Articoli di riviste: "Confocal imaging on living cells"

1

Williams, D. A., S. H. Cody, C. A. Gehring, R. W. Parish e P. J. Harris. "Confocal imaging of ionised calcium in living plant cells". Cell Calcium 11, n. 4 (aprile 1990): 291–97. http://dx.doi.org/10.1016/0143-4160(90)90006-g.

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Filić, Vedrana, e Igor Weber. "A young researcher’s guide to three-dimensional fluorescence microscopy of living cells". Periodicum Biologorum 125, n. 1-2 (25 ottobre 2023): 133–37. http://dx.doi.org/10.18054/pb.v125i1-2.25140.

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Abstract (sommario):

Three-dimensional imaging of fast intracellular processes by fluorescence microscopy should provide decent spatial and high temporal resolution while minimizing fluorophore bleaching and cytotoxicity. We give a condensed introductory overview of three contemporary methods mostly used for imaging of living cells in 3D and compare their performance in terms of temporal and spatial resolution, imaging flexibility and specimen photodamage: point-scanning confocal microscopy, spinning-disc confocal microscopy, and lattice light-sheet microscopy. While point-scanning instruments are unsurpassed in terms of confocal performance, flexibility and configurability of their optical path, spinning-disc and lattice light-sheet optical designs excel in acquisition speed and low levels of light-inflicted specimen deterioration.

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SCHWARZLÄNDER, M., M. D. FRICKER, C. MÜLLER, L. MARTY, T. BRACH, J. NOVAK, L. J. SWEETLOVE, R. HELL e A. J. MEYER. "Confocal imaging of glutathione redox potential in living plant cells". Journal of Microscopy 231, n. 2 (agosto 2008): 299–316. http://dx.doi.org/10.1111/j.1365-2818.2008.02030.x.

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He, Fang, Ze-Yu Ye, Li-Dong Zhao, Bin-Cheng Yin e Bang-Ce Ye. "Probing exosome internalization pathways through confocal microscopy imaging". Chemical Communications 55, n. 93 (2019): 14015–18. http://dx.doi.org/10.1039/c9cc07491k.

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Zoladek, A., F. Pascut, P. Patel e I. Notingher. "Development of Raman Imaging System for time-course imaging of single living cells". Spectroscopy 24, n. 1-2 (2010): 131–36. http://dx.doi.org/10.1155/2010/521962.

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Development of novel inverted Raman micro-spectrometer with the ability to perform multi-hours spectral measurements on living cells is presented. Our system combines a Confocal Raman Micro-Spectrometer and Fluorescence Microscope with cell incubator enclosure allowing measurement of cells in extended time period. To illustrate the feasibility of this Raman micro-spectroscopy system forin vitrotime-course studies of cells we performed an experiment where the same group of cells were scanned with the laser at 2 hours intervals between the scans over 8 hours to build Raman spectral images and ensure that no changes occur due to laser damage or environmental conditions. Cell viability test was performed with fluorescence microscopy on exactly the same cells at the end of the time-course Raman measurements.

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Skiba, Joanna, Aleksandra Kowalczyk, Marta A. Fik, Magdalena Gapińska, Damian Trzybiński, Krzysztof Woźniak, Valerije Vrček, Rafał Czerwieniec e Konrad Kowalski. "Luminescent pyrenyl-GNA nucleosides: synthesis, photophysics and confocal microscopy studies in cancer HeLa cells". Photochemical & Photobiological Sciences 18, n. 10 (2019): 2449–60. http://dx.doi.org/10.1039/c9pp00271e.

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He, Zhaoshuai, Yajie Chou, Hanxin Zhou, Han Zhang, Tanyu Cheng e Guohua Liu. "A nitroreductase and acidity detecting dual functional ratiometric fluorescent probe for selectively imaging tumor cells". Organic & Biomolecular Chemistry 16, n. 17 (2018): 3266–72. http://dx.doi.org/10.1039/c8ob00670a.

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A dual functional ratiometric fluorescent probe can obviously distinguish acidity, nitroreductase, and nitroreductase in an acidic environment. Confocal fluorescence imaging of A549 cells indicates the probe can detect acidity and expressed nitroreductase in living cells.

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WANG, XIAO-PING, HUAI-NA YU e TONG-SHENG CHEN. "QUANTITATIVE FRET MEASUREMENT BASED ON CONFOCAL MICROSCOPY IMAGING AND PARTIAL ACCEPTOR PHOTOBLEACHING". Journal of Innovative Optical Health Sciences 05, n. 03 (luglio 2012): 1250015. http://dx.doi.org/10.1142/s1793545812500150.

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Fluorescence resonance energy transfer (FRET) technology had been widely used to study protein–protein interactions in living cells. In this study, we developed a ROI-PbFRET method to real-time quantitate the FRET efficiency of FRET construct in living cells by combining the region of interest (ROI) function of confocal microscope and partial acceptor photobleaching. We validated the ROI-PbFRET method using GFPs-based FRET constructs including 18AA and SCAT3, and used it to quantitatively monitor the dynamics of caspase-3 activation in single live cells stably expressing SCAT3 during staurosporine (STS)-induced apoptosis. Our results for the first demonstrate that ROI-PbFRET method is a powerful potential tool for detecting the dynamics of molecular interactions in live cells.

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Okuno, Masanari, e Hiro-o. Hamaguchi. "Multifocus confocal Raman microspectroscopy for fast multimode vibrational imaging of living cells". Optics Letters 35, n. 24 (2 dicembre 2010): 4096. http://dx.doi.org/10.1364/ol.35.004096.

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Feofanov, Alexei V., Alexei I. Grichine, Larissa A. Shitova, Tatyana A. Karmakova, Raisa I. Yakubovskaya, Marguerite Egret-Charlier e Paul Vigny. "Confocal Raman Microspectroscopy and Imaging Study of Theraphthal in Living Cancer Cells". Biophysical Journal 78, n. 1 (gennaio 2000): 499–512. http://dx.doi.org/10.1016/s0006-3495(00)76612-4.

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11

Cooper, M. S. "Imaging intracellular membrane traffic and organelle dynamics using confocal microscopy". Proceedings, annual meeting, Electron Microscopy Society of America 49 (agosto 1991): 390–91. http://dx.doi.org/10.1017/s0424820100086258.

