of small-subunit rRNA-targeted fluorescence in situ hybridization and microscopy. The protocol is based on cryosections of whole specimens. In contrast to more commonly applied paraffin-embedding techniques, the protocol is quicker and reduces the number of manipulations which might damage the microscopic material. The method allowed the study of the bacterial colonization of Folsomia candida (Collembola) and the detection of bacteria in both the gut and tissue.

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Hannig, C., M. Hannig, O. Rehmer, G. Braun, E. Hellwig y A. Al-Ahmad. "Fluorescence microscopic visualization and quantification of initial bacterial colonization on enamel in situ". Archives of Oral Biology 52, n.º 11 (noviembre de 2007): 1048–56. http://dx.doi.org/10.1016/j.archoralbio.2007.05.006.

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Imaging, Contrast Media &. Molecular. "Retracted: Application of Fluorescence In Situ Hybridization Assisted by Fluorescence Microscope in Detection of Her2 Gene in Breast Cancer Patients". Contrast Media & Molecular Imaging 2023 (19 de julio de 2023): 1. http://dx.doi.org/10.1155/2023/9763619.

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21

Darriet, Florent, Paola Bernioles, Ahmed Loukil, Nadia Saidani, Carole Eldin y Michel Drancourt. "Fluorescence in situ hybridization microscopic detection of Bacilli Calmette Guérin mycobacteria in aortic lesions". Medicine 97, n.º 30 (julio de 2018): e11321. http://dx.doi.org/10.1097/md.0000000000011321.

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22

La Selva, R., M. Sanlorenzo, G. Rozzo, V. De Giorgi, M. T. Fierro y P. Broganelli. "Observation in vivo de la peau par microscopie à fluorescence : les premières applications". Annales de Dermatologie et de Vénéréologie 144, n.º 12 (diciembre de 2017): S174. http://dx.doi.org/10.1016/j.annder.2017.09.257.

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23

Brown, Garth R., Vindhya Amarasinghe, Gyula Kiss y John E. Carlson. "Preliminary karyotype and chromosomal localization of ribosomal DNA sites in white spruce using fluorescence in situ hybridization". Genome 36, n.º 2 (1 de abril de 1993): 310–16. http://dx.doi.org/10.1139/g93-043.

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We have localized the major ribosomal DNA (rDNA) loci on metaphase chromosomes and in interphase nuclei of white spruce (2n = 24) by fluorescence in situ hybridization. Hybridization sites of the biotin-labelled rDNA probe were detected using antibody–fluorochrome conjugates and a confocal laser scanning microscope. White spruce has at least 12, and possibly as many as 14, rDNA sites, 1 site present on each of seven separate chromosome pairs. This is one of the highest numbers of rDNA loci yet reported among plant species. The position of the rDNA loci together with secondary constriction patterns permit, for the first time, all homologous pairs of white spruce chromosomes to be distinguished. We discuss the application of molecular cytogenetics in studies relating to the organization and evolution of DNA sequences within conifer genomes.Key words: fluorescence in situ hybridization, Picea, rDNA, karyotype.

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Van Wijk, I. J., J. M. Van Vugt, A. A. Könst, M. A. Mulders, A. W. Nieuwint y C. B. Oudejans. "Multiparameter in situ analysis of trophoblast cells in mixed cell populations by combined DNA and RNA in situ hybridization." Journal of Histochemistry & Cytochemistry 43, n.º 7 (julio de 1995): 709–14. http://dx.doi.org/10.1177/43.7.7608525.

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We developed a non-radioactive assay for simultaneous detection of cytoplasmic mRNA and nuclear genomic DNA in fetal trophoblast cells by sequential in situ hybridization. Trophoblast-specific mRNA is detected with a digoxigenin-labeled RNA probe complementary to HLA-G, followed by visualization through the generation of stable contrast-rich DAB/Ni complexes. Genomic target DNA is subsequently visualized in labeled cells by fluorescent in situ hybridization using biotin-labeled chromosome-specific DNA probes. Simultaneous visualization of both targets is made possible using a fluorescence microscope with FITC filter and conventional brightfield light. This method allows detection of trophoblast cells within a mixed cell population and, at the same time, analysis of chromosome anomalies in the trophoblast cells identified. For prenatal diagnosis of fetal cells enriched from maternal peripheral blood during pregnancy, this multiparameter in situ analysis of immobilized fetal trophoblast cells will be very useful.

