Dissertations / Theses on the topic 'Microscopy'
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Payton, Oliver David. "High-speed atomic force microscopy under the microscope." Thesis, University of Bristol, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.574416.
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SINCE its invention in 1986, the atomic force microscope (AFM) has revolutionised the field of nanotechnology and nanoscience. It is a tool that has enabled research into areas of medicine, advanced materials, biology, chemistry and physics. However due to its low frame rate it is a tool that has been limited to imaging small areas using a time lapse technique. It has only been in recent years that the frame rate of the device has been increased in a tool known as high-speed AFM (HSAFM). This increased frame rate allows, for the first time, biological processes to be viewed in real time or macro sized areas to be imaged with nanoscale resolution. The research presented here concentrates on a specific type of high-speed AFM developed at the University of Bristol called contact mode HSAFM. This thesis explains how the microscope is able to function, and presents a leap in image quality due to an increased understanding of the dynamics of the system. The future of the device is also discussed. III
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Franklin, Thomas. "Scanning ionoluminescence microscopy with a helium ion microscope." Thesis, University of Southampton, 2012. https://eprints.soton.ac.uk/352281/.
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The ORIONR PLUS scanning helium ion microscope (HIM) images at sub nanometer resolution. Images of the secondary electron emission have superior resolution and depth of field compared to a scanning electron microscope (SEM). Ionoluminescent imaging is not an area that has been extensively explored by typical ion beam systems as they have large spot sizes in the region of microns, leading to poor spatial resolution. This thesis confirms that the ORIONR PLUS can form images from the ionoluminescent signal, resolutions of 20nm can be obtained for images of bright nanoparticles. Ionoluminescence spectra can also be obtained from some samples. The position of emission peaks in samples under the ORIONR PLUS does not deviate significantly from cathodoluminescence (CL) peaks under SEM. However, the relative heights of the emission peaks in a sample can vary between ionoluminescence (IL) and CL. In addition, It is found that there exists a proportional relationship between acceleration voltage and ionoluminescent signal in the ORIONR PLUS, this relationship is also exhibited in CL. However, when normalised for current and acceleration voltage there appears to be no samples that show greater luminescence under ionoluminescence than cathodoluminescence, with ionoluminescent intensities up to an order of magnitude lower. Ionoluminescence under the ORIONR PLUS is found to be a poor candidate for the analysis of direct band gap semiconductors, this is attributed to the smaller interaction volumes and achievable beam current of the ORIONR PLUS. It is also found that some direct band gap materials are very susceptible to beam damage under the ion beam at beam doses typically used for secondary electron (SE) imaging. It is possible to obtain simultaneous IL and SE images of organic fluorospores in a biological sample. However, the luminescence of the fluorospores was only just sufficient to form images with a 200nm resolution. Rare earth based nanoparticles show brighter luminescence and greater resistance to beam damage than organic fluorospores. If such particles could be utilised for immunofluorescence it would make combined secondary electron and immunofluorescence imaging under the ORIONR PLUS a viable technique.
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Szelc, Jedrzej. "THz imaging and microscopy : a multiplexed near-field TeraHertz microscope." Thesis, University of Southampton, 2011. https://eprints.soton.ac.uk/209643/.
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Wright, Adele Hart. "Design, development, and application of an automated precision scanning microscope stage with a controlled environment." Thesis, Georgia Institute of Technology, 1997. http://hdl.handle.net/1853/16409.
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Yu, Enhua. "Crossed and uncrossed retinal fibres in normal and monocular hamsters : light and electron microscopic studies /." [Hong Kong : University of Hong Kong], 1990. http://sunzi.lib.hku.hk/hkuto/record.jsp?B13014316.
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Toledo, Acosta Bertha Mayela. "Multimodal image registration in 2D and 3D correlative microscopy." Thesis, Rennes 1, 2018. http://www.theses.fr/2018REN1S054/document.
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Cette thèse porte sur la définition d'un schéma de recalage automatique en microscopie corrélative 2D et 3D, en particulier pour des images de microscopie optique et électronique (CLEM). Au cours des dernières années, la CLEM est devenue un outil d'investigation important et puissant dans le domaine de la bio-imagerie. En utilisant la CLEM, des informations complémentaires peuvent être collectées à partir d'un échantillon biologique. La superposition des différentes images microscopiques est généralement réalisée à l'aide de techniques impliquant une assistance manuelle à plusieurs étapes, ce qui est exigeant et prend beaucoup de temps pour les biologistes. Pour faciliter et diffuser le procédé de CLEM, notre travail de thèse est axé sur la création de méthodes de recalage automatique qui soient fiables, faciles à utiliser et qui ne nécessitent pas d'ajustement de paramètres ou de connaissances complexes. Le recalage CLEM doit faire face à de nombreux problèmes dus aux différences entre les images de microscopie électronique et optique et leur mode d'acquisition, tant en termes de résolution du pixel, de taille des images, de contenu, de champ de vision et d'apparence. Nous avons conçu des méthodes basées sur l'intensité des images pour aligner les images CLEM en 2D et 3D. Elles comprennent plusieurs étapes : représentation commune des images LM et EM à l'aide de la transformation LoG, pré-alignement exploitant des mesures de similarité à partir d'histogrammes avec une recherche exhaustive, et un recalage fin basé sur l'information mutuelle. De plus, nous avons défini une méthode de sélection robuste de modèles de mouvement, et un méthode de détection multi-échelle de spots, que nous avons exploitées dans le recalage CLEM 2D. Notre schéma de recalage automatisé pour la CLEM a été testé avec succès sur plusieurs ensembles de données CLEM réelles 2D et 3D. Les résultats ont été validés par des biologistes, offrant une excellente perspective sur l'utilité de nos développementsThis thesis is concerned with the definition of an automated registration framework for 2D and 3D correlative microscopy images, in particular for correlative light and electron microscopy (CLEM) images. In recent years, CLEM has become an important and powerful tool in the bioimaging field. By using CLEM, complementary information can be collected from a biological sample. An overlay of the different microscopy images is commonly achieved using techniques involving manual assistance at several steps, which is demanding and time consuming for biologists. To facilitate and disseminate the CLEM process for biologists, the thesis work is focused on creating automatic registration methods that are reliable, easy to use and do not require parameter tuning or complex knowledge. CLEM registration has to deal with many issues due to the differences between electron microscopy and light microscopy images and their acquisition, both in terms of pixel resolution, image size, content, field of view and appearance. We have designed intensity-based methods to align CLEM images in 2D and 3D. They involved a common representation of the LM and EM images using the LoG transform, a pre-alignment step exploiting histogram-based similarities within an exhaustive search, and a fine mutual information-based registration. In addition, we have defined a robust motion model selection method, and a multiscale spot detection method which were exploited in the 2D CLEM registration. Our automated CLEM registration framework was successfully tested on several real 2D and 3D CLEM datasets and the results were validated by biologists, offering an excellent perspective in the usefulness of our methods
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Battistella, Eliana. "Towards an improved photonic force microscope: a novel technique for biological microscopy." Master's thesis, Alma Mater Studiorum - Università di Bologna, 2017. http://amslaurea.unibo.it/14864/.
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Una delle tecniche più note nello studio topografico di campioni biologici è l’AFM. Ci sono però limitazioni dovute alla presenza del cantilever, il quale pone un limite nella forza minima applicabile su un campione per ottenere un’immagine topografica. Questa forza (ordine dei 10 pN) può essere sufficiente a danneggiare il campione e a deformare i dettagli topografici che si vorrebbero evidenziare. Per superare questo problema si può usare un Photonic Force Microscope, dove il cantilever è sostituito da Optical Tweezers. Questa tecnica permette di effettuare scansioni di campioni biologici applicando forze dell’ordine dei 100 fN. All’interno della trappola ottica viene posizionata una microparticella che agisce da sonda, attraverso la quale possono essere rilevati dettagli topografici del campione. La differenza rispetto al PFM tradizionale si trova proprio nel tipo di sonda utilizzata durante la scansione. Lo standard prevede l’utilizzo di una sonda sferica, di dimensioni dell’ordine dei 100 nm mentre l’ipotesi è che si possano utilizzare delle sonde cilindriche con alla base un dettaglio acuminato che richiama la punta dell’AFM. Questo tipo di sonda consentirebbe di raggiungere una risoluzione maggiore, rispetto al PFM tradizionale, che risente del limite dato dal diametro della sfera. Due differenti setup per la PFM sono stati costruiti e testati durante questo periodo di tesi. Sono state testate diverse microparticelle cilindriche, di dimensioni differenti in termini di aspect ratio con lo scopo di osservare la stabilità di questo tipo di sonda. Nei risultati viene proposto un metodo per controllare la stabilità e l’orientazione della microparticella cilindrica all’interno della trappola ottica. Viene inoltre fatta un’ipotesi su un metodo per stimare la lunghezza della punta che dovrà essere verificata da una misura sistematica. I risultati preliminari riguardanti la scansione di strutture note suggeriscono la validità dell’uso di questo nuovo tipo di sonda.