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Movements of exocytic and endocytic vesicles within cells are controlled in part by active transport along microtubules. The forces generated by microtubule-based transport are also known to strongly influence the distribution and shapes of membranous organelles, such as mitochondria and the endoplasmic reticulum. The ability to observe such dynamic interactions between cytoskeletal elements and fluorescently-labelled intracellular membranes in living cells has been improved recently by the advent of scanning laser confocal microscopy. Here we describe the use of this technique to study activities of the Golgi apparatus in living cells.Vesicular traffic, diffusive transport and morphological dynamics within the Golgi apparatus of cultured rat hippocampal astrocytes were imaged with a Bio-Rad MRC-500 scanning laser confocal microscope. Golgi elements were labelled with NBD-ceramide, a molecule which serves as a vital stain for the trans-most cisternae of Golgi stacks. Single scans of the specimen, obtained with the laser microscope's slow scan rate (4 sec/image), were stored sequentially on an optical memory disk recorder.

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Jester, J. V., H. D. Cavanagh e M. A. Lemp. "In vivo confocal imaging of the eye using tandem scanning confocal microscopy (TSCM)". Proceedings, annual meeting, Electron Microscopy Society of America 46 (1988): 56–57. http://dx.doi.org/10.1017/s0424820100102365.

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New developments in optical microscopy involving confocal imaging are now becoming available which dramatically increase resolution, contrast and depth of focus by optically sectioning through structures. The transparency of the anterior ocular structures, cornea and lens, make microscopic visualization and optical sectioning of the living intact eye an interesting possibility. Of the confocal microscopes available, the Tandem Scanning Reflected Light Microscope (referred to here as the Tandem Scanning Confocal Microscope), developed by Professors Petran and Hadravsky at Charles University in Pilzen, Czechoslovakia, permits real-time image acquisition and analysis facilitating in vivo studies of ocular structures.Currently, TSCM imaging is most successful for the cornea. The corneal epithelium, stroma, and endothelium have been studied in vivo and photographed in situ. Confocal scanning images of the superficial epithelium, similar to those obtained by scanning electron microscopy, show both light and dark surface epithelial cells.

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Nitschke, Roland, e Kenneth R. Spring. "Electro-optical Wavelength Selection Enables Confocal Ratio Imaging at Low Light Levels". Microscopy and Microanalysis 1, n. 1 (febbraio 1995): 1–11. http://dx.doi.org/10.1017/s1431927695110016.

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A confocal attachment (Odyssey) to an inverted microscope was modified to better study living cultured epithelial cells stained with fluorescent dyes. Improvements to the instrument included elimination of light leaks, improved electronic shielding, reduction of thermal effects, and use of low dark current detectors. In addition, rapid changes in illumination wavelength and power were accomplished by replacing the original mechanical filter changer by an acousto-optic tunable filter attached to the argon laser light source. The addition of a liquid crystal tunable filter to one of the two photomultiplier detectors also permitted rapid spectral scanning of the fluorescence emission. High-resolution, differential interference contrast transmitted light images were formed simultaneously by replacement of the photodiode-based transmitted light detector with a photomultiplier tube and dichroic mirror assembly. An illumination intensity of only 40 μW/cm2 at the back focal plane of the microscope objective allowed high-quality fluorescence and transmitted light images of living cells at video rates with minimal bleaching and photodynamic damage. Both excitation ratio imaging and emission spectral scanning of living epithelial cells were accomplished. The system performance was evaluated by optical sections of fluorescent beads and thin films.

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Quentin, Christine, Rūta Gerasimaitė, Alexandra Freidzon, Levon S. Atabekyan, Gražvydas Lukinavičius, Vladimir N. Belov e Gyuzel Y. Mitronova. "Direct Visualization of Amlodipine Intervention into Living Cells by Means of Fluorescence Microscopy". Molecules 26, n. 10 (18 maggio 2021): 2997. http://dx.doi.org/10.3390/molecules26102997.

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Amlodipine, a unique long-lasting calcium channel antagonist and antihypertensive drug, has weak fluorescence in aqueous solutions. In the current paper, we show that direct visualization of amlodipine in live cells is possible due to the enhanced emission in cellular environment. We examined the impact of pH, polarity and viscosity of the environment as well as protein binding on the spectral properties of amlodipine in vitro, and used quantum chemical calculations for assessing the mechanism of fluorescence quenching in aqueous solutions. The confocal fluorescence microscopy shows that the drug readily penetrates the plasma membrane and accumulates in the intracellular vesicles. Visible emission and photostability of amlodipine allow confocal time-lapse imaging and the drug uptake monitoring.

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Loren Buhle, E., Bernard Himpens, Avril V. Somlyo, Henry Shuman e Andrew P. Somlyo. "3D biological imaging using TSRLM confocal microscopy". Proceedings, annual meeting, Electron Microscopy Society of America 45 (agosto 1987): 638–39. http://dx.doi.org/10.1017/s0424820100127621.

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Tandem scanning reflected light (TSRLM) confocal microscopy permits the imaging of optical sections through translucent material in real time. We are currently using a TSRLM microscope to examine the morphological components of biological tissue. Optical sections of either stained or unstained biological specimens can be rapidly obtained by a through focus series of a potentially dynamic specimen. Each optical section shown here is an average of 256 512X512x8 bit frames collected from a silicon intensified target camera at video rates.Unstained living frog muscle fibers were imaged with sufficient contrast to obtain 1μ optical sections traversing a total distance of approximately 80μ. Fig. 1 shows 4μ sections through a blood vessel. Frog red cells can be clearly seen in panels 1c-e. Frog muscle fibers were mounted and stretched to a defined length. The total fiber volume was easily imaged by progressive 1μ sections.

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RoldÃ¯Â¿Â½n, M., F. Thomas, S. Castel, A. Quesada e M. HernÃ¯Â¿Â½ndez-MarinÃ¯Â¿Â½. "Noninvasive Pigment Identification in Single Cells from Living Phototrophic Biofilms by Confocal Imaging Spectrofluorometry". Applied and Environmental Microbiology 70, n. 6 (giugno 2004): 3745–50. http://dx.doi.org/10.1128/aem.70.6.3745-3750.2004.

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ABSTRACT A new imaging technique for the analysis of fluorescent pigments from a single cell is reported. It is based on confocal scanning laser microscopy coupled with spectrofluorometric methods. The setup allows simultaneous establishment of the relationships among pigment analysis in vivo, morphology, and three-dimensional localization inside thick intact microbial assemblages.

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Keene, Douglas R., Sara F. Tufa, Gregory P. Lunstrum, Paul Holden e William A. Horton. "Confocal/TEM Overlay Microscopy: A Simple Method for Correlating Confocal and Electron Microscopy of Cells Expressing GFP/YFP Fusion Proteins". Microscopy and Microanalysis 14, n. 4 (4 luglio 2008): 342–48. http://dx.doi.org/10.1017/s1431927608080306.

