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‣ Simultaneous observation of collagen and elastin in normal and pathological tissues: analysis of Sirius-red-stained sections by fluorescence microscopy

Borges, L. F.; Taboga, SR; Gutierrez, P. S.
Fonte: Springer Publicador: Springer
Tipo: Artigo de Revista Científica Formato: 551-552
Português
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In order to observe collagen and elastic fibers simultaneously, sections of human aorta, skin, lung, liver, and bladder were stained by Sirius red and analyzed by fluorescence microscopy. In all cases, the fibers of collagen presented the characteristic fluorescent red-orange color that results from the interaction of this extracellular protein with the dye, whereas elastic fibers showed strong green fluorescence (intrinsic fluorescence). This method efficiently detects collagen and elastic fibers when these two structures are present and could have valuable applications in processes that involves both fibers.

‣ The Application of Fluorescence Microscopy and Scanning Electron Microscopy in the Detection of Delayed Ettringite Formation in Concrete

Matos, L.; Silva, A.S.; Soares, D.; Salta, M.; Mirao, J.; Candeias, A.
Fonte: Trans Tech Publications Publicador: Trans Tech Publications
Tipo: Artigo de Revista Científica
Português
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57.3936%
The degradation of concrete structures caused by delayed ettringite formation (DEF) is a problem that nowadays affects many concrete structures worldwide. This pathology is due to the formation of an expansive compound – ettringite - inside the material. This is a hydrated calcium sulphoaluminate produced by the chemical reaction between sulphate ions, calcium hydroxide and alumina present in the Portland cement paste. This product, normally formed during the hydration of cement, presents an acicular morphology (needles) that can be observed by scanning electron microscopy (SEM). However, DEF can also be formed after the setting of the cement causing, in this case, a deleterious expansion of the concrete. This secondary ettringite can also be produced after an excessive heating of the concrete, caused by a high amount of cement or by the use of heat cure. SEM has been used to distinguish between expansive and non expansive ettringite based normally in morphology analysis, since the former is characterized by a compressed or compact nature where the needle shapes disappear or are welded together. Furthermore, the use of other techniques, like X-ray diffraction or micro-XRF, has been limited because the compressed or compact ettringite is badly crystallized or even amorphous and the elemental composition is similar and therefore it is difficult to detect. This article presents a methodology for the diagnosis of DEF using polished concrete thin sections and combining polarised and fluorescence light optical microscopy with SEM-EDS.

‣ High-resolution fluorescence microscopy based on a cyclic sequential multiphoton process

Isobe, Keisuke; Suda, Akira; Hashimoto, Hiroshi; Kannari, Fumihiko; Kawano, Hiroyuki; Mizuno, Hideaki; Miyawaki, Atsushi; Midorikawa, Katsumi
Fonte: Optical Society of America Publicador: Optical Society of America
Tipo: Artigo de Revista Científica
Publicado em 07/09/2010 Português
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57.36888%
We demonstrate high-resolution fluorescence microscopy based on a cyclic sequential multiphoton (CSM) process, which gives rise to fluorescence emission following a sequence of cyclic transitions between the bright and dark states of a fluorophore induced by pump and reverse light. By temporally modulating the reverse intensity, we can extract the fluorescence signal generated through the CSM process. We show that the demodulated fluorescence signal is nonlinearly proportional to the excitation intensities and it gives a higher spatial resolution than that of a confocal microscope.

