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Articoli di riviste sul tema "In-Situ fluorescence microscopy"

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Autore: Grafiati

Pubblicato: 25 maggio 2024

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1

Lu, Fang, Tingting Zhou, Yan Liu, Liying Song, Bin Zhang e 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 agosto 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.

2

SERWER, PHILIP, SHIRLEY J. HAYES, KAREN LIEMAN e GARY A. GRIESS. "In situ fluorescence microscopy of bacteriophage aggregates". Journal of Microscopy 228, n. 3 (dicembre 2007): 309–21. http://dx.doi.org/10.1111/j.1365-2818.2007.01855.x.

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3

Liv, Nalan, Daan S. B. van Oosten Slingeland, Jean-Pierre Baudoin, Pieter Kruit, David W. Piston e Jacob P. Hoogenboom. "Electron Microscopy of Living Cells During in Situ Fluorescence Microscopy". ACS Nano 10, n. 1 (8 dicembre 2015): 265–73. http://dx.doi.org/10.1021/acsnano.5b03970.

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4

Ballard, S. G., e D. C. Ward. "Fluorescence in situ hybridization using digital imaging microscopy." Journal of Histochemistry & Cytochemistry 41, n. 12 (dicembre 1993): 1755–59. http://dx.doi.org/10.1177/41.12.8245423.

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5

Bouffier, Laurent, e Thomas Doneux. "Coupling electrochemistry with in situ fluorescence (confocal) microscopy". Current Opinion in Electrochemistry 6, n. 1 (dicembre 2017): 31–37. http://dx.doi.org/10.1016/j.coelec.2017.06.015.

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6

Leger, I., M. Robert-Nicoud e 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. 2 (febbraio 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.

7

Reinhardt, Susanne C. M., Luciano A. Masullo, Isabelle Baudrexel, Philipp R. Steen, Rafal Kowalewski, Alexandra S. Eklund, Sebastian Strauss et al. "Ångström-resolution fluorescence microscopy". Nature 617, n. 7962 (24 maggio 2023): 711–16. http://dx.doi.org/10.1038/s41586-023-05925-9.

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AbstractFluorescence microscopy, with its molecular specificity, is one of the major characterization methods used in the life sciences to understand complex biological systems. Super-resolution approaches1–6 can achieve resolution in cells in the range of 15 to 20 nm, but interactions between individual biomolecules occur at length scales below 10 nm and characterization of intramolecular structure requires Ångström resolution. State-of-the-art super-resolution implementations7–14 have demonstrated spatial resolutions down to 5 nm and localization precisions of 1 nm under certain in vitro conditions. However, such resolutions do not directly translate to experiments in cells, and Ångström resolution has not been demonstrated to date. Here we introdue a DNA-barcoding method, resolution enhancement by sequential imaging (RESI), that improves the resolution of fluorescence microscopy down to the Ångström scale using off-the-shelf fluorescence microscopy hardware and reagents. By sequentially imaging sparse target subsets at moderate spatial resolutions of >15 nm, we demonstrate that single-protein resolution can be achieved for biomolecules in whole intact cells. Furthermore, we experimentally resolve the DNA backbone distance of single bases in DNA origami with Ångström resolution. We use our method in a proof-of-principle demonstration to map the molecular arrangement of the immunotherapy target CD20 in situ in untreated and drug-treated cells, which opens possibilities for assessing the molecular mechanisms of targeted immunotherapy. These observations demonstrate that, by enabling intramolecular imaging under ambient conditions in whole intact cells, RESI closes the gap between super-resolution microscopy and structural biology studies and thus delivers information key to understanding complex biological systems.

8

Collinson, Lucy M. "Smart Microscopy: Automation of CLEM using In situ Fluorescence Detection". Microscopy and Microanalysis 25, S2 (agosto 2019): 1018–19. http://dx.doi.org/10.1017/s1431927619005828.