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Rea, Nigel P. "Interference and laser feedback optical microscopy." Thesis, University of Oxford, 1995. http://ora.ox.ac.uk/objects/uuid:989c9fca-947d-490c-9f34-38065a7c57d9.
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This thesis concerns the development of simple, compact scanning optical microscopes which can obtain confocal and interference images. The effects of feeding the reflected signal back into the laser cavity of a confocal microscope are investigated and exploited. Monomode optical fibres are used to perform the spatial filtering required for confocal microscopy and, later, as the source of reference beams for interferometry. The theory describing the basic operation of the microscopes is developed. The optical systems are modelled using scalar diffraction theory and the effects of optical feedback into the laser cavity are described, with the practical implications emphasised. A fully reciprocal arrangement of the microscope is developed, in which a single mode optical fibre both launches the signal towards the object and then collects the reflected signal. The fibre is shown to exhibit the spatial filtering properties required for the source and detector in a confocal microscope. It is shown that a semiconductor laser can be used as a detector of the amplitude of the object signal. This is first demonstrated by directing the microscope signal back into the laser cavity and measuring the variation of the optical intensity in the cavity itself. Comparable results are obtained when the variation of the junction voltage across the cavity is measured. It is also shown that the optical fibre is redundant in this system, since the lasing mode of the cavity itself is sufficiently small to adequately spatially filter the reflected signal. When a Helium-Neon laser is used as the source of illumination the effect of the feedback on the laser is seen to be very different, resulting in interferometry. It is shown that high frequency modulation techniques can be used to obtain both confocal images and surface profiles from the same system. This is first demonstrated using an optical feedback scheme in which the modulation of the optical path length of the object beam is controlled electrooptically. In an alternative scheme the images are obtained by calculation, rather than by using a control loop system. In this case the modulation is achieved mechanically. The theoretical limits for the resolutions of the systems described are discussed. It is shown that the lateral resolution of the surface profile systems is inherently non-linear with feature height. Finally, a semiconductor laser based microscope is developed which can obtain confocal images and surface profiles independently. The dependence of the wavelength on the injection current is exploited as a convenient means of introducing a phase shift into the feedback signal by which profilometry can be achieved. All the systems are described theoretically and demonstrated experimentally.
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Romero, Leiro Freddy José. "Poly-articulated microrobotics for correlative AFM-in-SEM microscopy." Electronic Thesis or Diss., Sorbonne université, 2023. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2023SORUS520.pdf.
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La microscopie corrélative est le résultat de la combinaison de deux ou plusieurs techniques de microscopie pour fournir des informations complémentaires sur un échantillon. En utilisant un microscope électronique à balayage (MEB) et un microscope à force atomique (AFM), la microscopie corrélative AFM-in-SEM permet non seulement la caractérisation 3D d'échantillons observés à l'intérieur d'un MEB, mais aussi la manipulation de micro- et nanostructures avec une très grande précision. Cette technique peut être appliquée à divers échantillons dans les domaines de la biologie, de l'électronique et de la science des matériaux. Bien que les solutions AFM-in-SEM existantes dans l'état actuel soient puissantes, elles nécessitent des utilisateurs experts, elles ne sont pas assez polyvalentes pour être utilisées pour différents types de tâches et elles utilisent des robots AFM cartésiens qui limitent fortement la dextérité et la performance du système d'imagerie. L'objectif de cette thèse est d'étudier et d'expérimenter un concept original d'AFM basé sur la robotique poly-articulée pour la microscopie corrélative AFM-in-SEM. Un système robotique AFM à 6 ddls (3 translations et 3 rotations) est développé et intégré à l'intérieur d'un MEB. La capacité de contrôler 3 positions et 3 rotations d'une sonde AFM de taille micrométrique tout en maintenant le centre de rotation à proximité d'une micro-structure est un véritable défi. Cela est principalement dû aux incertitudes inhérentes à l'assemblage des systèmes micro-robotiques et aux jeux mécaniques dans les articulations du robot qui sont du même ordre de grandeur que la précision requise pour le positionnement de la sonde AFM. Les méthodes d'étalonnage des robots et la théorie du contrôle peuvent cependant surmonter ces limitations, comme le démontre cette thèse. Des stratégies de contrôle et une interface utilisateur sont étudiées pour faire fonctionner le système d'imagerie corrélative multi-ddl de manière polyvalente et intuitive. Plusieurs caractéristiques clés qui vont au-delà de l'état de l'art sont mises en œuvre, notamment - Le contrôle par vision électronique (MEB) permet l'atterrissage rapide et automatisé d'une sonde AFM sur un échantillon de taille micrométrique, avec une robustesse par rapport au grossissement du MEB. L'utilisateur peut sélectionner n'importe quelle région d'intérêt (ROI) sur un échantillon en cliquant simplement sur l'écran du MEB. Quel que soit le grossissement du MEB, l'algorithme de contrôle assure un atterrissage sûr de la sonde AFM sur la région d'intérêt. La surface de l'échantillon peut atteindre plusieurs centimètres carrés et le positionnement peut être réalisé avec une précision micrométrique. - Rotation dans le plan et hors du plan d'un échantillon par rapport à la sonde AFM tout en maintenant le centre de rotation autour de la pointe de l'AFM. Le centre de rotation est défini par l'utilisateur par un clic de souris sur l'écran du MEB. Cette fonction est utile pour les tâches de manipulation et de topographie, ainsi que pour les observations multi-angles d'un échantillon à l'intérieur d'un MEB. - Modes de sélection de la trajectoire et de la vitesse de la sonde AFM. Mode AFM à faible vitesse pour une imagerie topographique détaillée. Mode AFM rapide (4fps) pour des observations dynamiques à l'échelle nanométrique. Les utilisateurs ont également accès aux paramètres de contrôle. Ils peuvent être modifiés en fonction de leurs besoins. - Mode AFM mosaïque pour étendre la zone de balayage de la topographie à l'intérieur d'un MEB. Toutes ces caractéristiques s'appuient sur les travaux de recherche en robotique, mécatronique et contrôle réalisés au cours de la thèse. Ces derniers ont le potentiel d'ouvrir la porte à une nouvelle ère de microscopes à force atomique poly-articulés utilisés en microscopie corrélativeCorrelative microscopy is the result of the combination of two or more microscopy techniques to provide complementary information on a sample. When using a scanning electron microscope (SEM) and an atomic force microscope (AFM), AFM-in-SEM correlative microscopy not only enables the 3D characterization of samples observed inside a SEM, but also the manipulation of micro- and nanostructures with an extremely high precision. This technique can be applied to various samples in biology, electronics and materials science. Although existing AFM-in-SEM solutions in the current state of the art are powerful, they require expert users; they are not versatile enough to be used for different types of tasks; and they use Cartesian AFM robots that severely limit the dexterity and performance of the imaging system. The aim of this thesis is to study and experiment an original concept of an AFM based on poly- articulated robotics for AFM-in-SEM correlative microscopy. A homemade 6 DoF (3 translations and 3 rotations) robotic AFM system is developed and integrated inside a SEM. The ability to control 3 positions and 3 rotations of a micrometer sized AFM probe while keeping the center of rotation at the close proximity of a micro-structure is very challenging. This is mainly due to the uncertainties inherent to the assembly of micro-robotic systems and clearances in the joints of the robot that are of the same order of magnitude as the required AFM probe positioning accuracy. Robot calibration methods and control theory can however overcome these limitations as demonstrated in the thesis. Control strategies and a user interface are studied to operate the multi DoF correlative imaging system in a versatile and intuitive way for low-level end users while keeping it enough powerful for high-level end users. Several key features that go beyond the state of the art are implemented, including - Vision based control for fast and automated landing of an AFM probe on a micrometer sized sample with robustness with respect to the SEM magnification. The user can select any region of interest (ROI) on a sample by simply performing a mouse click on the SEM screen. Whatever the SEM magnification, the control algorithm ensures a safe landing of the AFM probe on the ROI. The surface of the sample can be as high as several square centimeters and the positioning can be achieved with a micrometric precision. - In-plane and out-of-plane rotation of a sample relatively to the AFM probe while keeping the center of rotation around the tip of the AFM. The center of rotation is defined by the user with a mouse click on the SEM screen. This feature is useful for manipulation and topography tasks, as well as for multi-angle observations of a sample inside a SEM. - Trajectory/speed selection modes. Low speed AFM mode for a detailed topography imaging. Fast AFM mode (4fps) for dynamic observations at the nanoscale. The users also have access to the control parameters. They can be modified to suit their needs. - Mosaic AFM mode to extend the topography scanning area inside a SEM. All these features rely on research works in robotics, mechatronics and control made during the thesis. The latter has the potential to opens the door to a new era of poly-articulated atomic force microscopes used in correlative microscopy
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Mattocks, Philip. "Scanning tunnelling microscopy and atomic force microscopy of semiconducting materials." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/scanning-tunnelling-microscopy-and-atomic-force-microscopy-of-semiconducting-materials(9bc10301-2c4d-4dfb-a374-f65ee37ae23a).html.