‣ Quantitative scheme for full-field polarization rotating fluorescence microscopy using a liquid crystal variable retarder

Lesoine, John F.; Youn Lee, Ji; Krogmeier, Jeffrey R.; Kang, Hyeonggon; Clarke, Matthew L.; Chang, Robert; Sackett, Dan L.; Nossal, Ralph; Hwang, Jeeseong
Fonte: U.S. Government Publicador: U.S. Government
Tipo: Artigo de Revista Científica
Português
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We present a quantitative scheme for full-field polarization rotating fluorescence microscopy. A quarter-wave plate, in combination with a liquid crystal variable retarder, provides a tunable method to rotate polarization states of light prior to its being coupled into a fluorescence microscope. A calibration of the polarization properties of the incident light is performed in order to correct for elliptical polarization states. This calibration allows the response of the sample to linear polarization states of light to be recovered. Three known polarization states of light can be used to determine the average fluorescent dipole orientations in the presence of a spatially varying dc offset or background polarization-invariant fluorescence signal. To demonstrate the capabilities of this device, we measured a series of full-field fluorescence polarization images from fluorescent analogs incorporated in the lipid membrane of Burkitts lymphoma CA46 cells. The fluorescent lipid-like analogs used in this study are molecules that are labeled by either a DiI (1,1′-Dioctadecyl 3,3,3′,3′-Tetramethylindocarbocyanine) fluorophore in its head group or a Bodipy (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) molecule in its acyl chain. A spatially varying contrast in the normalized amplitude was observed on the cell surface...

‣ Stimulated emission reduced fluorescence microscopy: a concept for extending the fundamental depth limit of two-photon fluorescence imaging

Wei, Lu; Chen, Zhixing; Min, Wei
Fonte: Optical Society of America Publicador: Optical Society of America
Tipo: Artigo de Revista Científica
Publicado em 22/05/2012 Português
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Two-photon fluorescence microscopy has become an indispensable tool for imaging scattering biological samples by detecting scattered fluorescence photons generated from a spatially confined excitation volume. However, this optical sectioning capability breaks down eventually when imaging much deeper, as the out-of-focus fluorescence gradually overwhelms the in-focal signal in the scattering samples. The resulting loss of image contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation efficiency. Herein we propose to extend this depth limit by performing stimulated emission reduced fluorescence (SERF) microscopy in which the two-photon excited fluorescence at the focus is preferentially switched on and off by a modulated and focused laser beam that is capable of inducing stimulated emission of the fluorophores from the excited states. The resulting image, constructed from the reduced fluorescence signal, is found to exhibit a significantly improved signal-to-background contrast owing to its overall higher-order nonlinear dependence on the incident laser intensity. We demonstrate this new concept by both analytical theory and numerical simulations. For brain tissues, SERF is expected to extend the imaging depth limit of two-photon fluorescence microscopy by a factor of more than 1.8.

‣ Three-color femtosecond source for simultaneous excitation of three fluorescent proteins in two-photon fluorescence microscopy

Wang, Ke; Liu, Tzu-Ming; Wu, Juwell; Horton, Nicholas G.; Lin, Charles P.; Xu, Chris
Fonte: Optical Society of America Publicador: Optical Society of America
Tipo: Artigo de Revista Científica
Publicado em 31/07/2012 Português
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We demonstrate a fiber-based, three-color femtosecond source for simultaneous imaging of three fluorescent proteins (FPs) using two-photon fluorescence microscopy (2PM). The three excitation wavelengths at 775 nm, 864 nm and 950 nm, are obtained through second harmonic generation (SHG) of the 1550-nm pump laser and the 1728-nm and 1900-nm solitons generated through soliton self-frequency shift (SSFS) in a large-mode-area (LMA) fiber. These energetic pulses are well matched to the two-photon excitation peaks of red, cyan and yellow fluorescent proteins (TagRFPs, TagCFPs, and TagYFPs) for efficient excitation. We demonstrate simultaneous 2PM of human melanoma cells expressing a “rainbow” combination of these three fluorescent proteins.