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9

Arend, J., A. Wetzel e B. Middendorf. "In-situ-investigation of superplasticizer-particle-interaction by fluorescence microscopy". Materials Today: Proceedings 5, n. 7 (2018): 15292–97. http://dx.doi.org/10.1016/j.matpr.2018.05.008.

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10

Fetni, Raouf, Patrick Scott, Frédérique Tihy, Claude-Lise Richer e Nicole Lemieux. "Increased resolution of in situ hybridization signal by electron microscopy: A comparison with fluorescence microscopy". Genome 42, n. 5 (1 ottobre 1999): 1001–7. http://dx.doi.org/10.1139/g99-071.

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Cytogenetic studies by in situ hybridization (ISH) have proven to be valuable for gene mapping on banded chromosomes when combined with fluorescence microscopy (FISH). However, even under the best conditions, FISH technology has a resolving power inherent to light of just 0.2 µm. Its utilization is further limited by the diffusion of light coming from the fluorescent signal which covers an area considerably larger than the target DNA sequence. The development of new ISH protocols applied to electron microscopy (EMISH) should increase the resolution for cytogenetic mapping and fine chromosomal structure studies. Despite these advances, few attempts have been made which exploit this increased resolution. Here we present a detailed analysis of ISH signals obtained by fluorescence and electron microscopy methodologies to demonstrate and define the higher sensitivity obtainable by electron microscopy. This comparative study was conducted with probes of different origins: telomeric, classical satellite, alpha satellite, and single-copy DNA sequences, which provide a good reference point for later studies. We were also able to map a 200-bp cDNA probe by EMISH. This study assesses the nature of the resolution and the better definition of the EMISH signal, which confirms the greater resolution of electron microscopy as compared with that achieved with light microscopy. It also indicates that better delineation of two closely linked sequences is achieved at the electron microscopy level.Key words: In situ hybridization, electron microscopy, fluorescence microscopy, localization, repetitive and small single-copy probes.

11

Deerinck, Thomas J., Maryann E. Martone, Varda Lev-Ram, David P. L. Green, Roger Y. Tsien, David L. Spector, Sui Huang e 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.

12

Hoogenboom, Jacob P. "Super-Resolution Fluorescence in Electron Micrographs Using In-Situ Integrated Microscopy". Microscopy and Microanalysis 22, S5 (novembre 2016): 42–43. http://dx.doi.org/10.1017/s143192761601223x.

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13

Macville, Merryn, Ernst-Jan Speel, Dirk Soenksen, Ton Hopman e ThomasRied. "Spectral Imaging of Chromogenic Dyes in Cytological Specimens". Microscopy and Microanalysis 3, S2 (agosto 1997): 143–44. http://dx.doi.org/10.1017/s1431927600007601.

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Recently, spectral imaging has been successfully applied to 24-color fluorescence in situ hybridization (FISH), and it has evolved into a new powerful method for the analysis of structural and numerical chromosomal aberrations on metaphase spreads (spectral karyotyping) For cytological specimens it is often impossible to use fluorescence microscopy because of (fixation-induced) auto-fluorescence or prior cytological staining. Also, re-examination of archived fluorescent specimens is hampered by fading. However, the need for multi-parameter cytochemical analysis remains when rare or unique material is to be studied in research or clinical diagnosis. Multi-color bright-field microscopy using enzyme precipitates has become feasible for in situ hybridization as well as immunohistochemical detection. Conventional transmission microscopy optics have allowed up to three targets to be detected simultaneously. In parallel to 24-color FISH, we explored the potential of spectral imaging to increase the number of parameters to be analyzed in bright-field microscopy.

14

Arend, Johannes, Alexander Wetzel e Bernhard Middendorf. "Fluorescence Microscopy of Superplasticizers in Cementitious Systems: Applications and Challenges". Materials 13, n. 17 (24 agosto 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.