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Carrington, Walter A. "3-D image restoration of fluorescence microscope images by regularization". Proceedings, annual meeting, Electron Microscopy Society of America 51 (1 de agosto de 1993): 148–49. http://dx.doi.org/10.1017/s0424820100146588.

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By combining optical sections of a fluorescently labelled cell with quantitative calibration of the microscope’s blurring, we are able to computationally reverse the blurring of the microscope. We apply this approach to images taken on a conventional wide field microscope using a cooled CCD camera. Our algorithm is flexible in its use of data, which enables us to obtain good results with a small number of optical sections. We use this flexibility to minimize photodamage and photobleaching, so that we are able to obtain 3-D movies of living cells with high spatial and temporal resolution. We obtain rejection of out of focus light, resolution and accuracy of quantitative fluorescent measurement superior to confocal microscopes with substantially less photobleaching and photodamage to the sample. Recent algorithmic advances have enabled us to achieve transverse resolution as good as 100nm and axial resolution as good as 400nm.The wide variety of cells and labels examined in 3-D in our lab illustrate the robustness and effectiveness of our image restoration approach. These applications range from samples as sparsely labelled as single copy genes in lymphocytes and fibroblasts labelled by in situ hybridization to densely labelled cells with fluorescently labelled dextran or Fura-2 that completely fill the cell volume.

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Zieba, Agata, Carolina Wählby, Fredrik Hjelm, Lee Jordan, Jonathan Berg, Ulf Landegren y Katerina Pardali. "Bright-Field Microscopy Visualization of Proteins and Protein Complexes by In Situ Proximity Ligation with Peroxidase Detection". Clinical Chemistry 56, n.º 1 (1 de enero de 2010): 99–110. http://dx.doi.org/10.1373/clinchem.2009.134452.

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Abstract Background: The in situ proximity ligation assay (PLA) allows a protein or protein complex to be represented as an amplifiable DNA molecule. Recognition is mediated by proximity probes consisting of antibodies coupled with oligonucleotides. Upon dual binding of the proximity probes, the oligonucleotides direct the formation of a circular DNA molecule, which is then amplified by rolling-circle replication. The localized concatemeric product is then detected with fluorescent probes. The in situ PLA enables localized detection of individual native proteins or interacting protein pairs in fixed cells or tissue sections, thus providing an important tool for basic and clinical research. Methods: We used horseradish peroxidase (HRP)-conjugated oligonucleotides to couple in situ PLA with enzymatic visualization of the localized detection event. Results: We demonstrate the detection of protein complexes, both in cells and in tissue sections, and show that we can quantify the complexes with image-analysis software specially developed for recognizing HRP signals in bright-field microscopy images. We show that fluorescence and HRP signals produce equivalent results, both in cultured cells and in tissue samples. Conclusions: The combination of in situ PLA with bright-field detection and automated image analysis allows the signals present to be counted in an automated fashion and thus provides a sensitive and specific method for quantification of proteins and protein complexes with bright-field microscopy. With this approach, in situ PLA can be used without the requirement for expensive fluorescence microscopes, thereby avoiding problems with nonspecific fluorescence while maintaining compatibility with conventional histologic staining.

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Hamkalo, B. A., S. Narayanswami y A. P. Kausch. "High-resolution nucleic acid sequence mapping via in situ hybridization at the Electron Microscope level". Proceedings, annual meeting, Electron Microscopy Society of America 53 (13 de agosto de 1995): 752–53. http://dx.doi.org/10.1017/s0424820100140130.

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The availability of nonradioactive methods to label nucleic acids an the resultant rapid and greater sensitivity of detection has catapulted the technique of in situ hybridization to become the method of choice to locate of specific DNA and RNA sequences on chromosomes and in whole cells in cytological preparations in many areas of biology. It is being applied to problems of fundamental interest to basic cell and molecular biologists such as the organization of the interphase nucleus in the context of putative functional domains; it is making major contributions to genome mapping efforts; and it is being applied to the analysis of clinical specimens. Although fluorescence detection of nucleic acid hybrids is routinely used, certain questions require greater resolution. For example, very closely linked sequences may not be separable using fluorescence; the precise location of sequences with respect to chromosome structures may be below the resolution of light microscopy(LM); and the relative positions of sequences on very small chromosomes may not be feasible.