‣ Combined versatile high-resolution optical tweezers and single-molecule fluorescence microscopy

Sirinakis, George; Ren, Yuxuan; Gao, Ying; Xi, Zhiqun; Zhang, Yongli
Fonte: American Institute of Physics Publicador: American Institute of Physics
Tipo: Artigo de Revista Científica
Português
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Optical trapping and single-molecule fluorescence are two major single-molecule approaches. Their combination has begun to show greater capability to study more complex systems than either method alone, but met many fundamental and technical challenges. We built an instrument that combines base-pair resolution dual-trap optical tweezers with single-molecule fluorescence microscopy. The instrument has complementary design and functionalities compared with similar microscopes previously described. The optical tweezers can be operated in constant force mode for easy data interpretation or in variable force mode for maximum spatiotemporal resolution. The single-molecule fluorescence detection can be implemented in either wide-field or confocal imaging configuration. To demonstrate the capabilities of the new instrument, we imaged a single stretched λ DNA molecule and investigated the dynamics of a DNA hairpin molecule in the presence of fluorophore-labeled complementary oligonucleotide. We simultaneously observed changes in the fluorescence signal and pauses in fast extension hopping of the hairpin due to association and dissociation of individual oligonucleotides. The combined versatile microscopy allows for greater flexibility to study molecular machines or assemblies at a single-molecule level.

‣ Fluorescence Microscopy Gets Faster and Clearer: Roles of Photochemistry and Selective Illumination

Wolenski, Joseph S.; Julich, Doerthe
Fonte: YJBM Publicador: YJBM
Tipo: Artigo de Revista Científica
Publicado em 05/03/2014 Português
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Significant advances in fluorescence microscopy tend be a balance between two competing qualities wherein improvements in resolution and low light detection are typically accompanied by losses in acquisition rate and signal-to-noise, respectively. These trade-offs are becoming less of a barrier to biomedical research as recent advances in optoelectronic microscopy and developments in fluorophore chemistry have enabled scientists to see beyond the diffraction barrier, image deeper into live specimens, and acquire images at unprecedented speed. Selective plane illumination microscopy has provided significant gains in the spatial and temporal acquisition of fluorescence specimens several mm in thickness. With commercial systems now available, this method promises to expand on recent advances in 2-photon deep-tissue imaging with improved speed and reduced photobleaching compared to laser scanning confocal microscopy. Superresolution microscopes are also available in several modalities and can be coupled with selective plane illumination techniques. The combination of methods to increase resolution, acquisition speed, and depth of collection are now being married to common microscope systems, enabling scientists to make significant advances in live cell and in situ imaging in real time. We show that light sheet microscopy provides significant advantages for imaging live zebrafish embryos compared to laser scanning confocal microscopy.

‣ System-Wide Studies of Gene Expression in Escherichia coli by Fluorescence Microscopy and High Throughput Sequencing

Chen, Huiyi
Fonte: Harvard University Publicador: Harvard University
Tipo: Thesis or Dissertation
Português
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Gene expression is a fundamental process in the cell and is made up of two parts – the information flow from DNA to RNA, and from RNA to protein. Here, we examined specific sub-processes in Escherichia coli gene expression using newly available tools that permit genome-wide analysis. We begin our studies measuring mRNA and protein abundances in single cells by single-molecule fluorescence microscopy, and then focus our attention to studying RNA generation and degradation by high throughput sequencing. The details of the dynamics of gene expression can be observed from fluctuations in mRNA and protein copy numbers in a cell over time, or the variations in copy numbers in an isogenic cell population. We constructed a yellow fluorescent fusion protein library in E. coli and measured protein and mRNA abundances in single cells. At below ten proteins per cell, a simple model of gene expression is sufficient to explain the observed distributions. At higher expression levels, the distributions are dominated by extrinsic noise, which is the systematic heterogeneity between cells. Unlike proteins which can be stable over many hours, mRNA is made and degraded on the order of minutes in E. coli. To measure the dynamics of RNA generation and degradation...