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Michael Faraday first documented semiconducting behaviour in 1833 whenhe observed that the resistance of silver sulphide decreased with temperature,contrary to the behaviour of normal conducting materials. Up untilthe middle of the twentieth century, semiconductors were used as photodetectors,thermisters and rectifiers. In 1947 the invention of the transistor byBardeen and Brattain lead to the integrated circuit and paved the way formodern electronics. The need to produce smaller and faster transistors hasdriven research into new semiconductors. This thesis will first introduce the physics of semiconductors, followed bya description of the experimental techniques employed; scanning tunnellingmicroscopy (STM) and atomic force microscopy (AFM). Chapter 3 is concernedwith explaining anomalous scanning tunnelling spectroscopy resultsobtained for Si(100) and GaAs(110). To this end, a one-dimensional planarmodel, in which surface states affect the charge distribution and tunnellingin the system is proposed. Graphene, a novel two-dimensional material,is introduced in Chapter 4. Scanning tunnelling microscopy measurementsof graphene suspended on a metal grid are presented in this chapter. Finally,Indium antimonide Schottky contacts are investigated using conductingatomic force microscopy in Chapter 5.
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BIAGINI, CLAUDIO. "Bead Mediated Microscopy: from high resolution microscopy to nano-Raman." Doctoral thesis, Università degli studi di Genova, 2020. http://hdl.handle.net/11567/1030687.
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Solid-state physics, material science, as well as biology, need continuously more and more information from their samples. High spatial resolution information such as optical or electrical properties, chemical species identification as well as topography are important information that optical microscopy or Scanning Probe Microscopy (SPM) can provide. Although electron microscopy (SEM and TEM) certainly assumes a position of absolute importance in the field, its cost and its need to be used by highly specialised personnel still make it an instrument of limited everyday use. On the contrary, probe microscopy has now become of very high diffusion in research labs. To develop my thesis I focused myself on three main and somehow related microscopy techniques: high resolution Raman microscopy, Scanning Near-field Optical Microscopy (SNOM), and Tip Enhanced Raman Spectroscopy (TERS). All of them are state-of-the-art on surface optical analysis techniques but still present relevant limits; among others, respectively: spatial resolution, local power density, complexity and field of applicability. My approach wants to combine some aspects of these techniques to go beyond their limits.
Raman spectroscopy is a powerful optical technique, which measures the inelastic scattering of an incoming EM radiation due to the vibrational modes of the molecules present on the surface of a sample. Thanks to its high specificity, it is very powerful in identifying the chemical components of a sample. Several organic and inorganic molecules have their typical Raman spectral peaks, hence, by the Raman spectra, it’s possible to provide a qualitative and quantitative analysis of the elements of a sample. High spatial resolution Raman setups uses the combination of a confocal microscope with a spectrometer assisted by a series of long pass and band pass filters. Despite its extreme versatility, basing Raman spectroscopy on a confocal system also constrains it to acquire its limit in spatial resolution determined by the limit of diffraction.
To overcome this limit the most used techniques in SPM are Scanning Near-field Optical Microscopy (SNOM) and Tip Enhanced Raman Spectroscopy (TERS).
Both of them exploits evanescent field, which is an electric field that is created by oscillating charges and/or currents and does not propagate in the far field as a classical electromagnetic wave, but is spatially concentrated very near to its source. This confinement allows to obtain field sources definitely smaller than in confocal systems.
In SNOM technique, the excitation light is focused through an aperture smaller than the wavelength, creating an evanescent field strongly localized near the aperture itself. Scanning the sample in this near range brings the spatial resolution down to the aperture dimension.
The main disadvantage of aperture SNOM is that the overall optical efficiency of probes is very low. The excitation power cannot be too high in order to prevent any damage of the probe, hence the energy that reaches the sample is usually not enough for Raman analysis.
TERS instead is more suitable for this purpose. It basically exploits Surface Enhanced Raman Spectroscopy (SERS) principles, using a laser irradiated gold sharp tip to obtain a local enhancement at its apex. Its good efficiency permits to analyze Raman effects with a spatial super-resolution, but, on the other hand, TERS probes usually lack of reprodubility and require very skilled and specialised users.
My PhD project has been focused to investigate and optimize an original approach to perform high resolution optical microscopy and Raman spectroscopy, well below the diffraction limit. The concept is to exploit the optical proprieties of a dielectric micro bead lens to achieve a powerful nanoscale near field confinement of light and the Scanning Probe Microscopy (SPM) technique to scan a sample to acquire optical maps. When a dielectric micro bead is hit by an Electromagnetic (EM) wave its effect is to transmit and concentrate the incident EM radiation in a specific area called nanojet, at first glance similar to that created with a standard lens. Some optical proprieties of the nanojets have been already introduced in the literature, but their application in the world of SPM, their employment in Raman microscopy and their combination with nanostructures to improve the spatial resolution are novel features whose investigation is promising. I gave to this technique the name of Beam Mediated Microscopy (BeMM).
The combination of super resolution bead mediated SPM with Raman spectroscopy opens interesting perspectives about powerful surface analysis for samples that need a versatile optical probe with a high spatial resolution and soft interaction with the sample, like soft matter substrates or biosamples.
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Zhang, Hao. "Functional photoacoustic microscopy." [College Station, Tex. : Texas A&M University, 2006. http://hdl.handle.net/1969.1/ETD-TAMU-1743.
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Elder, A. D. "Quantitative fluorescence microscopy." Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598801.
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The work presented here improves the level of quantification achievable with fluorescence microscopy by integrating novel technologies and developing new experimental and theoretical methodologies. Initial work focused on the use of fluorescence microscopy for the quantification of molecular interactions in living cells. This resulted in the development of an analysis routine for the quantification of Förster resonance energy transfer (FRET) by intensity-based sensitised acceptor emission measurements. The developed technique enabled quantification of the strength of interaction as well as the relative stoichiometry of free and bound fluorophores. The work culminated in the dynamic measurement of the cyclin – cyclin dependent kinase interaction through the course of the cell cycle. To improve the flexibility of microscopy techniques, a confocal microscopy system was designed and built which used novel fibre-based supercontinuum illumination technique and a prism-based spectrometer to provide wavelength resolved measurements. The multiparametric imaging approach which this system enabled was shown to aid in the quantification of complex systems. The remainder of this thesis considers the development of new frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) techniques. The advantages of lifetime imaging techniques were illustrated through their application to quantitative chemical analysis in microfluidic devices. Novel illumination technology was integrated into FD-FLIM systems; both in the form of inexpensive light emitting diodes and fibre-based supercontinuum technology. An in-depth theoretical analysis permitted the development of systems with much improved photon economy. Using extensions of the AB analysis technique, multicomponent lifetime data could be accurately quantified. finally, a new experimental technique was implemented, termed ø2FLIM, which enabled the rapid acquisition of alias-free fluorescence lifetime data.
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Naredi-Rainer, Nikolaus. "Advanced confocal microscopy." Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-168349.
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Confocal microscopy is known for its capability to produce exceptional 3D images, even in living tissue. At the same time, it is a powerful spectroscopic tool, facilitating fluores- cence methods such as Fluorescence Correlation Spectroscopy (FCS) or single-molecule Förster Resonance Energy Transfer (FRET). It is heavily used to investigate a wide range of biological problems. This holds true especially for protein properties such as ligand binding, complex formation, conformational changes, or the intracellular distribution of the species in question.
In this work, I will describe the assembly of two instruments: The first is a multi- parameter fluorescence detection (MFD) setup. It is a purely spectroscopic tool that offers the capability to characterize a fluorescent molecule, delivering information like fluorescence lifetime, anisotropy or the speed of its diffusion in free solution. When the molecule of interest is labelled with two fluorophores, additional information, like the energy transfer in-between them, becomes accessible and the correct distance between these two fluorophores can be calculated. If the two fluorophores are attached to different molecules, the MFD setup can detect interactions of these molecules in the range from pM up to μM with the help of Fluorescence Cross-Correlation Spectroscopy (FCCS).
The second instrument, a stimulated emission depletion setup, combines some of the mentioned techniques, like FCS, with the superior image capability of a confocal micro- scope. One particular problem of fluorescent microscopes, though, is that image resolution is always restricted to the diffraction limit of the wavelength of the laser light. The STED setup utilizes the effect of stimulated emission in order to circumvent the diffraction bar- rier and allows images with a three-fold resolution increase, down to 75nm.