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Genetic manipulation allows simultaneous expression of green fluorescent protein (GFP) and its derivatives with a wide variety of cellular proteins in a variety of living systems. Epifluorescent and confocal laser scanning microscopy (confocal) localization of GFP constructs within living tissue and cell cultures has become routine, but correlation of light microscopy and high resolution transmission electron microscopy (TEM) on components within identical cells has been problematic. In this study, we describe an approach that specifically localizes the position of GFP/yellow fluorescent protein (YFP) constructs within the same cultured cell imaged in the confocal and transmission electron microscopes. We present a simplified method for delivering cell cultures expressing fluorescent fusion proteins into LR White embedding media, which allows excellent GFP/YFP detection and also high-resolution imaging in the TEM. Confocal images from 0.5-μm-thick sections are overlaid atop TEM images of the same cells collected from the next serial ultrathin section. The overlay is achieved in Adobe Photoshop by making the confocal image somewhat transparent, then carefully aligning features within the confocal image over the same features visible in the TEM image. The method requires no specialized specimen preparation equipment; specimens are taken from live cultures to embedding within 8 h, and confocal transmission overlay microscopy can be completed within a few hours.

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Tao, Ran, e Tao Zhang. "Design of a Scanning Module in a Confocal Microscopic Imaging System for Live-Cell Imaging". Photonics 11, n. 1 (28 dicembre 2023): 26. http://dx.doi.org/10.3390/photonics11010026.

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This study proposes a Nipkow-based pinhole disk laser scanning confocal microscopic imaging system for ordinary optical microscopy, fluorescence microscopy, and confocal microscopy imaging of biological samples in order to realize the dynamic experimental monitoring of space-based life science experiments and the fine observation of biological samples. Confocal microscopic imaging is mainly completed by a scanning module that is composed of a spinning disk and other components. The parameters of the spinning disk directly determine the quality of the image. During the design process, the resolution and signal-to-noise ratios caused by different pinhole diameters in the spinning disk are the main considerations. Changes and image blurring caused by crosstalk due to the pinhole arrangement and different pinhole spacings are addressed. The high photon efficiency of the new EMCCD (electron-multiplying charge-coupled device) and CMOS (complementary metal-oxide-semiconductor) camera reduces the exposure time as much as possible, reduces damage to living cells, and achieves high-speed confocal imaging. It is shown in a confocal imaging experiment with a variable magnification of 1–40× that the imaging resolution of the system can reach a maximum of 2592 × 1944, the spatial resolution can reach 1 μm, and the highest sampling frequency is 10 fps, thus meeting the design requirements for high-speed live-cell imaging.

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Turner, J. N., D. H. Szarowski, M. Fejtl, S. N. Ayrapetyan e D. O. Carpenter. "Confocal light microscopy and electrophysiology of neurons in culture". Proceedings, annual meeting, Electron Microscopy Society of America 49 (agosto 1991): 400–401. http://dx.doi.org/10.1017/s0424820100086301.

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Cell volume changes and regulation are thought to be important in the physiology and pathology of neurons. Osmotic challenges alter a number of physiologic parameters including electrical properties, chemosensitivity, and Na pump activity. Thus, a good method for correlating images of the living cell's surface with electrophysiologic measures is needed, and we are exploring the use of confocal light microscopy for imaging neurons in culture.Isolated neurons from ganglia of Aplvsia California were plated on polylysine coated coverslips in L15 media with 20% hemolymph at room temperature. Cells stained with “Dil” were imaged with a Bio—Rad MRC—600 and an Olympus BH—2. Images were displayed using Bio—Rad's maximum projection procedure, or Vital Images’ Voxel View software run on an IBM RISC 6000 Powerstation 320. Electrophysiologic properties were assessed after imaging.

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Gonçalves, Raquel C. R., Efres Belmonte-Reche, João Pina, Milene Costa da Silva, Sónia C. S. Pinto, Juan Gallo, Susana P. G. Costa e M. Manuela M. Raposo. "Bioimaging of Lysosomes with a BODIPY pH-Dependent Fluorescent Probe". Molecules 27, n. 22 (20 novembre 2022): 8065. http://dx.doi.org/10.3390/molecules27228065.

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Fluorescence-based probes represent a powerful tool for noninvasive imaging of living systems in real time and with a high temporal and spatial resolution. Amongst several known fluorophores, 3-difluoroborodipyrromethene (BODIPY) derivatives have become a cornerstone for innovative fluorescent labelling applications, mainly due to their advantageous features including their facile synthesis, structural versatility and exceptional photophysical properties. In this context, we report a BODIPY-based fluorescent probe for imaging of lysosomes in living cells. The BODIPY derivative displayed a remarkable fluorescence enhancement at low pH values with a pKa* of 3.1. In vitro studies by confocal microscopy in HeLa cells demonstrated that the compound was able to permeate cell membrane and selectively label lysosome whilst remaining innocuous to the cell culture at the maximum concentration tested. Herein, the BODIPY derivative holds the promise of investigating lysosomal dynamics and function in living cells through fluorescence imaging.

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Cooper, M. S. "Imaging cellular dynamics using scanning laser confocal microscopy". Proceedings, annual meeting, Electron Microscopy Society of America 50, n. 1 (agosto 1992): 12–13. http://dx.doi.org/10.1017/s0424820100120461.

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In recent years, the ability to image morphological dynamics and physiological changes in living cells and tissues has been greatly advanced by the advent of scanning laser confocal microscopy. Confocal microscopes employ optical systems in which both the condenser and objective lenses are focused onto a single volume element of the specimen. In practice, galvanometer-driven mirrors or acousto-optical deflectors are used to scan a laser beam over the specimen in a raster-like fashion through an epifluorescence microscope. The incident laser beam, as well as the collected fluorescent light, are passed through pinhole or slit apertures in image planes that are conjugate to the plane of the specimen. This method of illumination and detection prevents fluorescent light which is generated above and below the plane-of-focus from impinging on the imaging system's photodetector, thus rejecting much of the fluorescent light which normally blurs the image of a three-dimensional fluorescent specimen.

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Roberti, M. Julia, Thomas M. Jovin e Elizabeth Jares-Erijman. "Confocal Fluorescence Anisotropy and FRAP Imaging of α-Synuclein Amyloid Aggregates in Living Cells". PLoS ONE 6, n. 8 (8 agosto 2011): e23338. http://dx.doi.org/10.1371/journal.pone.0023338.