15

Thimm, Torsten, e Christoph C. Tebbe. "Protocol for Rapid Fluorescence In Situ Hybridization of Bacteria in Cryosections of Microarthropods". Applied and Environmental Microbiology 69, n. 5 (maggio 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.

16

Zieba, Agata, Carolina Wählby, Fredrik Hjelm, Lee Jordan, Jonathan Berg, Ulf Landegren e Katerina Pardali. "Bright-Field Microscopy Visualization of Proteins and Protein Complexes by In Situ Proximity Ligation with Peroxidase Detection". Clinical Chemistry 56, n. 1 (1 gennaio 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.

17

Pernthaler, Jakob, Annelie Pernthaler e Rudolf Amann. "Automated Enumeration of Groups of Marine Picoplankton after Fluorescence In Situ Hybridization". Applied and Environmental Microbiology 69, n. 5 (maggio 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.

18

Arend, J., A. Wetzel e B. Middendorf. "In-situ investigation of superplasticizers: From fluorescence microscopy to concrete rheology". Cement and Concrete Research 113 (novembre 2018): 178–85. http://dx.doi.org/10.1016/j.cemconres.2018.08.011.

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19

Brown, Koshonna, Ted Thurn, Lun Xin, William Liu, Remon Bazak, Si Chen, Barry Lai et al. "Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection". Nano Research 11, n. 1 (19 luglio 2017): 464–76. http://dx.doi.org/10.1007/s12274-017-1654-8.

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20

Jovin, Thomas M., Michel Robert-Nicoud, Donna J. Arndt-Jovin e 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.

21

Smeets, Marit, Anna Bieber, Cristina Capitanio, Oda Schioetz, Thomas van der Heijden, Andries Effting, Éric Piel, Bassim Lazem, Philipp Erdmann e Juergen Plitzko. "Integrated Cryo-Correlative Microscopy for Targeted Structural Investigation In Situ". Microscopy Today 29, n. 6 (novembre 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.

22

Tawa, Keiko, Chikara Yasui, Chie Hosokawa, Hiroyuki Aota e Junji Nishii. "In Situ Sensitive Fluorescence Imaging of Neurons Cultured on a Plasmonic Dish Using Fluorescence Microscopy". ACS Applied Materials & Interfaces 6, n. 22 (23 ottobre 2014): 20010–15. http://dx.doi.org/10.1021/am505579u.

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23

Al-Ahmad, Ali, Marie Follo, Ann-Carina Selzer, Elmar Hellwig, Matthias Hannig e Christian Hannig. "Bacterial colonization of enamel in situ investigated using fluorescence in situ hybridization". Journal of Medical Microbiology 58, n. 10 (1 ottobre 2009): 1359–66. http://dx.doi.org/10.1099/jmm.0.011213-0.

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Oral biofilms are one of the greatest challenges in dental research. The present study aimed to investigate initial bacterial colonization of enamel surfaces in situ using fluorescence in situ hybridization (FISH) over a 12 h period. For this purpose, bovine enamel slabs were fixed on buccal sites of individual splints worn by six subjects for 2, 6 and 12 h to allow biofilm formation. Specimens were processed for FISH and evaluated with confocal laser-scanning microscopy, using probes for eubacteria, Streptococcus species, Veillonella species, Fusobacterium nucleatum and Actinomyces naeslundii. The number of adherent bacteria increased with time and all tested bacterial species were detected in the biofilm formed in situ. The general percentage composition of the eubacteria did not change over the investigated period, but the number of streptococci, the most frequently detected species, increased significantly with time (2 h: 17.7±13.8 %; 6 h: 20.0±16.6 %; 12 h: 24.7±16.1 %). However, ≤1 % of the surface was covered with bacteria after 12 h of biofilm formation in situ. In conclusion, FISH is an appropriate method for quantifying initial biofilm formation in situ, and the proportion of streptococci increases during the first 12 h of bacterial adherence.