These two setups will be used for several applications: The first will be centered around the molecular conformation of proteins, which are sensitive to the nature of the aqueous environment. In particular, the presence of ions can stabilize or destabilize (denature) protein secondary structure. The underlying mechanisms of these actions are still not fully understood. I will apply single-pair FRET to a small 29 amino acid long model peptide to investigate unfolding mechanisms of different unfolding reagents from the Hofmeister series, like sodium perchlorate or guanidinium chloride. The results show that certain salts, which are commonly summarized as denaturing agents, achieve the unfolding by either collapsing the molecule to a compressed state or swelling it to a denatured state.
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The second application of the MFD setup is the investigation of the enhanced green fluorescent protein (EGFP). Although highly used in biochemistry and biophysics, for example to read out the expression level of genes, it is still not fully known what percentage of EGFP is fluorescent. This lack of knowledge makes it nearly impossible to make quantitative statements. With the help of FCCS, it is shown that the folding efficiencies range from 40 − 90%, depending on the environment of the fluorescent protein and which particular mutant is used.
In the third application, the focus will be shifted to nucleation- and polymerization- behavior of actin. The actin cytoskeleton is a central mediator of cellular morphogenesis, and rapid actin reorganization drives essential processes such as cell migration and cell di- vision. In order to compare results of confocal spectroscopy methods with well-established bulk essays, we successfully ported the standard bulk essay to the confocal microscope, allowing for the first time to follow the decrease of monomer concentration and appear- ance of small filaments. Also, the formation of dimers or other small oligomers below the critical concentration is proven for the first time, using FCCS.
The last application will utilize the STED setup in order to carry out the first steps towards the investigation of the nucleation and branching behavior of actin in cooperation with the actin related protein 2/3 (ARP2/3). This protein complex preferentially attaches to actin filaments that are located at the leading edge of a cell and forms branched filamentous structures. The exact conditions under which this process occurs are not well characterized. This part of the work will deal with the steps that are necessary to follow the polymerization process on the STED setup.
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Davies, Eva Melari. "Single molecule microscopy." Diss., Ludwig-Maximilians-Universität München, 2013. http://nbn-resolving.de/urn:nbn:de:bvb:19-173355.
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PeÌrez, JoseÌ CristoÌbal Valera. "Electric force microscopy." Thesis, University of Exeter, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.400970.
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Leane, Robert B. "Scanning tunnelling microscopy." Thesis, University of Cambridge, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.291716.
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Li, Jing. "Ultrafast thermoreflectance microscopy." Thesis, Boston University, 2013. https://hdl.handle.net/2144/11118.
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Thesis (Ph.D.)--Boston UniversityAs electronic and photonic devices shrink to the nanoscale, heat dissipation becomes the bottleneck for performance. As a result, understanding and controlling nanoscale thermal transport in thin films and across interfaces is a critical issue requiring new experimental tools. In this thesis, the development of an ultrafast thermoreflectance microscope for high resolution thermal property imaging is described. It can function as a time domain thermoreflectance (TDTR) or frequency domain thermoreflectance (FDTR) system. Design and implementation of the optical system will be introduced in detail. A thermal model derived from heat transfer theory is used to analyze the experimental data and obtain quantitative property maps for bulk and thin-film samples. The system is used to obtain temperature dependent thermal properties of single crystal diamond and thin film VO2, as well as thermal property maps of several thin film samples.
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Mermelstein, Michael Stephen. "Synthetic aperture microscopy." Thesis, Massachusetts Institute of Technology, 1999. http://hdl.handle.net/1721.1/8178.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 1999.Includes bibliographical references (p. 134-136).
In the late 1800's, Ernst Abbe, research director of the Carl Zeiss Optical Works, wrote down the rules for a lens to form a sharp image. Advances in communications theory, signal processing, and computers have allowed us.finally to break those rules. Our "Synthetic Aperture Microscope" floods a large region with a richly complex, finely structured pattern of light-the interference pattern of a ring of n coherent sources. A target within the volume of the interference fluoresces (or scatters or transmits) an amount of lights that reveals correspondences with this "probing illumination." Modulating'fthe phases and amplitudes of the n beams with carefully chosen modulation signals causes the probe illumination to step through a predetermined or measured family of patterns. A sensor records the target's response in a time-sequence. This time-sequence contains each of order n2 complex Fourier coefficients of the target. Each of these coefficients is encrypted by a unique spread-spectrum key embedded in the amplitude and phase modulation signals. Signal processing picks out these coefficients to reconstruct an image of the target. Low resolution conventional imaging maps an array of "targets" (actually portions of a larger target) to a CCD array, thus allowing this sensing process to be done in parallel over a large region. The end result is to boost the resolution of a conventional imager by hundreds to thousands of sub-pixels per physical pixel. Both theoretical and experimental work on the engineering to make the concept practical are reported.
by Michael Stephen Mermelstein.
Ph.D.
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McKendry, Rachel Anne. "Chemical force microscopy." Thesis, University of Cambridge, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.624272.
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Schlachter, Simon Christopher. "Quantitative multidimensional microscopy." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609221.
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Fafchamps, Lionel. "Aperture correlation microscopy." Thesis, Imperial College London, 2014. http://hdl.handle.net/10044/1/45648.
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Microscopy as applied to the biological sciences has benefited tremendously in the last 50 years from the invention of optically sectioning microscopes. Many techniques for microscopy developed recently offer super-resolved imaging, but often at the cost of light efficiency, signal, acquisition speed or significant amounts of money. This thesis investigates further development of aperture correlation microscopy: a cheap, light-efficient, wide-field, optically sectioning, video-rate imaging technique until now incapable of super-resolution. An aperture correlation microscope was constructed, using a geometry consisting of 2 separate cameras permitting a larger field size than previous spinning disk aperture correlation systems. This microscope, operating at 16Hz (limited by the cameras) was then tested by imaging different samples and found to match reasonably well to the theory, while producing very clear images of biological samples. In order to power this aperture correlation microscope, an image registration algorithm must first register the frames from both cameras. We present a high-speed image registration algorithm suitable for fixed deformations, where a calibration pattern can first be imaged, by leveraging the graphics processing unit. In testing, this registration algorithm is capable of framerates of over 200Hz on modest hardware. A reflection correlation microscope was also constructed and characterised, operating at an illumination wavelength of 405nm, representing an advance into the UV for this technique. Synchronisation of camera acquisition and disk rotation allow for fast acquisition from a single camera. Measurements of its axial resolution match closely to the theory for 4 different illumination pattern pitches. Z-Stacks of transistor and Photovoltaic cell samples were taken in order to demonstrate this system's viability for imaging semiconductor devices. Finally, a novel type of aperture correlation microscope capable of super-resolution is designed and characterised, leveraging coherent illumination. It is demonstrated how tailoring the spatial frequencies present in the aperture mask can result in enhancement of high frequency information up to twice the diffraction limit. Transfer functions are measured, and found to match reasonably well with theory. Lateral resolution improvement of 1.5 times over conventional microscopy is seen here, paving the way for a new, highly efficient super-resolving microscope.
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Pacheco, Shaun, and Shaun Pacheco. "Array Confocal Microscopy." Diss., The University of Arizona, 2017. http://hdl.handle.net/10150/623252.
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Confocal microscopes utilize point illumination and pinhole detection to reject out-of-focus light. Because of the point illumination and detection pinhole, confocal microscopes typically utilize point scanning for imaging, which limits the overall acquisition speed. Due to the excellent optical sectioning capabilities of confocal microscopes, they are excellent tools for the study of three-dimensional objects at the microscopic scale. Fluorescence confocal microscopy is especially useful in biomedical imaging due to its high sensitivity and specificity. However, all designs for confocal microscopes must balance tradeoffs between the numerical aperture (NA), field of view (FOV), acquisition speed, and cost during the design process. In this dissertation, two different designs for an array confocal microscope are proposed to significantly increase the acquisition speed of confocal microscopes. An array confocal microscope scans an array of beams in the object plane to parallelize the confocal microscope to significantly reduce the acquisition time. If N beams are used in the array confocal microscope, the acquisition time is reduced by a factor of N. The first design scans an array of miniature objectives over the object plane to overcome the trade-off between FOV and NA. The array of objectives is laterally translated and each objective scans a small portion of the total FOV. Therefore, the number of objectives used in the array limits the FOV, and the FOV is increased without sacrificing NA. The second design utilizes a single objective with a high NA, large FOV, and large working distance designed specifically for whole brain imaging. This array confocal microscope is designed to speed up the acquisition time required for whole brain imaging. Utilizing an objective with a large FOV and scanning using multiple beams in the array significantly reduces the time required to image large three-dimensional volumes. Both array confocal microscope designs use beam-splitting gratings to efficiently split one laser beam into a number of equal energy outgoing beams, so this dissertation explores design methods and analyses of beam-splitting gratings to fabrication errors. In this dissertation, an optimization method to design single layer beam-splitting gratings with reduced sensitivity to fabrication errors is proposed. Beam-spitting gratings are typically only designed for a single wavelength, so achromatic beam-splitting grating doublets are also analyzed for possible use in array confocal microscopes with multiple excitation wavelengths. An analysis of the lateral shift between grating layers in the achromatic grating doublet proves grating profiles with constant first spatial derivatives are significantly less sensitive than continuous phase profiles. These achromatic grating doublets have designed performance at two wavelengths, but the diffraction angles at the two wavelengths differ. To overcome that limitation, scale-invariant achromatic gratings are designed, which not only provide designed performance at two wavelengths, but also equal diffraction angles at two wavelengths.