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Zhang, D., P. Wadsworth e P. K. Hepler. "Microtubule dynamics in living dividing plant cells: confocal imaging of microinjected fluorescent brain tubulin." Proceedings of the National Academy of Sciences 87, n. 22 (1 novembre 1990): 8820–24. http://dx.doi.org/10.1073/pnas.87.22.8820.

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SAUX, B. LE, B. CHALMOND, Y. YU, A. TROUVÉ, O. RENAUD e S. L. SHORTE. "Isotropic high-resolution three-dimensional confocal micro-rotation imaging for non-adherent living cells". Journal of Microscopy 233, n. 3 (marzo 2009): 404–16. http://dx.doi.org/10.1111/j.1365-2818.2009.03128.x.

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Skiba, Joanna, Claudia Schmidt, Petra Lippmann, Philipp Ensslen, Hans-Achim Wagenknecht, Rafał Czerwieniec, Fabian Brandl et al. "Substitution of Metallocenes with [2.2]Paracyclophane to Enable Confocal Microscopy Imaging in Living Cells". European Journal of Inorganic Chemistry 2019, n. 20 (27 maggio 2019): 2565. http://dx.doi.org/10.1002/ejic.201900533.

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Skiba, Joanna, Claudia Schmidt, Petra Lippmann, Philipp Ensslen, Hans-Achim Wagenknecht, Rafał Czerwieniec, Fabian Brandl et al. "Substitution of Metallocenes with [2.2]Paracyclophane to Enable Confocal Microscopy Imaging in Living Cells". European Journal of Inorganic Chemistry 2017, n. 2 (13 luglio 2016): 297–305. http://dx.doi.org/10.1002/ejic.201600281.

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Renaud, Olivier, Jose Viña, Yong Yu, Christophe Machu, Alain Trouvé, Hans Van der Voort, Bernard Chalmond e Spencer L. Shorte. "High-resolution 3-D imaging of living cells in suspension using confocal axial tomography". Biotechnology Journal 3, n. 1 (gennaio 2008): 53–62. http://dx.doi.org/10.1002/biot.200700188.

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Roberts, R. L., M. A. Barbieri, K. M. Pryse, M. Chua, J. H. Morisaki e P. D. Stahl. "Endosome fusion in living cells overexpressing GFP-rab5". Journal of Cell Science 112, n. 21 (1 novembre 1999): 3667–75. http://dx.doi.org/10.1242/jcs.112.21.3667.

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CHO and BHK cells which overexpress either wild-type rab5 or rab5:Q79L, a constitutively active rab5 mutant, develop enlarged cytoplasmic vesicles that exhibit many characteristics of early endosomes including immunoreactivity for rab5 and transferrin receptor. Time-lapse video microscopy shows the enlarged endosomes arise primarily by fusion of smaller vesicles. These fusion events occur mostly by a ‘bridge’ fusion mechanism in which the initial opening between vesicles does not expand; instead, membrane flows slowly and continuously from the smaller to the larger endosome in the fusing pair, through a narrow, barely perceptible membranous ‘bridge’ between them. The unique aspect of rab5 mediated ‘bridge’ fusion is the persistence of a tight constriction at the site where vesicles merge and we hypothesize that this constriction results from the relatively slow disassembly of a putative docking/fusion complex. To determine the relation of rab5 to the fusion ‘bridge’, we used confocal fluorescence microscopy to monitor endosome fusion in cells overexpressing GFP-rab5 fusion proteins. Vesicle docking in these cells is accompanied by recruitment of the GFP-rab5 into a brightly fluorescent spot in the ‘bridge’ region between fusing vesicles that persists throughout the entire length of the fusion event and which often persist for minutes following endosome fusion. Other endosomal membrane markers, including FM4-64, are not concentrated in fusion ‘bridges’. These results support the idea that the GFP-rab5 spots represent the localized accumulation of GFP-rab5 between fusing endosomes and not simply overlap of adjacent membranes. The idea that the GFP-rab5 spots do not represent membrane overlap is further supported by experiments using photobleaching techniques and confocal imaging which show that GFP-rab5 localized in spots between fusion couplets is resistant to diffusion while GFP-rab5 on endosomal membranes away from these spots rapidly diffuses with a rate constant of about 1.0 (+/-0.3) x10(-)(9)cm(2)/second.

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Swedlow, Jason R., Paul D. Andrews, Ke Hu, David S. RoosT e John M. Murray. "Defining the Tools: an Analysis of Laser Scanning Confocal and Wide-Field/Restoration Fluorescence Microscope Imaging". Microscopy and Microanalysis 7, S2 (agosto 2001): 1002–3. http://dx.doi.org/10.1017/s1431927600031081.

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Digital fluorescence microscopy is now a standard tool for determining the localization of cellular components in fixed and living cells. Two fundamentally different imaging technologies are available for imaging fluorescently labelled cells and tissues, in either the fixed or living state. The laser scanning microscope uses a diffraction-limited focused beam to scan the sample and develop an image point by point. in addition, a pinhole placed in a plane confocal to the specimen prevents emitted out-of focus fluorescence from reaching the photomultiplier tube (PMT) detector. By combining spot illumination and selection of infocus fluorescence signal, the laser scanning confocal microscope (LSCM) creates an image of the specimen largely free of out-of-focus blur. By contrast, a wide-field microscope (WFM) illuminates the whole specimen simultaneously and detects the signal with a spatial array of point detectors, usually a charge-coupled device camera (CCD). This approach collects an image of all points of the specimen simultaneously and includes all the out-of-focus blurred light. Subsequent restoration by iterative deconvolution generates an estimate of the specimen, largely free of out-of-focus blur. While many other fluorescence imaging modalities exist, these two methods represent the majority of the fluorescence imaging systems currently in use in biomedical research.

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Parenti, Niccoló, Ambra Del Grosso, Claudia Antoni, Marco Cecchini, Renato Corradetti, Francesco S. Pavone e Martino Calamai. "Direct imaging of APP proteolysis in living cells". PeerJ 5 (12 aprile 2017): e3086. http://dx.doi.org/10.7717/peerj.3086.