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Nielsen, Jeppe Lund, Marilena Aquino de Muro y Per Halkjær Nielsen. "Evaluation of the Redox Dye 5-Cyano-2,3-Tolyl-Tetrazolium Chloride for Activity Studies by Simultaneous Use of Microautoradiography and Fluorescence In Situ Hybridization". Applied and Environmental Microbiology 69, n.º 1 (enero de 2003): 641–43. http://dx.doi.org/10.1128/aem.69.1.641-643.2003.

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ABSTRACT Three microscopic in situ techniques were used simultaneously to investigate viability and activity on a single-cell level in activated sludge. The redox dye 5-cyano-2,3-tolyl-tetrazolium chloride (CTC) was compared with microautoradiography (MAR) and fluorescence in situ hybridization (FISH) to indicate activity of cells in Thiothrix filaments and in single floc-forming bacteria. The signals from MAR and FISH correlated well, whereas only 65% of the active Thiothrix cells and 41% of all single cells were detectable by CTC reduction, which mainly targeted the most active cells.

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Smeets, Marit, Anna Bieber, Cristina Capitanio, Oda Schioetz, Thomas van der Heijden, Andries Effting, Éric Piel, Bassim Lazem, Philipp Erdmann y Juergen Plitzko. "Integrated Cryo-Correlative Microscopy for Targeted Structural Investigation In Situ". Microscopy Today 29, n.º 6 (noviembre de 2021): 20–25. http://dx.doi.org/10.1017/s1551929521001280.

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Abstract:Cryo-electron tomography (cryo-ET) has the potential to revolutionize our understanding of the building blocks of life since it provides the unique opportunity to study molecules and membrane architectures in the context of cellular interaction. In particular, the combination of fluorescence imaging with focused ion beam (FIB) milling allows the targeting of specific structures in thick cellular samples by preparing thin lamellae that contain a specific fluorescence marker. This technique has conventionally been time-consuming, as it requires sample transfer to multiple microscopes and presents several technical challenges that currently limit its success. Here we describe METEOR, a FIB-integrated microscopy solution that streamlines the correlative cryo-ET workflow. It protects the sample from ice contamination by minimizing handling steps, thus increasing the likelihood of high-quality data. It also allows for monitoring of the milling procedure to ensure the molecule of interest is captured and can then be imaged by cryo-ET.

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30

Meng, Bo-yang y Xiao-yan Li. "In Situ Visualization of Concentration Polarization during Membrane Ultrafiltration Using Microscopic Laser-Induced Fluorescence". Environmental Science & Technology 53, n.º 5 (29 de enero de 2019): 2660–69. http://dx.doi.org/10.1021/acs.est.8b05741.

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31

Ye, Xin, Yang Liu, Yun Lv, Guangfeng Liu, Xiaoxin Zheng, Quanxiang Han, Kenneth A. Jackson y Xutang Tao. "In Situ Microscopic Observation of the Crystallization Process of Molecular Microparticles by Fluorescence Switching". Angewandte Chemie International Edition 54, n.º 27 (14 de mayo de 2015): 7976–80. http://dx.doi.org/10.1002/anie.201503052.

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32

Ye, Xin, Yang Liu, Yun Lv, Guangfeng Liu, Xiaoxin Zheng, Quanxiang Han, Kenneth A. Jackson y Xutang Tao. "In Situ Microscopic Observation of the Crystallization Process of Molecular Microparticles by Fluorescence Switching". Angewandte Chemie 127, n.º 27 (14 de mayo de 2015): 8087–91. http://dx.doi.org/10.1002/ange.201503052.

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33

Schwarzacher, Trude y J. S. Heslop-Harrison. "In situ hybridization to plant telomeres using synthetic oligomers". Genome 34, n.º 3 (1 de junio de 1991): 317–23. http://dx.doi.org/10.1139/g91-052.