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Kielhorn, Martin. "Spatio-angular microscopy." Thesis, King's College London (University of London), 2013. http://kclpure.kcl.ac.uk/portal/en/theses/spatioangular-microscopy(dfcbd278-ea47-40cb-be90-76a0e5c150c2).html.
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Photobleaching and phototoxicity pose a problem in live cell imaging. Fluorescence imaging induces reactive oxygen species in observed organisms which can alter the behaviour of the sample. Hence, minimising the light exposure is an important goal. We augment a widefield epifluorescence microscope with two spatial light modulators. By controlling the spatial excitation pattern and the angle of illumination, we can adapt the illumination to the specimen. In many cases, this technique will create exposures with reduced excitation of the out-of-focus fluorophores, resulting in better image quality and less phototoxicity. My custom software is used to obtain an initial image stack of the specimen. Subsequent image sections are exposed with excitation patterns that account for the previous image stack. Depending upon the distribution of fluorophores, this adaptive exposure can considerably reduce photobleaching and phototoxicity.
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Ye, Peng. "Compressive confocal microscopy." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 50 p, 2009. http://proquest.umi.com/pqdweb?did=1889084501&sid=3&Fmt=2&clientId=8331&RQT=309&VName=PQD.
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Kuhn, William Paul. "Multiplexed acoustic microscopy." Diss., The University of Arizona, 1995. http://hdl.handle.net/10150/187389.
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There is much biological evidence that mechanical forces play a significant role in controlling normal cell growth and proliferation. This evidence has motivated many researchers to use scanning acoustic microscopes to study the mechanical properties of cells. Multiplexing techniques are used in a variety of imaging systems. This dissertation presents the results of the application of multiplexing concepts to acoustic microscopy. The introduction to this dissertation reviews evidence for the biological role of mechanical forces and relevant acoustic imaging techniques. This is followed by an introduction to multiplexing techniques that leads to the conceptual design of a multiplexed acoustic microscope (MAM). The results from simulating and prototyping a small MAM are used to perform a simulation of a large MAM. Data from a large MAM is either impractical to process or requires assumptions that produce unacceptable results. The ultimate solution requires the design of a MAM having a small point spread function.
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Ulrich, Elaine. "Hydrodynamic Force Microscopy." Diss., The University of Arizona, 2008. http://hdl.handle.net/10150/195004.
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Microfluidic networks and microporous materials have long been of interest in areas such as hydrology, petroleum engineering, chemical and electrochemical engineering, medicine and biochemical engineering. With the emergence of new processes in gas separation, cell sorting, ultrafiltration, and advanced materials synthesis, the importance of building a better qualitative and quantitative understanding of these key technologies has become apparent. However, microfluidic measurement and theory is still relatively underdeveloped, presenting a significant obstacle to the systematic design of microfluidic devices and materials. Theoretical challenges arise from the breakdown of classical viscous flow models as the flow dimensions approach the mean free path of individual molecules. Experimental challenges arise from the lack of flow profilometry techniques at sub-micron length scales. Here we present an extension of scanning probe microscopy techniques, which we have termed Hydrodynamic Force Microscopy (HFM). HFM exploits fluid drag to profile microflows and to map the permeability of microporous materials. In this technique, an atomic force microscope (AFM) cantilever is scanned close to a microporous sample surface. The hydrodynamic interactions arising from a pressure-driven flow through the sample are then detected by mapping the deflection of an AFM cantilever. For gas flows at atmospheric pressure, HFM has been shown to achieve a velocity sensitivity of 1 cm/s with a spatial resolution of ~ 10 nm. This compares very favorably to established techniques such as hot-wire and laser Doppler anemometry, whose spatial resolutions typically exceed 1 μm and which may rely on the use of tracer particles or flow markers. We demonstrate that HFM can successfully profile Poiseuille flows inside pores as small as 100 nm and can distinguish Poiseuille flow from uniform flow for short entry lengths. HFM detection of fluid jets escaping from porous samples can also reveal a "permeability map" of a sample’s pore structure, allowing us to distinguish between clear and blocked pores, even in cases where the subsurface fouling is undetectable by conventional AFM. The experimental data is discussed in context with theoretical aspects of HFM microflow measurement and practical limits of this technique. Finally, we conclude with variations of standard HFM techniques that show some promise for investigation of smaller nanometer-scale flows of gases and liquids.
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Li, Jianbo. "Microlens Assisted Microscopy." OpenSIUC, 2013. https://opensiuc.lib.siu.edu/theses/1298.
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In recent years, microlenses (ML), which are micro-scale spheres, have been used to overcome physical diffraction limit of optical microscopy (~200 nm). Although the use of such ML has provided highly resolved images of objects beyond the Abbe optical diffraction limit, the process needs to be refined before it can be applied widespread in materials, biological and clinical research. In this research work, we have implemented experiments on super-resolution imaging utilizing MLs of different refractive indices (n) and diameters to provide the scientific and engineering communities with practical guidelines for obtaining high resolution images with ease. With the support from experimental imaging data as well as FDTD simulations, we have shown that optimal super-resolution imaging with microspheres was accomplished under specific parameter range. We have identified ML with n=1.51 as a preferable choice over those MLs with n=1.4, 1.93, and 2.2, because of high reliability and high magnification for ML with n=1.51. With n=1.51 in mind, we have identified a diameter range from 15 μm to 50 μm provides high resolution and magnification for practical purposes. We show that other ML diameters provided high resolution as well; we believe that ML diameters between 15 μm and 50 μm are practically preferred. We were able to achieve <150 nm resolution and further refinement of this tool can potentially yield higher quality imaging results. Ideally, MLs will eventually be directly incorporated as a modular device in an optical microscope providing the researchers an effective, noninvasive, and economical alternative to complex super resolution microscopy techniques. To improve scanning efficiency, we also proposed microtubule (MT) based imaging. With the demonstration of theoretical optics, we conclude, at present time, that there are some practical concerns for MT-based imaging technique that may limit its application as super-resolution imaging technique. For example, MT-based imaging appears to possess a lower contrast than ML-based technique. Thus, although the concept of MT-based imaging is theoretically possible, we think that more work is needed to utilization of this tool for practical applications.
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Rogers, Stuart Craig. "Defect Detection Microscopy." BYU ScholarsArchive, 2010. https://scholarsarchive.byu.edu/etd/2256.
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The automotive industry's search for stronger lighter materials has been hampered in its desire to make greater use of Magnesium alloys by their poor formability below 150°C. One current challenge is to identify the complex structure and deformation mechanisms at work and determine which of these are primary contributors to the nucleation of defects. Orientation Imaging Microscopy has been the most accessible tool for microstructural analysis over the past 15 years. However, using OIM to analyze defect nucleation sites requires prior knowledge of where the defects will occur because once the defects nucleate the majority of microstructural information is destroyed. This thesis seeks to contribute to the early detection of nucleation sites via three mechanisms: 1. Detection of cracks that have already nucleated, 2. Detection of surface topography changes that may indicate imminent nucleation and 3. Beam control strategies for efficiently finding areas of interest in a scan. Successive in-situ OIM scans of a consistent sample region while strain is increased, while using the three techniques developed in this thesis, will be employed in future work to provide a powerful defect analysis tool. By analyzing retrieved EBSD patterns we are able to locate defect / crack sites via shadowing on the EBSD patterns. Furthermore, topographical features (and potentially regions of surface roughening) can be detected via changes in intensity metrics and image quality. Topographical gradients are currently only detectable in line with the beam incidence. It is therefore suggested that the tensile specimens to be examined are orientated such that the resulting shear bands occur preferentially to this direction. The ability to refine the scan around these areas of interest has been demonstrated via an off-line adaptive scan routine that is implemented via the custom scan tool. A first attempt at a defect detection framework has been outlined and coded into MATLAB. These tools offer a first step to accessing the information about defect nucleation that researchers are currently seeking.
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Frozoni, Marcos Roberto dos Santos 1969. "Efeito do fluor na organização supramolecular da matriz organica do esmalte dentario em camundongos." [s.n.], 2008. http://repositorio.unicamp.br/jspui/handle/REPOSIP/290014.