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Alzheimer’s disease is a multifactorial disorder caused by the interaction of genetic, epigenetic and environmental factors. The formation of cytotoxic oligomers consisting of Aβ peptide is widely accepted as being one of the main key events triggering the development of Alzheimer’s disease. Aβ peptide production results from the specific proteolytic processing of the amyloid precursor protein (APP). Deciphering the factors governing the activity of the secretases responsible for the cleavage of APP is still a critical issue. Kits available commercially measure the enzymatic activity of the secretases from cells lysates, in vitro. By contrast, we have developed a prototypal rapid bioassay that provides visible information on the proteolytic processing of APP directly in living cells. APP was fused to a monomeric variant of the green fluorescent protein and a monomeric variant of the red fluorescent protein at the C-terminal and N-terminal (mChAPPmGFP), respectively. Changes in the proteolytic processing rate in transfected human neuroblastoma and rat neuronal cells were imaged with confocal microscopy as changes in the red/green fluorescence intensity ratio. The significant decrease in the mean red/green ratio observed in cells over-expressing the β-secretase BACE1, or the α-secretase ADAM10, fused to a monomeric blue fluorescent protein confirms that the proteolytic site is still accessible. Specific siRNA was used to evaluate the contribution of endogenous BACE1. Interestingly, we found that the degree of proteolytic processing of APP is not completely homogeneous within the same single cell, and that there is a high degree of variability between cells of the same type. We were also able to follow with a fluorescence spectrometer the changes in the red emission intensity of the extracellular medium when BACE1 was overexpressed. This represents a complementary approach to fluorescence microscopy for rapidly detecting changes in the proteolytic processing of APP in real time. In order to allow the discrimination between the α- and the β-secretase activity, we have created a variant of mChAPPmGFP with a mutation that inhibits the α-secretase cleavage without perturbing the β-secretase processing. Moreover, we obtained a quantitatively robust estimate of the changes in the red/green ratio for the above conditions by using a flow cytometer able to simultaneously excite and measure the red and green fluorescence. Our novel approach lay the foundation for a bioassay suitable to study the effect of drugs or particular conditions, to investigate in an unbiased way the the proteolytic processing of APP in single living cells in order, and to elucidate the causes of the variability and the factors driving the processing of APP.

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Squirrell, J. M., D. L. Wokosin, B. D. Bavister e J. G. White. "Imaging Subcellular Changes in Living Mammalian Embryos Using 1047 Nm Two Photon Excitation Fluorescence Microscopy". Microscopy and Microanalysis 5, S2 (agosto 1999): 1060–61. http://dx.doi.org/10.1017/s1431927600018626.

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A major challenge for fluorescence imaging of living cells is maintaining viability during and following prolonged exposure to excitation illumination, especially when imaging over hours or even days, as when studying mammalian embryonic development. The use of specific fluorescently labeled components in living embryos promises to reveal the roles of organelles and molecules in a native and reproducible context. However, to gain a thorough understanding of dynamic biological systems, events of interest must be recorded as they occur, while limiting perturbations caused by the observation technique. Therefore, establishing long-term fluorescence imaging methods that maintain viability is critical for advancing our understanding of cell and developmental biology.One promising technique for imaging living cells is two photon laser scanning microscopy (TPLSM). The lower energy per photon and the restriction of fluorophore excitation to the imaged focal plane should reduce the total photodamage to thick specimens when compared to conventional laser scanning confocal microscopy (LSCM).

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Bénard, Magalie, Christophe Chamot, Damien Schapman, Aurélien Debonne, Alexis Lebon, Fatéméh Dubois, Guénaëlle Levallet, Hitoshi Komuro e Ludovic Galas. "Combining sophisticated fast FLIM, confocal microscopy, and STED nanoscopy for live-cell imaging of tunneling nanotubes". Life Science Alliance 7, n. 7 (22 aprile 2024): e202302398. http://dx.doi.org/10.26508/lsa.202302398.

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Cell-to-cell communication via tunneling nanotubes (TNTs) is a challenging topic with a growing interest. In this work, we proposed several innovative tools that use red/near-infrared dye labeling and employ lifetime-based imaging strategies to investigate the dynamics of TNTs in a living mesothelial H28 cell line that exhibits spontaneously TNT1 and TNT2 subtypes. Thanks to a fluorescence lifetime imaging microscopy module being integrated into confocal microscopy and stimulated emission depletion nanoscopy, we applied lifetime imaging, lifetime dye unmixing, and lifetime denoising techniques to perform multiplexing experiments and time-lapses of tens of minutes, revealing therefore structural and functional characteristics of living TNTs that were preserved from light exposure. In these conditions, vesicle-like structures, and tubular- and round-shaped mitochondria were identified within living TNT1. In addition, mitochondrial dynamic studies revealed linear and stepwise mitochondrial migrations, bidirectional movements, transient backtracking, and fission events in TNT1. Transfer of Nile Red–positive puncta via both TNT1 and TNT2 was also detected between living H28 cells.

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Delattre, Sabrina. "Igniting New Confocal Imaging Potential – Nikon AX R Series with NSPARC". Microscopy Today 31, n. 6 (novembre 2023): 23–27. http://dx.doi.org/10.1093/mictod/qaad088.

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Abstract The Nikon AX/AX R microscope with the recently developed Nikon Spatial Array Detector (NSPARC) provides rapid, super-resolution image acquisition of fixed and living cells. Here, we discuss the structure and function of the NSPARC, which provides excellent resolution and signal-to-noise (S/N) capabilities, and show applications related to in vitro neutrophil movement within pancreatic blood vessels and dynamic changes in mitochondrial morphology occurring over a matter of seconds. The large field-of-view and associated AI software facilitates acquisition and analysis of large 3D samples.

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Haupt, B. J., A. E. Pelling e M. A. Horton. "Integrated Confocal and Scanning Probe Microscopy for Biomedical Research". Scientific World JOURNAL 6 (2006): 1609–18. http://dx.doi.org/10.1100/tsw.2006.269.

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Atomic force microscopy (AFM) continues to be developed, not only in design, but also in application. The new focus of using AFM is changing from pure material to biomedical studies. More frequently, it is being used in combination with other optical imaging methods, such as confocal laser scanning microscopy (CLSM) and fluorescent imaging, to provide a more comprehensive understanding of biological systems. To date, AFM has been used increasingly as a precise micromanipulator, probing and altering the mechanobiological characteristics of living cells and tissues, in order to examine specific, receptor-ligand interactions, material properties, and cell behavior. In this review, we discuss the development of this new hybrid AFM, current research, and potential applications in diagnosis and the detection of disease.

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Kolmogorov, Vasilii, Nikita Savin, Aleksei Iakovlev, Alexander Vaneev, Yuri Efremov, Svetlana Lavrushkina, Helena Lopatukhina et al. "CORRELATIVE QUANTITATIVE NANOMECHANICAL MAPPING AND CONFOCAL IMAGING OF LIVING CELLS BY SCANNING ION-CONDUCTANCE MICROSCOPY". Microscopy and Microanalysis 27, S1 (30 luglio 2021): 570–71. http://dx.doi.org/10.1017/s1431927621002464.