24

Zhang, Wenbo, Zihe Zhai, Shifen Li, Xue Lin, Wei Bai, Ning Ding, Yue Zhang, Jiaqi Tong, Jingzhi Sun e Changyou Gao. "In situ formation of tetraphenylethylene nano-structures on microgels inside living cells via reduction-responsive self-assembly". Nanoscale 13, n. 1 (2021): 138–49. http://dx.doi.org/10.1039/d0nr06661c.

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25

Deerinck, T. J., M. E. Martone, V. Lev-Ram, D. P. Green, R. Y. Tsien, D. L. Spector, S. Huang e 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. 4 (15 agosto 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.

26

Huang, Z., W. You, R. P. Haugland, V. B. Paragas, N. A. Olson e R. P. Haugland. "A novel fluorogenic substrate for detecting alkaline phosphatase activity in situ." Journal of Histochemistry & Cytochemistry 41, n. 2 (febbraio 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.

27

Pelicci, Simone, Laura Furia, Pier Giuseppe Pelicci e Mario Faretta. "Correlative Multi-Modal Microscopy: A Novel Pipeline for Optimizing Fluorescence Microscopy Resolutions in Biological Applications". Cells 12, n. 3 (17 gennaio 2023): 354. http://dx.doi.org/10.3390/cells12030354.

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The modern fluorescence microscope is the convergence point of technologies with different performances in terms of statistical sampling, number of simultaneously analyzed signals, and spatial resolution. However, the best results are usually obtained by maximizing only one of these parameters and finding a compromise for the others, a limitation that can become particularly significant when applied to cell biology and that can reduce the spreading of novel optical microscopy tools among research laboratories. Super resolution microscopy and, in particular, molecular localization-based approaches provide a spatial resolution and a molecular localization precision able to explore the scale of macromolecular complexes in situ. However, its use is limited to restricted regions, and consequently few cells, and frequently no more than one or two parameters. Correlative microscopy, obtained by the fusion of different optical technologies, can consequently surpass this barrier by merging results from different spatial scales. We discuss here the use of an acquisition and analysis correlative microscopy pipeline to obtain high statistical sampling, high content, and maximum spatial resolution by combining widefield, confocal, and molecular localization microscopy.

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Shah, Jyotsna S., e Ranjan Ramasamy. "Fluorescence In Situ Hybridization (FISH) Tests for Identifying Protozoan and Bacterial Pathogens in Infectious Diseases". Diagnostics 12, n. 5 (21 maggio 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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De Vries, J. E., F. H. Kornips, J. Wiegant, P. M. Moerkerk, N. Senden, B. Schutte, J. P. Geraedts, F. T. Bosman e J. Ten Kate. "Chromosomal localization of transfected genes by a combination of hot banding and fluorescence in situ hybridization." Journal of Histochemistry & Cytochemistry 40, n. 7 (luglio 1992): 1053–58. http://dx.doi.org/10.1177/40.7.1607638.

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We describe the combination of hot banding with fluorescence in situ hybridization as a rapid and efficient method to identify integration sites of transfected DNA sequences in chromosomes. As a test system we used SW480 EJ2, a clonal cell line obtained after transfection of SW480 with pSV2neoEJ, a plasmid containing a point-mutated, c-Ha-RAS oncogene. Nick-translated probes were compared with random primed-labeled probes to evaluate their relative efficiency in fluorescence in situ hybridization. The fluorescence signals were quantified in interphase nuclei by confocal scanning laser microscopy. Nick-translated probes were found to yield better results. Hot banding followed by fluorescence in situ hybridization localized the integration site of pSV2neoEJ in SW480 EJ2 at the site of a translocation on a marker chromosome Xp+. The combination of fluorescence in situ hybridization and hot banding can be used to (a) rapidly and efficiently analyze integration sites in large numbers of transfectants, (b) assess the clonality of transfected cell lines, and (c) localize the site of integration of transfected genes in the recipient genome.