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Orientador: Sergio Roberto Peres LineDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: A biossíntese do esmalte dentário inicia-se pela secreção, processamento proteolítico e auto-agregação de uma complexa mistura de proteínas, sintetizadas pelos ameloblastos, conhecida como matriz orgânica do esmalte. A formação desta matriz ocorre em três estágios: secreção (inicial), transição e maturação e parece ser fundamental para o controle da orientação e morfologia dos cristais de hidroxiapatita, que constituem a fase mineral do esmalte em desenvolvimento. No estágio de secreção da amelogênese, a matriz orgânica do esmalte apresenta uma organização supramolecular birrefringente, dessa forma, a referida matriz pode ser observada e quantificada por meio de microscopia de luz polarizada. Alterações genéticas e ambientais podem induzir a distúrbios na organização molecular da matriz orgânica extracellular do esmalte (MOECE) dentário no estágio secretório, gerando modificações em sua birrefringência, tais distúrbios podem contribuir para alterações na estrutura do esmalte maduro. Altos níveis de ingestão de flúor causam mudanças na estrutura e concentração das proteínas da matriz orgânica do esmalte, induzindo a falhas na mineralização e formação desorganizada dos cristais do esmalte. Estas alterações caracterizam a fluorose de esmalte e incluem aumento da porosidade, redução do conteúdo mineral e diminuição da microdureza do esmalte maduro. O objetivo deste estudo foi analisar os efeitos do flúor sobre a birrefringência da MOECE no estágio secretório. Quinze camundongos da linhagem A/J foram divididos em 3 grupos e submetidos a um tratamento de 30 dias com dieta exclusiva de ração e água deionizada ad libitum. A água ingerida continha 0, 25, e 50 ppm de flúor (NaF) nos grupos A/J-Controle, A/J-Flúor 25 ppm e A/J-Flúor 50 ppm, respectivamente. Os mesmos procedimentos foram aplicados a quinze camundongos da linhagem NOD (Non Obese Diabetic), caracterizando os grupos NOD-Controle, NOD-Flúor 25 ppm e NOD-Flúor 50 ppm. Após o período acima mencionado, todos os animais foram perfundidos com uma mistura de paraformaldeído 2% com glutaral deído 0,5% em tampão fosfato 0,2 M e suas hemimaxilas foram extraídas e mantidas na mesma solução fixadora por 16h, as amostras foram então descalcificadas em mistura de ácido nítrico 5% com formaldeído 4 % por 6 h sob agitação. Após desidratação e inclusão em parafina, obtive-se cortes longitudinais de 5µm de espessura que foram desparafinizados, hidratados, montados em solução aquosa de glicerina 80% e analisados em microscopia de luz polarizada. Realizou-se a análise da matriz orgânica dos incisivos superiores de modo a se determinar o retardo óptico em nanômetros (nm) na área de maior birrefringência no estágio de secreção da amelogênese. Os valores de retardo ótico foram submetidos à análise estatística (Kruskal-Wallis) e os grupos A/J e NOD foram comparados separadamente. Observou-se um aumento, estatisticamente significante, dos valores de retardo ótico nos grupos A/J-Flúor 25 ppm e A/J-Flúor 50 ppm, quando comparados ao grupo A/J-Controle (p<0,01). O mesmo aconteceu com os grupos NOD-Flúor 25 ppm e NOD-Flúor 50 ppm que mostraram aumento, estatisticamente significante, dos valores de retardo ótico quando comparados ao grupo NOD-Controle (p<0,01). Os grupos A/J-Flúor apresentaram valores semelhantes (p>0,05) o que também ocorreu com os grupos NOD-Flúor. Os resultados do presente estudo mostram que o flúor induz a um aumento da birrefringência da MOECE no estágio de secreção, podendo estar associado ao mecanismo de desenvolvimento da fluorose de esmalte
Abstract: Dental enamel biosynthesis begins with secretion, proteolytic processing and self-assembly of a highly complex mixture of proteins, synthesised by ameloblast, which is known as the enamel organic matrix. This matrix formation occurs at three stages: secretion (initial), transition and maturation and seems to be essential for controlling orientation and morphology of the hydroxyapatite crystals that comprise mineral phase of developing enamel. In the secretory stage of amelogenesis, the enamel organic matrix presents a birefringent supramolecular organization. Therefore, it can be observed and quantified by polarizing microscopy. Genetic and environmental alterations may induce disturbances in the molecular organization of the secretory-stage enamel organic extracellular matrix (EOECM), producing birefringence changes, these disturbances may contribute to mature enamel alterations. High levels of ingested fluoride cause modifications in the structure and concentration of proteins of the enamel organic matrix inducing failures in the mineralization and disorganized enamel crystals formation. These alterations characterize enamel fluorosis, include increased porosity, mineral content reduction and diminished mature enamel micro hardness. The aim of the present study was to analyse the effects of fluoride on the birefringence of secretory stage EOECM. Fifteen A/J inbred mice strain were divided into 3 groups and submitted to a treatment during 30 days with exclusive diet of food and deionized water ad libitum. The ingested water contained 0, 25 and 50 ppm fluoride (NaF) in the groups A/J Control, A/J 25 ppm fluoride and A/J 50 ppm fluoride, respectively. The same procedures were applied to fifteen NOD (Non Obese Diabetic) mice, which formed the groups NOD Control, NOD 25 ppm fluoride and NOD 50 ppm fluoride. After the abovementioned period, all the animals were perfused with 2% paraformaldehyde, 0.5% glutaraldehyde in 0.2 M phosphate buffered solution. Its hemimaxillae were then extracted and maintained in the same fixative solution for 16 h, the samples were decalcified under stirring in 5% nitric acid, 4% formaldehyde for 6 h. After dehydration and embedded in paraffin, longitudinal 5-µm-thick sections were obtained and deparaffined, hydrated and mounted with aqueous 80% glycerine as imbibing medium and analyzed with polarizing microscopy. Optical retardation (nm) of the area that showed the highest birefringence brightness in the EOECM of upper incisors was determined. Optical retardation values were submitted to statistical analysis (Kruskal-Wallis) and A/J and NOD groups were separately compared. An statistically significant increase in optical retardations values was observed in A/J 25 ppm Fluoride and A/J 50 ppm Fluoride, when compared to A/J Control group (p<0.01). The same happened with NOD 25 ppm Fluoride and NOD 50 ppm Fluoride groups which exhibited statistically significant increase in optical retardations values when compared to NOD Control group (p<0.01). A/J Fluoride groups presented similar optical retardation values (p>0.05) which occurred with NOD fluoride groups. The results presented here show the fluoride induces an increase in the birefringence of secretory stage EOECM, which may be associated with enamel fluorosis development. Key words: Enamel, Amelogenesis, Enamel Organic Matrix, Birefringence, Fluoride
Mestrado
Histologia e Embriologia
Mestre em Biologia Buco-Dental
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Pini, Núbia Inocencya Pavesi 1987. "In vitro and in situ evaluation of microabrasion technique on enamel microhardness and morphology = Avaliação in vitro e in situ da técnica de microabrasão sobre a microdureza e morfologia do esmalte dental." [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/290365.