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36

Weidemann, Thomas, Malte Wachsmuth, Tobias A. Knoch, Gabriele Müller, Waldemar Waldeck e Jörg Langowski. "Counting Nucleosomes in Living Cells with a Combination of Fluorescence Correlation Spectroscopy and Confocal Imaging". Journal of Molecular Biology 334, n. 2 (novembre 2003): 229–40. http://dx.doi.org/10.1016/j.jmb.2003.08.063.

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WANG, XIAO-PING, TONGSHENG CHEN, LONGXIANG WANG e LEI SUN. "LIVE IMAGING OF XIAO-AI-PING-INDUCED CELL DEATH IN HUMAN LUNG ADENOCARCINOMA CELLS". Journal of Innovative Optical Health Sciences 01, n. 01 (giugno 2008): 151–56. http://dx.doi.org/10.1142/s1793545808000133.

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Abstract (sommario):

Xiao-Ai-Ping (XAP), a traditional oriental medicinal herb isolated from the stem of Marsdenia tenacissima (Roxb.) Wight et Arn., has been shown to induce tumor cell apoptosis. In this study, we used confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET) techniques to study the molecular mechanism of XAP-induced apoptosis in single living human lung adenocarcinoma (ASTC-a-1) cells. The efficacious apoptosis was observed at 6 h after of 100 μl XAP treatment. Further monitoring the dynamics of caspase-3 activation using FRET imaging in single living ASTC-a-1 cell expressing stably with SCAT3, a FRET probe, showed that XAP activated the caspase-3 at about 2 h after XAP treatment. These data suggest that caspase-3 activation was involved in the XAP-induced apoptosis in ASTC-a-1 cells.

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Zhu, Dan, Jiaxuan Huang, Yanting Xia, Shao Su, Xiaolei Zuo, Qian Li e Lianhui Wang. "DNAzymes-Embedded Framework Nucleic Acids (FNAzymes) for Metal Ions Imaging in Living Cells". Chemosensors 11, n. 7 (25 giugno 2023): 358. http://dx.doi.org/10.3390/chemosensors11070358.

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Abstract (sommario):

Simultaneous and non-destructive quantitative detection of intracellular metal ions holds great promise for improving the accuracy of diagnosis and biological research. Herein, novel multicolor DNAzymes-embedded framework nucleic acids (FNAzymes) were presented, which can easily enter cells and achieve simultaneous and quantitative detection of intracellular physiologically related Cu2+ and Zn2+. Two types of DNAzymes, specific to Cu2+ and Zn2+, were encoded in the framework nucleic acids (FNAs) via self-assembly. With the formation of a well-ordered FNAzyme nanostructure, the fluorophore and the quencher were close to each other; therefore, the fluorescence was quenched. In the presence of Cu2+ and Zn2+, the integrated FNAzymes would be specifically cleaved, resulting in the release of fluorophores in cells. Consequently, the fluorescence in living cells could be observed by a confocal microscope and semi-quantitatively analyzed by flow cytometry with low-nanomolar sensitivity for both metal ions. The FNAzymes have high uniformity and structural accuracy, which are beneficial for intracellular detection with excellent reproducibility. This proposed method offers new opportunities for non-destructive, semi-quantitative, multi-target detection in living cells.

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Xiao, Feng-Lian, Wei-Jie Gao, Cheng-Yi Liu, Xiao-Ping Wang e Tong-Sheng Chen. "Artemisinin induces caspase-8/9-mediated and Bax/Bak-independent apoptosis in human lung adenocarcinoma (ASTC-a-1) cells". Journal of X-Ray Science and Technology: Clinical Applications of Diagnosis and Therapeutics 19, n. 4 (gennaio 2011): 545–55. http://dx.doi.org/10.3233/xst-2011-031300313.

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Abstract (sommario):

Artemisinin (ARTE), an antimalarial phytochemical component from the sweet wormwood plant, has been shown a potential anticancer activity by inducing cell apoptosis. The aim of this report is to explore the mechanism of ARTE-induced human lung adenocarcinoma (ASTC-a-1) cell apoptosis. Cell counting kit (CCK-8) assay showed that ARTE induced cytotoxcity in a dose- and time-dependent manner. Confocal microscopy fluorescence imaging of cells stained with Hoechst 33258 and flow cytometry (FCM) analysis of cells stained with Annexin V-FITC/propidium iodide (PI) showed that ARTE induced reactive oxygen species (ROS)-dependent apoptosis. Confocal fluorescence resonance energy transfer (FRET) imaging of single living cells expressing SCAT3, SCAT9 or CFP-Bid-YFP and fluorometic substrate assay showed that ARTE induced the activation of caspase-3, -8 and -9. Moreover, inhibition of caspase-8 or -9 completely blocked ARTE-induced apoptosis which was only partially attenuated by caspase-3 inhibitor. Interestingly, silencing Bax and Bak by RNA interference (RNAi) did not attenuate ARTE-induced apoptosis. Collectively, ARTE induces caspase-dependent but Bax/Bak-independent apoptosis in ASTC-a-1 cells.

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40

Ahmadian, Somaieh, Patrick J. Lindsey, Hubert J. M. Smeets, Florence H. J. van Tienen e Marc A. M. J. van Zandvoort. "Spinning Disk Confocal Microscopy for Optimized and Quantified Live Imaging of 3D Mitochondrial Network". International Journal of Molecular Sciences 25, n. 9 (28 aprile 2024): 4819. http://dx.doi.org/10.3390/ijms25094819.

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Abstract (sommario):

Mitochondria are the energy factories of a cell, and depending on the metabolic requirements, the mitochondrial morphology, quantity, and membrane potential in a cell change. These changes are frequently assessed using commercially available probes. In this study, we tested the suitability of three commercially available probes—namely 5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolo-carbocyanine iodide (JC-1), MitoTracker Red CMX Rox (CMXRos), and tetramethylrhodamine methyl ester (TMRM)—for assessing the mitochondrial quantity, morphology, and membrane potential in living human mesoangioblasts in 3D with confocal laser scanning microscope (CLSM) and scanning disk confocal microscope (SDCM). Using CLSM, JC-1, and CMXRos—but not TMRM—uncovered considerable background and variation. Using SDCM, the background signal only remained apparent for the JC-1 monomer. Repetitive imaging of CMXRos and JC-1—but not TMRM—demonstrated a 1.5–2-fold variation in signal intensity between cells using CLSM. The use of SDCM drastically reduced this variation. The slope of the relative signal intensity upon repetitive imaging using CLSM was lowest for TMRM (−0.03) and highest for CMXRos (0.16). Upon repetitive imaging using SDCM, the slope varied from 0 (CMXRos) to a maximum of −0.27 (JC-1 C1). Conclusively, our data show that TMRM staining outperformed JC-1 and CMXRos dyes in a (repetitive) 3D analysis of the entire mitochondrial quantity, morphology, and membrane potential in living cells.