‣ Self-interference fluorescence microscopy: three dimensional fluorescence imaging without depth scanning

de Groot, Mattijs; Evans, Conor L.; de Boer, Johannes F.
Fonte: Optical Society of America Publicador: Optical Society of America
Tipo: Artigo de Revista Científica
Português
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We present a new method for high-resolution, three-dimensional fluorescence imaging. In contrast to beam-scanning confocal microscopy, where the laser focus must be scanned both laterally and axially to collect a volume, we obtain depth information without the necessity of depth scanning. In this method, the emitted fluorescence is collected in the backward direction and is sent through a phase plate that encodes the depth information into the phase of a spectrally resolved interference pattern. We demonstrate that decoding this phase information allows for depth localization accuracy better than 4 µm over a 500 µm depth-of-field. In a high numerical aperture configuration with a much smaller depth of field, a localization accuracy of tens of nanometers can be achieved. This approach is ideally suited for miniature endoscopes, where space limitations at the endoscope tip render depth scanning difficult. We illustrate the potential for 3D visualization of complex biological samples by constructing a three-dimensional volume of the microvasculature of ex vivo murine heart tissue from a single 2D scan.

‣ A robust algorithm for segmenting fluorescence images and its application to single-molecule counting

Boisvert, Jacques
Fonte: Université de Montréal Publicador: Université de Montréal
Tipo: Thèse ou Mémoire numérique / Electronic Thesis or Dissertation
Português
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57.51002%
La microscopie par fluorescence de cellules vivantes produit de grandes quantités de données. Ces données sont composées d’une grande diversité au niveau de la forme des objets d’intérêts et possèdent un ratio signaux/bruit très bas. Pour concevoir un pipeline d’algorithmes efficaces en traitement d’image de microscopie par fluorescence, il est important d’avoir une segmentation robuste et fiable étant donné que celle-ci constitue l’étape initiale du traitement d’image. Dans ce mémoire, je présente MinSeg, un algorithme de segmentation d’image de microscopie par fluorescence qui fait peu d’assomptions sur l’image et utilise des propriétés statistiques pour distinguer le signal par rapport au bruit. MinSeg ne fait pas d’assomption sur la taille ou la forme des objets contenus dans l’image. Par ce fait, il est donc applicable sur une grande variété d’images. Je présente aussi une suite d’algorithmes pour la quantification de petits complexes dans des expériences de microscopie par fluorescence de molécules simples utilisant l’algorithme de segmentation MinSeg. Cette suite d’algorithmes a été utilisée pour la quantification d’une protéine nommée CENP-A qui est une variante de l’histone H3. Par cette technique...

‣ New Policies, New Technologies : Modelling the Potential for Improved Smear Microscopy Services in Malawi

Ramsay, A.; Cuevas, L. E.; Mundy, C. J.; Nathanson, C. M.; Chirambo, P.; Dacombe, R.; Squire, S. B.; Salaniponi, F. M.; Munthali, S.
Fonte: Banco Mundial Publicador: Banco Mundial
Tipo: Artigo de Revista Científica
Português
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BACKGROUND: To quantify the likely impact of recent WHO policy recommendations regarding smear microscopy and the introduction of appropriate low-cost fluorescence microscopy on a) case detection and b) laboratory workload. METHODOLOGY/PRINCIPAL FINDINGS: An audit of the laboratory register in an urban hospital, Lilongwe, Malawi, and the application of a simple modelling framework. The adoption of the new definition of a smear-positive case could directly increase case detection by up to 28%. Examining Ziehl-Neelsen (ZN) sputum smears for up to 10 minutes before declaring them negative has previously been shown to increase case detection (over and above that gained by the adoption of the new case definition) by 70% compared with examination times in routine practice. Three times the number of staff would be required to adequately examine the current workload of smears using ZN microscopy. Through implementing new policy recommendations and LED-based fluorescence microscopy the current laboratory staff complement could investigate the same number of patients, examining auramine-stained smears to an extent that is equivalent to a 10 minutes ZN smear examination. CONCLUSIONS/SIGNIFICANCE: Combined implementation of the new WHO recommendations on smear microscopy and LED-based fluorescence microscopy could result in substantial increases in smear positive case-detection using existing human resources and minimal additional equipment.