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The distribution of telomeric repeats in Hordeum vulgare (barley) and Secale cereale (rye) was studied by DNA–DNA in situ hybridization to root-tip chromosome preparations. Biotinylated synthetic oligomers, (TTTAGGG)6 and (CCCTAAA)6, homologous to the consensus sequence of the Arabidopsis thaliana telomeric repeat, were used as probes, and hybridization sites were detected by Texas red fluorescence or horseradish peroxidase catalyzed precipitation of diaminobenzidine. After examination in the light microscope, the same preparations were transferred to the electron microscope for high-resolution analysis. Sites of hybridization were visualized as single or double dots at the end of most chromosome arms. The sizes of the signal dots varied widely, indicating that individual telomeres may contain different numbers of repeats of the telomeric sequence. In contrast to many mammals, no telomere repeats were detected other than at the ends of the chromosomes. At interphase, signals were concentrated in one region of the nucleus, as would be expected from the Rabl orientation typical for dividing cells from cereal root tips.Key words: barley, rye, nuclear architecture, DNA–DNA in situ hybridization, telomeres.

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Zhou, Xu, Jin Zhao, Li Zheng y Jin Wang. "Fluorescence in situ hybridization detection of cytogenetic abnormalities and prognosis in multiple myeloma". Archives of Biological Sciences 67, n.º 2 (2015): 437–43. http://dx.doi.org/10.2298/abs140608008z.

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We evaluated the prognosis of patients with newly diagnosed multiple myeloma (MM) and attempted to find a suitable treatment strategy for them. Interphase fluorescence in situ hybridization (FISH) detection was performed on 57 patients with MM. The following probes: IgH, p53, 1q21, RB1, and D13S319 specific for the rearrangements of 14q32, 17p13, 1q21 and 13q14 were used. Fluorescent hybridization signals were observed using an Olympus BX60 epifluorescence microscope equipped with filters for detecting fluoroisothiocyanate (FITC), Texas red, and 4'-6-Diamidino-2-phenylindole (DAPI). Triple color clone-specific images were captured using a Quips XL genetic workstation. The mortalities in patients with moderate prognosis (66.7%) and poor prognosis (50%) were significantly higher compared with that in patients with good prognosis (15%, P<0.05). All the patients in good and moderate prognosis groups achieved complete remission (CR)/very good partial remission (VGPR)/partial remission (PR), whereas only half of the cases in the poor prognosis group reached this level. Patients 2 supported by autologous hematopoietic stem-cell transplantation presented CR/PR and long survival. For those with poor prognosis, a proper therapeutic regimen and timely transplantation, especially tandem transplantation, was necessary due to the rapid progression and complications.

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SATO, Naoya, Takehiro MORITA, Tetsuo YAMAGUCHI y Yoshinori SAWAE. "321 In-situ Observation of transfer film formation of polymer materials with fluorescence microscope". Proceedings of Conference of Kyushu Branch 2015.68 (2015): 123–24. http://dx.doi.org/10.1299/jsmekyushu.2015.68.123.

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36

Li, S., R. N. Spear y J. H. Andrews. "Quantitative fluorescence in situ hybridization of Aureobasidium pullulans on microscope slides and leaf surfaces." Applied and environmental microbiology 63, n.º 8 (1997): 3261–67. http://dx.doi.org/10.1128/aem.63.8.3261-3267.1997.

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37

Wu Yin, 吴寅, 梁永 Liang Yong, 张洁 Zhang Jie y 李辉 Li Hui. "荧光原位杂交扩增探针的结构光照明超分辨成像与计数". Laser & Optoelectronics Progress 61, n.º 4 (2024): 0411009. http://dx.doi.org/10.3788/lop231182.

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38

Pezzotti, Giuseppe, Hiroyuki Suenobu, Toshihiko Nishida y Orfeo Sbaizero. "Measurement of Microscopic Bridging Stresses in an Alumina/Molybdenum Composite by In Situ Fluorescence Spectroscopy". Journal of the American Ceramic Society 82, n.º 5 (21 de diciembre de 2004): 1257–62. http://dx.doi.org/10.1111/j.1151-2916.1999.tb01904.x.

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39

Loukil, A., S. A. Baron, X. Argemi, T. Maubon y C. Eldin. "Microscopic detection of bacillus Calmette–Guérin mycobacteria in bladder biopsy using fluorescence in situ hybridization". New Microbes and New Infections 39 (enero de 2021): 100826. http://dx.doi.org/10.1016/j.nmni.2020.100826.