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Orientadores: José Roberto Lovadino, Débora Alves Nunes Leite LimaTexto em português e inglês
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: Objetivo: Avaliar, in vitro, a influência dos ácidos utilizados para microabrasão e, in situ, o efeito do tempo de contato com a saliva na microdureza e morfologia do esmalte abrasionado. Metodologia: In vitro: Setenta blocos dentais bovinos foram divididos em 7 grupos (n=10). Os grupos experimentais foram tratados com aplicação ativa/passiva dos ácidos H3PO4 35% (E1/E2) ou HCl 6,6% (E3/E4); e controles, tratados com microabrasão com H3PO4+pedra-pomes (C5), HCl+silica (C6) ou nenhum tratamento (C7). In situ: Nove grupos (n=19) de blocos dentais bovinos foram divididos de acordo com o tratamento e o tempo de exposição salivar, sendo 4 grupos tratados com H3PO4+pedra-pomes, 4 com HCl+sílica e 1 grupo controle. Os grupos tratados foram subdivididos em: sem exposição salivar, 1 hora, 24 horas ou 7 dias de exposição em ambiente intrabucal. A microdureza superficial (SMH) foi avaliada antes e após a microabrasão, e após exposição salivar (in situ). A microdureza subsuperficial (CSMH - 10, 25, 50 e 75 ?m) foi analisada após a microabrasão (in vitro) e após a exposição salivar (in situ). Espécimes representativos foram selecionados para a avaliação da morfologia do esmalte por meio da microscopia confocal de varredura a laser (MCVL - in vitro) e por microscopia eletrônica de varredura (MEV - in situ). Para a análise estatística foi realizada análise de variância para medidas repetidas (Proc Mixed), e os testes de Tukey-Kramer e Dunnet (SMH) e ANOVA (parcelas subdivididas) e Tukey-Kramer (CSMH - in situ) (p<0.05). Resultados: In vitro: Não foram encontradas diferenças entre as análises pré e pós-microabrasão entre os grupos controles para SMH. Entre os grupos experimentais, a aplicação ativa demonstrou os maiores valores de SMH, sem diferença entre os ácidos, com a mesma forma de aplicação. A maioria dos grupos apresentou redução do valor de CSMH conforme aumento da profundidade, com diferenças entre os grupos com microabrasão (C5 e C6) e o C7; e entre todos os grupos experimentais e o C7. Comparando a aplicação dos ácidos, a aplicação ativa do H3PO4 (E1) mostrou maior CSMH com diferença estatística em relação ao HCl (E3). A MCVL demonstrou diferentes padrões de condicionamento para cada grupo. In situ: Para as análises de SMH, todos os grupos tratados apresentaram redução na microdureza, com diferenças em relação ao controle e a leitura inicial. Após exposição salivar, os resultados demonstraram que o tratamento com HCl+sílica foi mais propenso à remineralização, já que, com 1 hora foi verificado aumento na SMH, com diferença significante em relação à análise pós-microabrasão. Apenas o tratamento com HCl+sílica foi eficiente em reestabelecer tal propriedade em relação ao controle. A análise de CSMH confirmou a maior capacidade de remineralização do esmalte tratado com HCl+sílica, uma vez que após 7 dias de exposição salivar, os valores de microdureza foram restabelecidos para as camadas mais superficiais do esmalte (10 e 25 ?m). A MEV demonstrou o efeito remineralizador da saliva para ambos os tratamentos. Conclusões: Os ácidos utilizados para microabrasão apresentaram alto poder erosivo quando aplicados individualmente. O tratamento com HCl+sílica resultou em uma superfície de esmalte mais propensa à remineralização
Abstract: Objective: To evaluate, in vitro, the effect of acids used in microabrasion on enamel microhardness, and, in situ, the effects of remineralizing time on enamel surface after microabrasion. Methods: In vitro: Seven groups (n=10) of enamel blocks from bovine incisors were divided in: Experimental groups treated by active/passive application of 35% H3PO4 (E1/E2) or 6.6% HCl (E3/E4); and control groups treated by microabrasion with H3PO4+pumice (C5), HCl+silica (C6), or no treatment (C7). In situ: Nine groups (n=19) of same specimens were divided in according to microabrasion and salivary exposition being 1 control (no treatment) and 4 groups with microabrasion using 35% H3PO4+pumice and 4 groups using 6.6% +silica. One group of each treatment was submitted to 4 frames of salivary exposition, being without exposition and with 1 hour, 24 hours or 7 days of presence on in situ regimen. Surface microhardness (SMH) was evaluated before and after microabrasion, and after salivary exposition (in situ). Cross-sectional microhardness (CSMH) was analyzed after microabrasion (in vitro) and after salivary exposition (in situ). For confocal laser scanning microscopy (CLSM - in vitro) and scanning electron microscopy (SEM - in situ), representative specimens group were selected. Statistical analysis used Proc Mixed, Tukey-Kramer and Dunnet tests (SMH) e ANOVA (subdivided parcels) and Tukey-Kramer tests (CSMH - in situ) (p<0.05). Results: In vitro: For SMH, it was not found statistically differences between the control groups after treatment. Active application resulted in significantly higher microhardness results than passive application, with no difference between acids. For most groups, the CSMH decreased as the depth increased, with differences between the groups treated with microabrasion (C5 and C6) and C7; and between all of experimental groups and C7. A significantly higher mean CSMH result was obtained with active application of H3PO4 compared to HCl. CLSM revealed the conditioning pattern for each group. In situ: For SMH, the groups treated with microabrasion presented reducing in mineral content, with statistical difference in relation to the control and to the initial analysis. The treatment HCl+silica presented lower reduction and were statistically different from the treatment with H3PO4+pumice. After salivary exposition SMH results revealed that surface treated with HCl+silica was more prone to remineralizing effect of saliva, once it was verified since with 1 hour of presence in in situ regimen, with significant differences between the treatments after 7 days of salivary exposition. Just for SMH, the HCl+silica reached values obtained in control group. CSMH analysis showed that 7 days of salivary exposition were efficient in reestablish de values for the outer layers (10 e 25 ?m) of enamel treated with HCl+silica. SEM analysis presented the remineralizing effect in the course of the time. Conclusions: Acids used for enamel microabrasion presented a higher erosive action when solely applicated. Data suggested that enamel surface treated with HCl+silica presented more susceptibility for remineralizing action of saliva than that treated with phosphoric acid and pumice
Mestrado
Dentística
Mestra em Clínica Odontológica
32
Sfalcin, Ravana Angelini 1985. "Avaliação de propriedades físico-químicas de infiltrantes experimentais com adição de partículas de vidro bioativas = Evaluation of the physical-chemical properties of experimental infiltrants incorporated with bioactive glass particles." [s.n.], 2015. http://repositorio.unicamp.br/jspui/handle/REPOSIP/288423.
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Orientador: Americo Bortolazzo CorrerTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: O objetivo neste trabalho foi avaliar as propriedades físico-químicas de infiltrantes resinosos com adição de partículas bioativas, bem como sua capacidade de penetração e dureza da profundidade em lesões subsuperficiais de esmalte. Uma blenda contendo TEGDMA (75% em peso) e BisEMA (25% em peso) foi manipulada e a partir dela foram incorporados 5 tipos de partículas bioativas (10% em peso): hidroxiapatita (HAp), fosfato de cálcio amorfo (ACP), vidro bioativo policarboxilato de zinco (BAG Zn), vidro bioativo 45S5 (BAG 45S5), cimento de silicato de cálcio modificado por ?-TCP (HCAT-?). Um material comercial foi utilizado (ICON®) como controle. Dez espécimes foram confeccionados para cada grupo de cada teste: rugosidade superficial (Ra) antes e após a escovação; Resistência à flexão por 3 pontos (RF) e módulo de elasticidade (ME); resistência coesiva à tração (RC); dureza Knoop (KHN); densidade de ligação cruzada (DLC); grau de conversão (GC); sorção (S) e solubilidade (SL) em água; e micro-dureza (KHN). Os dados foram submetidos a ANOVA e teste Tukey (?=0.05). A penetração dos infiltrantes resinosos no esmalte humano desmineralizado foi qualitativamente avaliada em Microscopia Confocal de Varredura a Laser (n=5). Os resultados mostraram que os menores valores de rugosidade (antes e após a escovação foram apresentados pelo ACP. Com relação à resistência a flexão e módulo de elasticidade, T+B apresentou o maior valor e ICON® mostrou o menor valor. ICON® também mostrou o menor valor de resistência coesiva à tração; não houve diferença significativa entre os grupos T+B, HAp, ACP, BAG Zn, BAG 45S5 e HCAT-?. Para o teste de dureza Knoop, ICON® obteve o menor valor e BAG Zn mostrou o maior valor. Para densidade de ligação cruzada, ICON® apresentou maior quantidade de ligação cruzada e HAp, menor quantidade de ligação cruzada. ICON® apresentou grau de conversão significantemente menor que os infiltrantes experimentais, que não diferiram entre eles. ICON® apresentou a maior sorção de água e HAp a menor. Não houve diferença significativa entre os demais grupos. Para solubilidade, ICON® apresentou os maiores valores, mas sem diferença de ACP. BAG 45S5 apresentou a menor solubilidade. Com relação a micro-dureza, não houve diferença estatisticamente significante entre as profundidades avaliadas (50 µm, 200 µm, 350 µm e 500 µm). BAG 45S5, BAG Zn e HCAT-? não mostraram diferença estatística entre eles. Entretanto, HCAT-? e BAG Zn foram similares ao ICON® e ACP. O grupo cariado mostrou menor valor quando comparado a todos os grupos testados. A análise em microscopia confocal mostrou que todos os materiais apresentaram boa capacidade de penetração nas lesões iniciais, exceto para FCA. Pôde ser concluído que adição de partículas bioativas em um infiltrante experimental melhorou as propriedades mecânicas e não afetou a capacidade de penetração dos infiltrantes. O infiltrante resinoso contendo fosfato de cálcio amorfo foi o que apresentou o melhor desempenho no teste de rugosidade de superfície antes e após a escovação
Abstract: The aim of this study was to evaluate the physical-chemical properties of the experimental infiltrants with the addition of bioactive particles as well as their capability of penetration and depth Knoop hardness into caries-like lesions. A control blend was made with TEGDMA (75 wt%) and BisEMA (25 wt%). Five bioactive fillers were added in the control blend (10 wt%): Hydroxyapatite (Hap), amorphous calcium phosphate (ACP), Zinc-polycarboxylated bioactive glass (BAG-Zn), bioactive glass 45S5 (BAG 45S5), and ?-TCP modified calcium silicate cements (HCAT-?). An available commercially material was used (ICON®). Ten specimens were comprised by each group for the following tests: Surface roughness (Ra) before and after brushing abrasion; flexural strength (FS) and elastic modulus (E-Modulus); tensile cohesive strength (TCS); Knoop hardness (KHN); softnening ratio (SR); degree of conversion (DC); water sorption (WS) and solubility (SL); and micro-hardness (micro-KHN). Data were subjected to ANOVA and Tukey¿s test (?=0.05). Confocal Scanning Laser Microscopy was used to evaluate qualitatively the penetration capability of resin infiltrants into demineralized human enamel. Results showed that ACP had the lowest Ra before and after brushing abrasion. Regarding to the FS and E-modulus, T+B showed the higher value and ICON® showed the lower value. Also, ICON® showed the lower value of TCS, but there was no significant statistically difference among the groups T+B, HAp, ACP, BAG Zn, BAG 45S5 e HCAT-?. To the KHN, ICON® obtained the lower value and BAG Zn showed the higher value. According to the SR, ICON® showed lower SR and HAp, the higher SR. ICON showed DC significantly lower than experimental resin infiltrants. Regarding to the WS, ICON® presented the highest water sorption and HAp the lowest one. There was no significant statistically difference among the other groups. ICON showed the highest SL results; however, the results were similar to ACP. The lowest SL was found for BAG 45S5. Regarding to the micro-KHN, there was no statistically difference among the analyzed depths (50 µm, 200 µm, 350 µm and 500 µm). BAG 45S5, BAG Zn and HCAT- ? did not show statistical difference among them. However, HCAT- ? and BAG Zn were similar to ICON® and ACP. Carious group showed lower value when compared to all the tested groups. Confocal microscopy analysis showed good capability of penetration into the initial lesions for all materials, except for ACP. It could be concluded that the addition of bioactive particles into an experimental infiltrant improved the mechanical properties and did not affect the capability of penetration into the experimental infiltrants. The resin infiltrant with amorphous calcium phosphate presented the best performance to the roughness surface before and after brushing abrasion
Doutorado
Materiais Dentarios
Doutora em Materiais Dentários
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Rodrigues, Sergio Gasques. "Estudo de técnicas de microscopia para caracterização estrutural de heteroestruturas semicondutoras." Universidade de São Paulo, 1997. http://www.teses.usp.br/teses/disponiveis/88/88131/tde-22082015-113608/.