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Ma, Xiaohua, Yuanqiang Hao, Jiaxiang Liu, Guoguang Wu e Lin Liu. "A Green-emitting Fluorescent Probe Based on a Benzothiazole Derivative for Imaging Biothiols in Living Cells". Molecules 24, n. 3 (23 gennaio 2019): 411. http://dx.doi.org/10.3390/molecules24030411.

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A new green-emitting fluorescent probe 1 was developed for biothiol detection. The sensing mechanism was considered to be biothiol-induced cleavage of the 2,4-dinitrobenzene- sulfonate group in probe 1 and resulting inhibition of the probe’s photoinduced electron transfer (PET) process. Probe 1 exhibited favorable properties such as excellent selectivity, highly sensitive (0.12 µM), large Stokes shift (117 nm) and a remarkable turn-on fluorescence signal (148-fold). Furthermore, confocal fluorescence imaging indicated that probe 1 was membrane-permeable and suitable for visualization of biothiols in living A549 cells.

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42

Grigoryeva, Natalia Yu, e Dina D. Snarskaya. "Fluorescence methods for investigation of cyanobacterial communities during environmental monitoring of water bodies". Issues of modern algology (Вопросы современной альгологии), n. 2(23) (2020): 8–16. http://dx.doi.org/10.33624/2311-0147-2020-2(23)-8-16.

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Abstract (sommario):

The applicability of confocal laser scanning microscopy (CLSM) for environmental monitoring of water bodies is demonstrated on several examples. Such CLSM methods as spectral imaging and microscopic spectroscopy of living cyanobacterial cells are considered. It is shown that fluorescence spectroscopy application can facilitate time-consuming process of taxonomic analysis of field samples and to make monitoring of water bodies during cyanobacterial blooms, on-line.

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Campagnola, Paul, Aaron Lewis e Leslie M. Loew. "Second Harmonic Imaging Microscopy: A New Non-Linear Optical Modality for Cell Membrane Physiology". Microscopy and Microanalysis 6, S2 (agosto 2000): 810–11. http://dx.doi.org/10.1017/s1431927600036540.

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Abstract (sommario):

Confocal microscopy is an excellent high resolution method to image fluorescently labeled cells. However, the use of confocal microscopy to monitor physiological events, such as membrane potential changes, in living cells is hampered by photobleaching and phototoxicity. To reduce the collateral damage from excitation of fluorescent probes outside the optical slice, Webb and co-workers introduced the use of two-photon excited (TPE) fluorescence in laser scanning microscopy.1 Two-photon absorption depends on the square of the incident light intensity; this has the effect of confining excitation to the plane of focus where the photon flux density is greatest. The wavelength of the exciting light is in the near infrared facilitating penetration of thick tissues. Due to these significant advantages this methodology is rapidly gaining popularity as a tool for live cell and tissue imaging.To further exploit non-linear optical processes in laser scanning microscopy, we have developed surface second harmonic generation (SHG) as a powerful new imaging modality.

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Rossi, Ethan A., Charles E. Granger, Robin Sharma, Qiang Yang, Kenichi Saito, Christina Schwarz, Sarah Walters et al. "Imaging individual neurons in the retinal ganglion cell layer of the living eye". Proceedings of the National Academy of Sciences 114, n. 3 (3 gennaio 2017): 586–91. http://dx.doi.org/10.1073/pnas.1613445114.

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Abstract (sommario):

Although imaging of the living retina with adaptive optics scanning light ophthalmoscopy (AOSLO) provides microscopic access to individual cells, such as photoreceptors, retinal pigment epithelial cells, and blood cells in the retinal vasculature, other important cell classes, such as retinal ganglion cells, have proven much more challenging to image. The near transparency of inner retinal cells is advantageous for vision, as light must pass through them to reach the photoreceptors, but it has prevented them from being directly imaged in vivo. Here we show that the individual somas of neurons within the retinal ganglion cell (RGC) layer can be imaged with a modification of confocal AOSLO, in both monkeys and humans. Human images of RGC layer neurons did not match the quality of monkey images for several reasons, including safety concerns that limited the light levels permissible for human imaging. We also show that the same technique applied to the photoreceptor layer can resolve ambiguity about cone survival in age-related macular degeneration. The capability to noninvasively image RGC layer neurons in the living eye may one day allow for a better understanding of diseases, such as glaucoma, and accelerate the development of therapeutic strategies that aim to protect these cells. This method may also prove useful for imaging other structures, such as neurons in the brain.

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Manders, Erik M. M., Hiroshi Kimura e Peter R. Cook. "Direct Imaging of DNA in Living Cells Reveals the Dynamics of Chromosome Formation". Journal of Cell Biology 144, n. 5 (8 marzo 1999): 813–22. http://dx.doi.org/10.1083/jcb.144.5.813.

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Individual chromosomes are not directly visible within the interphase nuclei of most somatic cells; they can only be seen during mitosis. We have developed a method that allows DNA strands to be observed directly in living cells, and we use it to analyze how mitotic chromosomes form. A fluorescent analogue (e.g., Cy5-dUTP) of the natural precursor, thymidine triphosphate, is introduced into cells, which are then grown on the heated stage of a confocal microscope. The analogue is incorporated by the endogenous enzymes into DNA. As the mechanisms for recognizing and removing the unusual residues do not prevent subsequent progress around the cell cycle, the now fluorescent DNA strands can be followed as they assemble into chromosomes, and segregate to daughters and granddaughters. Movies of such strands in living cells suggest that chromosome axes follow simple recognizable paths through their territories during G2 phase, and that late replicating regions maintain their relative positions as prophase chromosomes form. Quantitative analysis confirms that individual regions move little during this stage of chromosome condensation. As a result, the gross structure of an interphase chromosome territory is directly related to that of the prophase chromosome.