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Pasulka, Alexis L., Amy L. Howes, Julia G. Kallet, Jennifer VanderKelen e Clayton Villars. "Visualization of probiotics via epifluorescence microscopy and fluorescence in situ hybridization (FISH)". Journal of Microbiological Methods 182 (marzo 2021): 106151. http://dx.doi.org/10.1016/j.mimet.2021.106151.

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Franco, Juliana C., Grasiele Gonçalves, Monique S. Souza, Samantha B. C. Rosa, Larissa M. Thiegue, Teresa D. Z. Atvars, Paulo T. V. Rosa e René A. Nome. "Towards in situ fluorescence spectroscopy and microscopy investigations of asphaltene precipitation kinetics". Optics Express 21, n. 25 (6 dicembre 2013): 30874. http://dx.doi.org/10.1364/oe.21.030874.

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Kneen, Malea M., Damien G. Harkin, Lesley L. Walker, Daine Alcorn e P. J. Harris. "Imaging of renal medullary interstitial cells in situ by confocal fluorescence microscopy". Anatomy and Embryology 200, n. 1 (20 maggio 1999): 117–21. http://dx.doi.org/10.1007/s004290050265.

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Bizzotto, Dan. "In situ spectroelectrochemical fluorescence microscopy for studying electrodes modified by molecular adsorbates". Current Opinion in Electrochemistry 7 (gennaio 2018): 161–71. http://dx.doi.org/10.1016/j.coelec.2017.11.019.

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Lin, Ran, Donald C. Chang e Yi-Kuen Lee. "Single-cell electroendocytosis on a micro chip using in situ fluorescence microscopy". Biomedical Microdevices 13, n. 6 (29 luglio 2011): 1063–73. http://dx.doi.org/10.1007/s10544-011-9576-9.

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Tawa, Keiko, e Kenichi Morigaki. "In situ imaging of micropatterned phospholipid membranes by surface plasmon fluorescence microscopy". Colloids and Surfaces B: Biointerfaces 81, n. 2 (dicembre 2010): 447–51. http://dx.doi.org/10.1016/j.colsurfb.2010.07.038.

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Yang, Fan, Xiaodong Ju, Yanhong Zeng, Xiaoke Tian, Xin Zhang, Jianquan Wang e Hongjie Huang. "In situ observation of cartilage matrix based on two-photon fluorescence microscopy". Biochemical and Biophysical Research Communications 682 (novembre 2023): 64–70. http://dx.doi.org/10.1016/j.bbrc.2023.09.057.

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Tanaka, Hideaki, Takahisa Akatsuka, Toru Ohe, Yoshiro Ogoma, Koji Abe e Yoshiyuki Kondo. "In situ observation of protein-adsorbed stearic acid monolayer by Brewster angle microscopy and fluorescence microscopy". Polymers for Advanced Technologies 9, n. 2 (febbraio 1998): 150–54. http://dx.doi.org/10.1002/(sici)1099-1581(199802)9:2<150::aid-pat743>3.0.co;2-n.

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Moraru, Cristina, e Rudolf Amann. "Crystal ball: Fluorescence in situ hybridization in the age of super-resolution microscopy". Systematic and Applied Microbiology 35, n. 8 (dicembre 2012): 549–52. http://dx.doi.org/10.1016/j.syapm.2012.10.001.

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Suh, Youngjoon, Hamsa Gowda e Yoonjin Won. "In situ investigation of particle clustering dynamics in colloidal assemblies using fluorescence microscopy". Journal of Colloid and Interface Science 576 (settembre 2020): 195–202. http://dx.doi.org/10.1016/j.jcis.2020.04.054.

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Dibbern-Brunelli, D., e T. D. Z. Atvars. "In situ chemical analysis of domains in polymer biends by optical fluorescence microscopy". Journal of Applied Polymer Science 58, n. 4 (24 ottobre 1995): 779–86. http://dx.doi.org/10.1002/app.1995.070580410.