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40

Arend, Johannes, Alexander Wetzel y Bernhard Middendorf. "Fluorescence Microscopy of Superplasticizers in Cementitious Systems: Applications and Challenges". Materials 13, n.º 17 (24 de agosto de 2020): 3733. http://dx.doi.org/10.3390/ma13173733.

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In addition to the desired plasticizing effect, superplasticizers used in high and ultra-high performance concretes (UHPC) influence the chemical system of the pastes and for example retardation of the cement hydration occurs. Thus, superplasticizers have to be chosen wisely for every material composition and application. To investigate the essential adsorption of these polymers to particle surfaces in-situ to overcome several practical challenges of superplasticizer research, fluorescence microscopy is useful. In order to make the superplasticizer polymers visible for this microscopic approach, they are stained with fluorescence dyes prior the experiment. In this work, the application of this method in terms of retardation and rheological properties of sample systems is presented. The hydration of tricalcium oxy silicate (C3S) in combination with different polycarboxylate ether superplasticizers is observed by fluorescence microscopy and calorimetry. Both methods can identify the retarding effect, depending on the superplasticizer’s chemical composition. On the other hand, the influence of the superplasticizers on the slump of a ground granulated blast furnace slag/cement paste is correlated to fluorescence microscopic adsorption results. The prediction of the efficiency by microscopic adsorption analysis succeeds roughly. At last, the possibility of high-resolution imaging via confocal laser scanning microscopy is presented, which enables the detection of early hydrates and their interaction with the superplasticizers.

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41

Powell, Richard D., Vishwas N. Joshi, Carol M. R. Halsey, James F. Hainfeld, Gerhard W. Hacker, Cornelia Hauser-Kronberger, Wolfgang H. Muss y Peter M. Takvorian. "Combined Cy3 / Nanogold Conjugates for ImmunocytoChemistry and in Situ Hybridization". Microscopy and Microanalysis 5, S2 (agosto de 1999): 478–79. http://dx.doi.org/10.1017/s1431927600015713.

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Fluorescein and the 1.4 nm Nanogold® gold cluster label may be incorporated into a single Fab’ immunoprobe by separate cross-linking reactions, to give a probe which labels antigenic sites in a single step for correlative fluorescence and electron microscope visualization. These probes show high labeling density, labeling a pre-mRNA splicing factor in the HeLa cell nucleus; Microtubules were also densely labeled using fluorescence, other optical modalities, and electron microscopy; in a parallel experiment, a 5 nm colloidal gold probe gave only occasional labeling. We now describe Fab’ and streptavidin probes containing both Nanogold® and the fluorescent cyanine dye, Cy3.F(ab’)2 Goat anti-Mouse IgG and F(ab’)2 goat anti-rabbit IgG fragments were reductively cleaved to Fab’ fragments using dithiothreitol (DTT) or mercaptoethylamine hydrochloride (MEA), which selectively reduce the F(ab’)2 hinge disulfide bonds, with 5 mm EDTA to prevent reoxidation. Fab’ fragments were isolated by gel filtration (coarse gel: GH25, Amicon) then labeled with Monomaleimido- Nanogold® which reacts site-specifically with thiols. Streptavidin was labeled using Mono- Sulfo-NHS-Nanogold® at pH 7.5. Nanogold® conjugates were isolated by gel filtration (Superose-12 column, Pharmacia), then reacted with excess Cy3 monofunctional NHS ester (labeling kit, Amersham Life Sciences) at pH 7.5; dual-labeled conjugates were isolated by gel filtration (Superose-12).

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42

Smail, Harem Othman. "The Roles of the Fluorescent In Situ Hybridization (FISH) and Comparative Genomic Hybridization (CGH) Techniques in the Detection of the Breast Cancer". Biology, Medicine, & Natural Product Chemistry 11, n.º 1 (24 de marzo de 2022): 45–54. http://dx.doi.org/10.14421/biomedich.2022.111.45-54.