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Campagnola, P. J., e L. M. Loew. "Second Harmonic Generation Imaging (SHG) in the Non-Linear Optical Microscopy of Living Cells". Microscopy and Microanalysis 4, S2 (luglio 1998): 414–15. http://dx.doi.org/10.1017/s1431927600022194.

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Abstract (sommario):

In recent years there has been considerable interest in two and three-photon excited fluorescence in laser scanning optical microscopy. Because absorption is confined tot he focal plane of the objective, these techniques provide intrinsic optical sectioning without the use of a confocal aperture. In addition, photobleaching and phototoxicity are greatly reduced above and below the focal plane. We have adapted a two-photon microscope to utilize surface second harmonic generation (SHG) as a new contrast mechanism for nonlinear optical biological imaging.Surface SHG was first described by Shen [1] and arises from the second order nonlinear susceptibility, χ(2). Signal will only arise from a non-centrosymmetric environment such as an interfacial region. Thus this technique has the potential to probe cellular membranes at high specificity. Further, since SHG results from an induced polarization and not absorption, photobleaching considerations are greatly reduced over fluorescence based methods.

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Baba, Koichi, Hitoshi Kasai, Akito Masuhara, Hidetoshi Oikawa e Hachiro Nakanishi. "Organic Solvent-Free Fluorescence Confocal Imaging of Living Cells Using Pure Nanocrystal Forms of Fluorescent Dyes". Japanese Journal of Applied Physics 48, n. 11 (20 novembre 2009): 117002. http://dx.doi.org/10.1143/jjap.48.117002.

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48

Yamaguchi, Yoshifumi, Naomi Shinotsuka, Keiko Nonomura, Kiwamu Takemoto, Keisuke Kuida, Hiroki Yosida e Masayuki Miura. "Live imaging of apoptosis in a novel transgenic mouse highlights its role in neural tube closure". Journal of Cell Biology 195, n. 6 (12 dicembre 2011): 1047–60. http://dx.doi.org/10.1083/jcb.201104057.

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Abstract (sommario):

Many cells die during development, tissue homeostasis, and disease. Dysregulation of apoptosis leads to cranial neural tube closure (NTC) defects like exencephaly, although the mechanism is unclear. Observing cells undergoing apoptosis in a living context could help elucidate their origin, behavior, and influence on surrounding tissues, but few tools are available for this purpose, especially in mammals. In this paper, we used insulator sequences to generate a transgenic mouse that stably expressed a genetically encoded fluorescence resonance energy transfer (FRET)–based fluorescent reporter for caspase activation and performed simultaneous time-lapse imaging of apoptosis and morphogenesis in living embryos. Live FRET imaging with a fast-scanning confocal microscope revealed that cells containing activated caspases showed typical and nontypical apoptotic behavior in a region-specific manner during NTC. Inhibiting caspase activation perturbed and delayed the smooth progression of cranial NTC, which might increase the risk of exencephaly. Our results suggest that caspase-mediated cell removal facilitates NTC completion within a limited developmental window.

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Piston, David W. "Two-Photon Excitation Microscopy in Cellular Biophysics". Proceedings, annual meeting, Electron Microscopy Society of America 54 (11 agosto 1996): 276–77. http://dx.doi.org/10.1017/s0424820100163848.

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Abstract (sommario):

Two-photon excitation microscopy (TPEM) provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging and photochemistry. Two-photon excitation arises from the simultaneous absorption of two photons in a single quantitized event whose probability is proportional to the square of the instantaneous intensity. For example, two red photons can cause the transition to an excited electronic state normally reached by absorption in the ultraviolet. In practice, two-photon excitation is made possible by the very high local instantaneous intensity provided by a combination of diffraction-limited focusing of a single laser beam in the microscope and the temporal concentration of 100 femtosecond pulses generated by a mode-locked laser. Resultant peak excitation intensities are 106 times greater than the CW intensities used in confocal microscopy, but the pulse duty cycle of 10 5 limits the average input power to less than 10 mW, only slightly greater than the power normally used in confocal microscopy.Three properties TPEM give this method a tremendous advantage over conventional optical sectioning microscopies for the study of thick samples: 1) The excitation is limited to the focal volume because of the intensity-squared dependence of the two-photon absorption. This inherent localization provides three-dimensional resolution and eliminates background equivalent to an ideal confocal microscope without requiring a confocal spatial filter, whose absence enhances fluorescence collection efficiency. Confinement of excitation to the focal volume also minimizes photobleaching and photo damage - the ultimate limiting factors in fluorescence microscopy of living cells and tissues. 2) The two-photon technique allows imaging of UV fluorophores with conventional visible light optics in both the scanning and imaging systems, because both the red excitation light (~700 nm) and the blue fluorescence (>400 nm) are within the visible spectrum. 3) Red or infrared light is far less damaging to most living cells and tissues than bluer light because fewer biological molecules absorb at the higher wavelengths. Longer wavelength excitation also reduces scattering of the incident light by the specimen, thus allowing more of the input power to reach the focal plane. This relative transparency of biological specimens to 700 nm light permits deeper sectioning, since both absorbance and scattering are reduced.

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Maddox, Paul, Julie Canman, Sonia Grego, Wendy Salmon, Clare Waterman-Storer e E. D. Salmon. "A spinning disk confocal microscope system for rapid high resolution, multimode, fluorescence speckle microscopy and GFP imaging in living cells". Microscopy and Microanalysis 7, S2 (agosto 2001): 8–9. http://dx.doi.org/10.1017/s1431927600026118.

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Abstract (sommario):

High resolution fluorescent speckle microscopy (FSM) and green fluorescent protein (GFP) imaging in living cells can require image recording at low densities of fluorophores (10 or less/resolvable unit) with low light excitation to prevent photobleaching. This needs efficient optical components, a high quantum efficiency detector, and a digital image acquisition and display system for time-lapse recording of multiple channels. Recently, Shinya and Ted Inoue have described the advantages of the Yokogawa CSU-10 spinning-disk confocal scanning unit for obtaining high quality fluorescent images with brief exposures and low fluorescence bleaching. Based on their findings, we have combined the CSU-10 unit with a high sensitivity pan-chromatic CCD camera to facilitate high spatial and temporal resolution imaging of fluorescence in living cells. in addition, the high signal-to-noise in images obtained with this instrument provides the opportunity to obtain 3-D views of extraordinary resolution and image quality after iterative constrained de-convolution.Our imaging system is constructed around a Nikon TE300 inverted microscope equipped with either a 60X or 100X Plan Apochromat objective, and standard epi-fluorescence optics for visual inspection of the specimen to locate cells for recording.

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