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41

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

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Abstract (sommario):

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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Tian, Rui, Kaitao Li, Wenying Shi, Caifeng Ding e Chao Lu. "In situ visualization of hydrophilic spatial heterogeneity inside microfluidic chips by fluorescence microscopy". Lab on a Chip 19, n. 6 (2019): 934–40. http://dx.doi.org/10.1039/c8lc01336e.

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43

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

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Abstract (sommario):

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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Tao, W., M. Soonpaa, G. Keller, H. Reinecke, C. Murry, L. Field e M. Rubart. "In Situ Multi-Photon Fluorescence Microscopy for Functional Screening of Intracardiac Cell Implants". Microscopy and Microanalysis 19, S2 (agosto 2013): 4–5. http://dx.doi.org/10.1017/s1431927613002018.

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Thakur, Deepa, Pawan Kumar e Viswanath Balakrishnan. "Phase selective CVD growth and photoinduced 1T → 1H phase transition in a WS2 monolayer". Journal of Materials Chemistry C 8, n. 30 (2020): 10438–47. http://dx.doi.org/10.1039/d0tc02037k.

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Abstract (sommario):

We report the direct chemical vapour deposition (CVD) growth of the metastable 1T phase of a WS₂ monolayer and the in situ phase transition characteristics with the aid of Raman, photoluminescence and fluorescence microscopy.

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Gruβmayer, K. S., K. Yserentant e D.-P. Herten. "Photons in - numbers out: perspectives in quantitative fluorescence microscopy for in situ protein counting". Methods and Applications in Fluorescence 7, n. 1 (15 gennaio 2019): 012003. http://dx.doi.org/10.1088/2050-6120/aaf2eb.

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Ma, Jianfeng, Zhe Ji, Xia Zhou, Zhiheng Zhang e Feng Xu. "Transmission Electron Microscopy, Fluorescence Microscopy, and Confocal Raman Microscopic Analysis of Ultrastructural and Compositional Heterogeneity of Cornus alba L. Wood Cell Wall". Microscopy and Microanalysis 19, n. 1 (febbraio 2013): 243–53. http://dx.doi.org/10.1017/s1431927612013906.

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Abstract (sommario):

AbstractTransmission electron microscopy (TEM), fluorescence microscopy, and confocal Raman microscopy can be used to characterize ultrastructural and compositional heterogeneity of plant cell walls. In this study, TEM observations revealed the ultrastructural characterization of Cornus alba L. fiber, vessel, axial parenchyma, ray parenchyma, and pit membrane between cells, notably with the ray parenchyma consisting of two well-defined layers. Fluorescence microscopy evidenced that cell corner middle lamella was more lignified than adjacent compound middle lamella and secondary wall with variation in lignification level from cell to cell. In situ Raman images showed that the inhomogeneity in cell wall components (cellulose and lignin) among different cells and within morphologically distinct cell wall layers. As the significant precursors of lignin biosynthesis, the pattern of coniferyl alcohol and aldehyde (joint abbreviation Lignin-CAA for both structures) distribution in fiber cell wall was also identified by Raman images, with higher concentration occurring in the fiber secondary wall where there was the highest cellulose concentration. Moreover, noteworthy was the observation that higher concentration of lignin and very minor amounts of cellulose were visualized in the pit membrane areas. These complementary microanalytical methods provide more accurate and complete information with regard to ultrastructural and compositional characterization of plant cell walls.

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Robinson, J. M., e T. Takizawa. "Biological Labeling and Correlative Microscopy". Microscopy and Microanalysis 5, S2 (agosto 1999): 474–75. http://dx.doi.org/10.1017/s1431927600015695.