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This paper aimed to understand and compare the two popular cytogenetic techniques of fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) in detecting breast cancer chromosomal abnormality. Several chromosomal anomalies play a role in the development of breast cancer, and the two above approaches play an important role in confirming fluorescence in situ hybridization in particular (FISH). However, comparative genomic hybridization has developed DNA copy number profiles for most of the publicly available breast cancer cell lines for the FISH methods rely on the fluorescent probes. Chromosomal profiles can be generated for the suspected chromosomal abnormality, copy number changes between the tumour and the DNA control can be compared, and the results can be registered. Today, modern cytogenetic tools such as fluorescence in situ hybridization (FISH) are more commonly used to detect any microdeletion that cannot be detected by conventional cytogenetic karyotypes that involve a high rate of cell division and good chromosomal morphology, which pose challenges for cytogeneticists, and a long period of testing and research. Usually, this is a problem for physicians, and there are still many drawbacks and disadvantages concerning the high benefits, such as false findings. Normal chromosome in situ hybridization requires the hybridization of a labelled DNA probe into denatured chromosomal DNA present in metaphase chromosomes in an air-dried microscope slide preparation. Metaphase spreads are used for traditional chromosome FISH (metaphase FISH). Positive and positive signs of hybridization also appear as a double spot, corresponding to the hybridized probe for both sister chromatids. A further extension of chromosome painting is comparative genomic hybridization (CCI-I). CCH involves simultaneous chromosome painting in two different colours using complete DNA from two similar sources as probes, which reveal variations concerning the benefit or loss of sub-chromosomal regions or even entire chromosomes.

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43

Deerinck, T. J., M. E. Martone, V. Lev-Ram, D. P. Green, R. Y. Tsien, D. L. Spector, S. Huang y M. H. Ellisman. "Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy." Journal of Cell Biology 126, n.º 4 (15 de agosto de 1994): 901–10. http://dx.doi.org/10.1083/jcb.126.4.901.

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A simple method is described for high-resolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other conventional fluorescent compounds. The technique allows for precise correlative immunolocalization studies on the same sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin is smaller in size than other conventional markers, this method results in improved penetration of labeling reagents compared to gold or enzyme based procedures. The improved penetration allows for three-dimensional immunolocalization using high voltage electron microscopy. Fluorescence photooxidation can also be used for high resolution light and electron microscopic localization of specific nucleic acid sequences by in situ hybridization utilizing biotinylated probes followed by an eosin-streptavidin conjugate.

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44

Cho, Soohee, Argel Islas-Robles, Ariana M. Nicolini, Terrence J. Monks y Jeong-Yeol Yoon. "In situ, dual-mode monitoring of organ-on-a-chip with smartphone-based fluorescence microscope". Biosensors and Bioelectronics 86 (diciembre de 2016): 697–705. http://dx.doi.org/10.1016/j.bios.2016.07.015.

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45

Montenegro, M. A. P., J. C. Araujo y R. F. Vazoller. "Microbial community evaluation of anaerobic granular sludge from a hybrid reactor treating pentachlorophenol by using fluorescence in situ hybridization". Water Science and Technology 48, n.º 6 (1 de septiembre de 2003): 65–73. http://dx.doi.org/10.2166/wst.2003.0359.

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We used in situ hybridization with fluorescently labeled rRNA-targeted oligonucleotide probes concurrently with microscopic examinations and methane measurements to characterize the microbial community of an anaerobic hybrid reactor treating pentachlorophenol (PCP) with a mixture of fatty acids (propionic, butyric, acetic and lactic) and methanol. Archaeal cells detected with probe ARC915 prevailed in anaerobic granular sludge without and with the addition of PCP in a range of 2.0 to 21.0 mg/L to the reactor. This group accounted for 81 and 90% of the DAPI-stained cells before and after the addition of 21 mg/L of PCP, respectively. In these conditions, cells detected with the Methanosarcinales specific probe (MSMX860) were the only methanogenic Archaea found and accounted for 59 to 87.6% of the DAPI-stained cells. No cells were detected by the Methanomicrobiales (MG1200), Methanobacteriaceae (MB1174) and Methanococcaceae (MC1109) specific probes. Bacterial cells detected with probe EUB338 were found in very low numbers, which ranged from 5.7 to 1.0% of the DAPI-stained cells. This finding agrees with the scanning electron microscope examinations, in which cells morphologically resembling Methanosaeta and Methanosarcina were predominantly observed in the granular sludge. Results contributed to the investigation of the importance of the methanogens during PCP degradation.