‣ Bayesian multi-target tracking: application to total internal reflection fluorescence microscopy

Rezatofighi, Seyed Hamid
Fonte: Universidade Nacional da Austrália Publicador: Universidade Nacional da Austrália
Tipo: Thesis (PhD)
Português
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This thesis focuses on the problem of automated tracking of tiny cellular and sub-cellular structures, known as particles, in the sequences acquired from total internal reflection fluorescence microscopy (TIRFM) imaging technique. Our primary biological motivation is to develop an automated system for tracking the sub-cellular structures involving exocytosis (an intracellular mechanism) which is helpful for studying the possible causes of the defects in diseases such as diabetes and obesity. However, all methods proposed in this thesis are generalized to be applicable for a wide range of particle tracking applications. A reliable multi-particle tracking method should be capable of tracking numerous similar objects in the presence of high levels of noise, high target density and complex motions and interactions. In this thesis, we choose the Bayesian filtering framework as our main approach to deal with this problem. We focus on the approaches that work based on detections. Therefore, in this thesis, we first propose a method that robustly detects the particles in the noisy TIRFM sequences with inhomogeneous and time-varying background. In order to evaluate our detection and tracking methods on the sequences with known and reliable ground truth...

‣ In situ analysis of foliar zinc absorption and short-distance movement in fresh and hydrated leaves of tomato and citrus using synchrotron-based X-ray fluorescence microscopy

Du, Y.; Kopittke, P.M.; Noller, B.N.; James, S.A.; Harris, H.H.; Xu, Z.P.; Li, P.; Mulligan, D.R.; Huang, L.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Publicado em //2015 Português
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BACKGROUND AND AIMS: Globally, zinc deficiency is one of the most important nutritional factors limiting crop yield and quality. Despite widespread use of foliar-applied zinc fertilizers, much remains unknown regarding the movement of zinc from the foliar surface into the vascular structure for translocation into other tissues and the key factors affecting this diffusion. METHODS: Using synchrotron-based X-ray fluorescence microscopy (µ-XRF), absorption of foliar-applied zinc nitrate or zinc hydroxide nitrate was examined in fresh leaves of tomato (Solanum lycopersicum) and citrus (Citrus reticulatus). KEY RESULTS: The foliar absorption of zinc increased concentrations in the underlying tissues by up to 600-fold in tomato but only up to 5-fold in citrus. The magnitude of this absorption was influenced by the form of zinc applied, the zinc status of the treated leaf and the leaf surface to which it was applied (abaxial or adaxial). Once the zinc had moved through the leaf surface it appeared to bind strongly, with limited further redistribution. Regardless of this, in these underlying tissues zinc moved into the lower-order veins, with concentrations 2- to 10-fold higher than in the adjacent tissues. However, even once in higher-order veins...

‣ X-ray fluorescence microscopy of zinc localization in wheat grains biofortified through foliar zinc applications at different growth stages under field conditions

Ajiboye, B.; Cakmak, I.; Paterson, D.; de Jonge, M.D.; Howard, D.L.; Stacey, S.P.; Torun, A.A.; Aydin, N.; McLaughlin, M.J.
Fonte: Kluwer Academic Publishers Publicador: Kluwer Academic Publishers
Tipo: Artigo de Revista Científica
Publicado em //2015 Português
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Aim: Biofortification of wheat with zinc (Zn) through foliar Zn application has been proposed as an agronomic strategy to increase grain Zn concentration, which could serve as a nutritional intervention in regions with dietary Zn deficiency. Methods: Bread wheat (Triticum aestivum L.) was biofortified through foliar Zn applications at different growth stages. The concentration of Zn and associated micronutrient in harvested whole grains was determined by ICP-OES. Synchrotron-based X-ray fluorescence microscopy (XFM) was then used to investigate the localization of Zn and associated micronutrients in cross sections of these grains. Results: The concentration of Zn and other micronutrients (Mn, Fe, and Cu) was higher in grains treated with foliar Zn during grain-filling (early milk/dough) than those treated at stem elongation. The increase in Zn concentration of wheat grain with foliar application during grain-filling can be attributed to the intense localization of Zn in the aleurone layer, modified aleurone, crease tissue, vascular bundle, and endosperm cavity, and to a modest localization in endosperm, which is the most dominant grain tissue. These tissues and the Zn they contain are presumed to remain after milling and can potentially increase the Zn concentration in wheat flour. Conclusions: By using XFM...