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Abstract (sommario):

A variety of biological labeling techniques has been developed in order to obtain specific chemical and spatial information from cells and tissues. Traditionally theses labeling techniques have been categorized as cytochemistry, immunocytochemistry, and in situ hybridization. Another special category relates to fluorescence analog cytochemistry in which specific fluorescently-labeled molecules become incorporated into the pool of endogenous molecules of the cell. They can thus serve as reporters for analysis of the dynamic properties of the population of molecules of interest. Such molecules are usually introduced into cells by microinjection or expressed within the cell (e.g., green fluorescent protein derivatives).The past few years have witnessed a renaissance in biological optical microscopy. Many of the advances in the elucidation of cell structure-function relationships made through the use of optical microscopy have relied upon fluorescence labeling technology. These advances notwithstanding there remain experimental situations in cell biology that require the higher spatial resolution afforded by electron microscopy. Combining fluorescence and electron microscopy to study the same structures would be very useful in many experimental situations in cell biology. Such an examination of the same structures with more than one imaging modality can be referred to as correlative or integrated microscopy. The number of such studies is relatively small; this is probably due to technical difficulties encountered by various investigators.

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CALLEJA, Véronique, Simon M. AMEER-BEG, Borivoj VOJNOVIC, Rudiger WOSCHOLSKI, Julian DOWNWARD e Banafshé LARIJANI. "Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy". Biochemical Journal 372, n. 1 (15 maggio 2003): 33–40. http://dx.doi.org/10.1042/bj20030358.

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Abstract (sommario):

To be able to detect in situ changes in protein conformation without perturbing the physiological environment would be a major step forward in understanding the precise mechanism occurring in protein interaction. We have developed a novel approach to monitoring conformational changes of proteins in intact cells. A double-labelled fluorescent green fluorescent protein–yellow fluorescent protein (GFP–YFP) fusion protein has been constructed, allowing the exploitation of enhanced-acceptor-fluorescence (EAF)-induced fluorescence resonance energy transfer (FRET). Additionally, a novel fusion partner, YFPdark, has been designed to act as a sterically hindered control for EAF-FRET. Any conformational changes will cause a variation in FRET, which, in turn, is detected by fluorescence lifetime imaging microscopy (‘FLIM’). Protein kinase B (PKB)/Akt, a key component of phosphoinositide 3-kinase-mediated signalling, was selected for this purpose. Although conformational changes in PKB/Akt consequent to lipid binding and phosphorylation have been proposed in models, its behaviour in intact cells has not been tractable. We report here that platelet-derived-growth-factor (‘PDGF’) stimulation of NIH3T3 cells expressing the GFP–Akt–YFP construct resulted in a loss of FRET at the plasma membrane and hence a change in PKB/Akt conformation. We also show that the GFP–Akt–YFP construct conserves fully its functional integrity. This novel approach of monitoring the in situ conformational changes has broad application for other members of the AGC kinase superfamily and other proteins.

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Caldwell, Kyle, e John C. Berg. "Direct observation of nanoparticle migration in epoxy based fiber reinforced composites using fluorescence microscopy". Journal of Composite Materials 51, n. 28 (17 febbraio 2017): 3877–85. http://dx.doi.org/10.1177/0021998317694704.

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Abstract (sommario):

The preferential accumulation of nanoparticles at carbon fiber surfaces, induced by the addition of a thermoplastic “migrating agent” to an epoxy resin, was monitored via in situ fluorescence microscopy. (3-Glydidyloxypropyl)trimethoxysilane functionalized fluorescent silica nanoparticles (GFCSP) were synthesized by a modified Stöber method to track the spatiotemporal abundance of nanoparticles. Single carbon fibers were embedded in an uncured epoxy mixture consisting of tetraglycidyl-4,4-diaminodiphenylmethane and 4,4’-diamnodiphenyl sulfone, as well as thermoplastic migrating agent, poly(ether sulfone), and GFCSP. A heated microscope stage was used to monitor the fluorescence in the local vicinity of the fiber as the epoxy begins to cross-link and solidify upon heating. Our results show that the synthesized GFCSP accumulate at fiber surfaces only in the presence of poly(ether sulfone), as verified using scanning electron micrographs of Mode I fiber fracture surfaces.