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46

Collins, Kim A., Stephen J. Cina, Mark J. Pettenati y Matthew Fitts. "Identification of Female Cells in Postcoital Penile Swabs Using Fluorescence In Situ Hybridization". Archives of Pathology & Laboratory Medicine 124, n.º 7 (1 de junio de 2000): 1080–82. http://dx.doi.org/10.5858/2000-124-1080-iofcip.

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Abstract Traditionally, the finding of semen, that is, spermatozoa and acid phosphatase, in cervicovaginal specimens has been considered the laboratory evidence needed to prove recent sexual contact. Recent research with fluorescence in situ hybridization (FISH) has shown that in the absence of semen, male epithelial and inflammatory cells can be found within the female genital tract. A striking paucity of literature exists pertaining to the examination of the penis of an alleged assailant for potential evidence indicative of sexual assault. The current study uses FISH to analyzepostcoital swabs of the penis for such laboratory evidence. A male and female volunteer couple consented to participate in this study. Following coitus, the male partner presented to one of the investigators for penile swabbing. Swabs were taken at varying postcoital intervals (1–24 hours) subsequent to 10 coital episodes. The male participant was instructed not to shower following coitus, but to otherwise go about daily activities until specimen collection. To obtain each sample, 4 sterile cotton-tipped applicators were slightly moistened in sterile saline and swabbed along the length of the penile shaft and around the base of the penis. From the swabs, 3 air-dried slides were prepared, coded, and blinded. As controls, swabs were taken from the buccal surfaces of both volunteers. Multicolor FISH was performed using dual X- and Y-chromosome probes, and slides were counterstained with 4′-6-diamidino-2-phenylindole (DAPI). Cells were easily visualized under a fluorescent microscope, but only cells with 2 nonoverlapping fluorescent signals were counted. Fluorescence in situ hybridization is highly sensitive and specific, and the dual probes easily distinguished between male and female cells. Female cells were identified on smears from every penile swab over the entire 1- to 24-hour postcoital interval. The FISH technique, previously successful in identifying male cells within the female genital tract, may also be employed on penile swabs. Once the presence of female cells is confirmed by FISH, the identity of the female can be confirmed by DNA analysis. Potentially, with such current molecular analyses, both the assailant and the victim can be positively identified.

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47

Durm, Markus, Frank-Martin Haar, Michael Hausmann, Horst Ludwig y Christoph Cremer. "Non-Enzymatic, Low Temperature Fluorescence in situ Hybridization of Human Chromosomes with a Repetitive α-Satellite Probe". Zeitschrift für Naturforschung C 52, n.º 1-2 (1 de febrero de 1997): 82–88. http://dx.doi.org/10.1515/znc-1997-1-215.

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Abstract In all DNA-DNA in situ hybridization (ISH) procedures described so far in the literature, the production of single-stranded target DNA sequences plays a decisive role. This can be achieved either by enzymatic treatment at physiological temperatures or by the separation of double-stranded DNA sequences. Denaturation by heat and chemical agents (e.g. formamide) is regarded as a prerequisite for the non-enzymatic ISH process. However, additional mechanisms of a non-enzymatic ISH procedure are conceivable which do not require high temperature treatment combined with formamide. Here, we report on a non-enzymatic, non-formamide, low temperature, fluorescence in situ hybridization (FISH) procedure which allowed a microscopic visualization and quantitative fluorescence analysis of the binding sites of a repetitive DNA probe. Following only probe denaturation at 94 °C, hybridization was performed at 52 °C for 30 min, i.e., at nearly physiological temperatures. Moreover, increasing the hybridization time to 3 hours indicated that hybridization sites became also visible at 37 °C. Since the protocols are based on recently described Fast FISH developments, the technique will be called Low Temperature Fast-FISH (LTFF).

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48

Jong, Hans de. "Visualizing DNA domains and sequences by microscopy: a fifty-year history of molecular cytogenetics". Genome 46, n.º 6 (1 de diciembre de 2003): 943–46. http://dx.doi.org/10.1139/g03-107.