‣ Bewertung der Leuchtdioden-Fluoreszenz-Mikroskopie im Vergleich zu konventioneller Fluoreszenz-Mikroskopie, Lichtmikroskopie und Polymerase-Kettenreaktion zur Diagnose von Plasmodium falciparum-Infektionen in Gabun; Assessment of LED fluorescence microscopy for the diagnosis of Plasmodium falciparum infections in Gabon

Lenz, Dominic
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
Português
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Hintergrund: Mit etwa 300 – 500 Mio. Infektionsfällen und 1 Mio. Toten durch die Infektion mit Plasmodien bleibt die Malaria eine der Hauptursachen für Morbidität und Mortalität weltweit. Über 90% der Todesfälle treten im tropischen Afrika auf, wo die Mehrheit der Infektionen durch Plasmodium falciparum hervorgerufen wird. Doch Malaria ist eine heilbare Krankheit. Eine schnelle und akkurate Diagnostik ist hierbei die zentrale Voraussetzung für die klinische Versorgung der Patienten. Zudem verhindert sie Fehlbehandlungen, welche zu Resistenzentwicklungen, Toxizität und wirtschaftlichen Verlusten führen können. Goldstandard ist seit 1904 die Lichtmikroskopie von mit Giemsa-Lösung gefärbter Dicker Tropfen. Unter optimalen Bedingungen ist dieses Verfahren schnell und zuverlässig, nichtsdestotrotz wäre ein schnelleres Ergebnis von großem Nutzen. Schnelltests als denkbare Alternative sind teuer und liefern nur qualitative Ergebnisse. Jedoch ist in Zeiten abnehmender Inzidenzen sowie schnellen Resistenzentwicklungen und teuren Medikamenten eine kostengünstige schnelle und zuverlässige Malaria-Diagnostik unabdingbar. Die auf Leuchtdioden basierende Fluoreszenz-Mikroskopie wurde als mögliches alternatives Diagnostik-Instrument in dieser Arbeit untersucht. Methoden: In Lambaréné (Gabun) wurden über ein Jahr die Leuchtdioden (LED)-Fluoreszenz-Mikroskopie in 400-facher (ledFM 400x) und 1000-facher Vergrößerung (ledFM 1000x) sowie die konventionelle Fluoreszenz-Mikroskopie (uvFM) in 1000facher Vergrößerung mit konventioneller Lichtmikroskopie (LM) anhand von Proben von 210 Patienten...

‣ Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy

Gualda, Emilio J.; Simão, Daniel; Pinto, Catarina; Alves, Paula M.; Brito, Catarina
Fonte: Frontiers Research Foundation Publicador: Frontiers Research Foundation
Tipo: Artigo de Revista Científica
Publicado em 06/08/2014 Português
Relevância na Pesquisa
67.48335%
The development of three dimensional (3D) cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex 3D matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy (LSFM) is becoming an excellent tool for fast imaging of such 3D biological structures. We demonstrate the potential of this technique for the imaging of human differentiated 3D neural aggregates in fixed and live samples, namely calcium imaging and cell death processes, showing the power of imaging modality compared with traditional microscopy. The combination of light sheet microscopy and 3D neural cultures will open the door to more challenging experiments involving drug testing at large scale as well as a better understanding of relevant biological processes in a more realistic environment.; FCT grants: (SFRH/BD/80717/2011, EXPL/BBB-IMG/0363/2013, PTDC/EBB-BIO/119243/2010, PTDC/EBB-BIO/112786/2009, SFRH/BD/52202/2013, SFRH/BD/78308/2011).