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This short review presents a historical perspective of chromosome research during the last 50 years. It shows how molecular knowledge and technology of DNA entered cytogenetics step by step making it now daily practice in almost every modern chromosome lab. A crucial milestone in these decades has been the development of in situ protocols by Pardue and Gall, among others, initially only with isotopic labels, and without fluorescence microscopy and sophisticated detection systems. But these very first in situ hybridizations played a decisive role in the discovery of chromosome banding profiles, which were obtained under specific chemical, physical, or enzymatic conditions, thus effecting stainability of specific chromosome regions. In the decades thereafter, numerous technical improvements were achieved leading to complex multi-colour fluorescence in situ hybridization (FISH) protocols for mammals, plants, and insects. Highly improved detection systems of the FISH signals further allowed detection of DNA targets of up to 50 bp, whereas other protocols, which were developed to stretch chromatin fibres to the full length of native DNA, improved spatial resolution of adjacent targets in the light microscope to 1 kb.Key words: historical review, chromosome banding, FISH technology.

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49

Bekelis, Kimon, Pablo A. Valdés, Kadir Erkmen, Frederic Leblond, Anthony Kim, Brian C. Wilson, Brent T. Harris, Keith D. Paulsen y David W. Roberts. "Quantitative and qualitative 5-aminolevulinic acid–induced protoporphyrin IX fluorescence in skull base meningiomas". Neurosurgical Focus 30, n.º 5 (mayo de 2011): E8. http://dx.doi.org/10.3171/2011.2.focus1112.

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Object Complete resection of skull base meningiomas provides patients with the best chance for a cure; however, surgery is frequently difficult given the proximity of lesions to vital structures, such as cranial nerves, major vessels, and venous sinuses. Accurate discrimination between tumor and normal tissue is crucial for optimal tumor resection. Qualitative assessment of protoporphyrin IX (PpIX) fluorescence following the exogenous administration of 5-aminolevulinic acid (ALA) has demonstrated utility in malignant glioma resection but limited use in meningiomas. Here the authors demonstrate the use of ALA-induced PpIX fluorescence guidance in resecting a skull base meningioma and elaborate on the advantages and disadvantages provided by both quantitative and qualitative fluorescence methodologies in skull base meningioma resection. Methods A 52-year-old patient with a sphenoid wing WHO Grade I meningioma underwent tumor resection as part of an institutional review board–approved prospective study of fluorescence-guided resection. A surgical microscope modified for fluorescence imaging was used for the qualitative assessment of visible fluorescence, and an intraoperative probe for in situ fluorescence detection was utilized for quantitative measurements of PpIX. The authors assessed the detection capabilities of both the qualitative and quantitative fluorescence approaches. Results The patient harboring a sphenoid wing meningioma with intraorbital extension underwent radical resection of the tumor with both visibly and nonvisibly fluorescent regions. The patient underwent a complete resection without any complications. Some areas of the tumor demonstrated visible fluorescence. The quantitative probe detected neoplastic tissue better than the qualitative modified surgical microscope. The intraoperative probe was particularly useful in areas that did not reveal visible fluorescence, and tissue from these areas was confirmed as tumor following histopathological analysis. Conclusions Fluorescence-guided resection may be a useful adjunct in the resection of skull base meningiomas. The use of a quantitative intraoperative probe to detect PpIX concentration allows more accurate determination of neoplastic tissue in meningiomas than visible fluorescence and is readily applicable in areas, such as the skull base, where complete resection is critical but difficult because of the vital structures surrounding the pathology.

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50

Lawrence, Jeanne Bentley. "Probing the spatial organization of nucleic acids within cells by nonisotopic in situ hybridization". Proceedings, annual meeting, Electron Microscopy Society of America 50, n.º 1 (agosto de 1992): 10–11. http://dx.doi.org/10.1017/s042482010012045x.

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In situ hybridization is a powerful experimental approach that directly couples molecular and cytological information in a visual context. Advances in hybridization procedures over recent years, coupled with previously described non-isotopic labelling methods developed in a number of laboratories, now provide a way to detect nucleic acids within cells with a high degree of resolution and sensitivity. Adaptations of this technology allow either DNA or RNA to be detected and visualized either with the light microscope, using fluorescence or colorimetric methods, or with the electron microscope using antibodies conjugated to gold or peroxidase. The potential applications of this technology are relevant to numerous areas of biomedical research and range from the more straightforward study of differential gene expression in single cells within a population to the precise localization of individual genes or RNAs within the cytoplasm or nucleus of a cell.

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