‣ Confocal fluorescence microscopy: a powerful tool in the study of Chagas' disease

Mortara,Renato Arruda; Silva,Solange da; Taniwaki,Noemi Nosomi
Fonte: Sociedade Brasileira de Medicina Tropical - SBMT Publicador: Sociedade Brasileira de Medicina Tropical - SBMT
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/02/2000 Português
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57.07249%
Confocal scanning fluorescence microscopy has become widely used in cell biology and pathology. In conjunction with monoclonal antibodies it may turn out to be a powerful diagnostic tool that also enables detailed studies of tissue forms of Trypanosoma cruzi.

‣ Effect of 2′-OH acetylation on the bioactivity and conformation of 7-O-[N-(4′-fluoresceincarbonyl)-l-alanyl]taxol. A NMR-fluorescence microscopy study

Barbero, José Luis; Souto, André A.; Abal, Miguel; Barasoain, Isabel; Evangelio, Juan A.
Fonte: Pergamon Press Publicador: Pergamon Press
Tipo: Artículo Formato: 6080 bytes; image/gif
Português
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57.308086%
The relationship between conformation, 2′-OH acetylation, and bioactivity of two fluorescent taxoids has been investigated by a combination of NMR and fluorescence microscopy techniques. These taxoids present the structure of taxol with the 7-OH group esterified with the N-(4′-fluoresceincarbonyl)-l-alanine group and with the 2′-OH group free (taxoid 2) or acetylated (taxoid 3). The larger water solubility of 2 and 3 compared with taxol allowed a detailed NMR study in DMSO-d6/D2O (3/7), showing that both taxoids adopt a similar collapsed conformation in which the hydrophobic groups 2-O-benzoyl, 3′-phenyl and 4-O-acetyl are in close proximity, with the fluorescein group displaying unrestricted motion. On the other hand, while taxoid 2 retains essentially the ability of taxol to induce in vitro microtubule assembly and to bind to cell microtubules, the 2′-acetylated derivative 3 does not show immediate activity. However, when taxoid 3 is left in the cell culture, the slow hydrolysis of the 2′-acetate group in the medium liberates the cytotoxic, microtubule-specific taxoid 2. The intense emission of this active derivative (2) allows the accurate recording of the drug-cell interaction from the very initial steps using fluorescence microscopy. These experiments show conclusively...

‣ Applying single-molecule localisation microscopy to achieve virtual optical sectioning and study T-cell activation

Palayret, Matthieu Gr?goire Simon
Fonte: University of Cambridge; Department of Chemistry Publicador: University of Cambridge; Department of Chemistry
Tipo: Thesis; doctoral; PhD
Português
Relevância na Pesquisa
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Single-molecule localisation microscopy (SMLM) allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. As a single-molecule technique, it has also introduced a new quantitative approach to fluorescence microscopy. In the Part A of this thesis, the design and building of three SMLM instruments, the implementation of a custom-developed image analysis package and the characterisation of the photo-physical properties of the photo-activable fluorescent protein used in this thesis (mEos), are discussed. Then, a new post-processing method for SMLM analysis is characterised: axial optical sectioning of SMLM images is demonstrated by thresholding fitted localisations using their fitted width and amplitude to reject fluorophores that emit from above or below a virtual ?light-sheet?, a thin volume centred on the focal plane of the microscope. This method provides qualitative and quantitative improvements to SMLM. In the Part B of this thesis, SMLM is applied to study T cell activation. Although the T cell receptor plays a key role in immunity, its stoichiometry in the membrane of resting T cells is still a matter of debate. Here, single-molecule counting methods are implemented to compare the stoichiometry of TCRs fused with mEos2 in resting T cells to monomeric and dimeric controls. However...