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Este trabalho tem como objetivo principal, o estudo de técnicas de microscopia para a caracterização estrutural de semicondutores, visando o desenvolvimento das técnicas de preparação de amostras, visto que a caracterização estrutural é de suma importância para a obtenção de melhores resultados no processo de produção de filmes de semicondutores do grupo III-V. Dentre as técnicas mais utilizadas na caracterização estrutural, destacam-se as técnicas de microscopia eletrônica de varredura e de transmissão, juntamente com a microscopia de força atômica. Foram utilizadas amostras semicondutoras de InGaAs/GaAs e InAs/GaAs, crescidas pela técnica de MBE (epitaxia por feixe molecular), contendo pontos quânticos, estruturas estas ricas em detalhes. Tais amostras foram preparadas e caracterizadas em cada uma das técnicas em estudo. A microscopia de varredura e de força atômica apresentam fácil preparação. Os resultados obtidos, porém mostram que a técnica de microscopia eletrônica de varredura não oferece resolução suficiente para visualização das heteroestruturas; já a técnica de microscopia de força atômica mostra resultados excelentes da topografia dos pontos quânticos. Para a microscopia de transmissão a preparação de amostras mostra-se muito difícil e demorada, entretanto, o resultado obtido foi muito satisfatório. O processo de preparação passa por etapas de clivagem, \"dimpling\" e \"ion milling\". As imagens obtidas revelam com clareza a estrutura de pontos quântico. Com o estudo realizado, foi possível determinar as principais características de cada técnica, assim como determinar uma metodologia que pode vir a ser aplicada a outros tipos de heteroestruturas semicondutorasThis work has as main objective, the study of microscopy techniques for structural characterization of semiconductors and the development of the techniques of sample preparation, because the structural characterization is of highest importance for the obtaining of better results in the process of production of semiconductors thin films. The techniques more used in the structural characterization, are the techniques of electronic microscopy (Scanning and Transmission), together with the Atomic Force Microscopy. Samples of InGaAs/GaAs and InAs/GaAs were used, grown by the technique of MBE (Molecular Beam Epitaxy), with quantum dots, structures these rich ones in details. Such samples were prepared and characterized in each one of the techniques in study. The Scanning Microscopy and Atomic Force present easy preparation. The obtained results even so they show that the technique of Scanning Microscopy doesn\'t offer enough resolution for visualization of the heteroestructures; already the technique of Atomic Force shows excellent results of the topography of the quantum dots. For the Transmission Microscopy the preparation of samples is shown very difficult and delayed, however, the obtained result was very satisfactory. The preparation process goes by cutting stages, dimpling and ion milling. The obtained images reveal with clarity the quantum structure of points. With the accomplished study, it was possible to determine the main characteristics of each technique, as well as determining a methodology that can come to be applied to the other types of semiconductors heterostructures
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Kuhn, Jeffrey Russell. "Modulated polarization microscopy : a new instrument for visualizing cytoskeletal dynamics in living cells /." Digital version accessible at:, 2000. http://wwwlib.umi.com/cr/utexas/main.
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Kim, Doory. "Ultrastructural Studies by Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy." Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17463150.
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Fluorescence light microscopy (LM) and electron microscopy (EM) are two of the most widely used imaging modalities for probing cellular structures. In this dissertation I present our works in both developing methods of several correlative super-resolution fluorescence light microscopy (LM) and electron microscopy (EM) assays by combining stochastic optical reconstruction microscopy (STORM), a super-resolution imaging technique with several different EM imaging modalities and applying super-resolution microscopy to investigate the distributions and interactions of purine biosynthetic enzymes organization complex called purinosomes within the cell.
The first work contained in this dissertation is to develop Correlative fluorescence light microscopy and electron microscopy allows the imaging of spatial distributions of specific biomolecules in the context of cellular ultrastructure. Recent development of super-resolution fluorescence microscopy allows the location of molecules to be determined with nanometer-scale spatial resolution. However, correlative super-resolution fluorescence microscopy and electron microscopy (EM) still remains challenging because the optimal specimen preparation and imaging conditions for super-resolution fluorescence microscopy and EM are often not compatible. Here, we have developed several experiment protocols for correlative stochastic optical reconstruction microscopy (STORM) and EM methods, both for un-embedded samples by applying EM-specific sample preparations after STORM imaging and for embedded and sectioned samples by optimizing the fluorescence under EM fixation, staining and embedding conditions. We demonstrated these methods using a variety of cellular targets.
In the second part of this dissertation, I focus on the study of dynamic purine biosynthetic enzymes organization complex called purinosomes. Purine biosynthetic enzymes are assembled into dynamic multi-enzyme complex called purinosomes. However, spatial or temporal control of these structures remains unknown. Here, we explored the endogenous purinosomes in medically important HGPRT-deficient LND fibroblasts in order to understand the de novo purine biosynthesis. Using super-resolution microscopy we investigated the interaction of purinosomes and mitochondria or microtubules using photoactivatable fluorescent protein, mMaple3 and LND fibroblast as an ideal model system for the endogenous purinosomes formation in order to avoid possible protein aggregation problems. The STORM images with this ideal model system revealed a highly correlated spatial distribution of endogenous purinosomes with mitochondria or microtubules, suggesting direct physical associations between two structures. In addition to identifying endogenous purinosome association with other cellular components, we also demonstrated that mTOR directly influenced the purinosome association with mitochondria. Inhibition of mTOR decouples spatial correlation of purinosomes with mitochondria. These data provide strong evidences for physical and functional association of endogenous purinosomes with mitochondria and microtubules.Chemistry and Chemical Biology
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Bethge, Philipp. "Development of a two-photon excitation STED microscope and its application to neuroscience." Thesis, Bordeaux, 2014. http://www.theses.fr/2014BORD0018/document.
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L’avènement de la microscopie STED (Stimulated Emission Depletion) a bouleversé le domaine desneurosciences du au fait que beaucoup de structures neuronale, tels que les épines dendritiques, lesaxones ou les processus astrocytaires, ne peuvent pas être correctement résolu en microscopiephotonique classique. La microscopie 2-photon est une technique d’imagerie photonique très largement utilisée dans le domaine des neurosciences car elle permet d’imager les événements dynamique en profondeur dans le tissu cérébral, offrant un excellent sectionnement optique et une meilleure profondeur de pénétration. Cependant, la résolution spatiale de cette approche est limitée autour de 0.5 μm, la rendant inappropriée pour étudier les détails morphologiques des neurones et synapses. Le but de mon travail de thèse était à A) développer un microscope qui permet d'améliorer l'imagerie 2-photon en la combinant avec la microscopie STED et B) démontrer son potentiel pour l'imagerie à l'échelle nanométrique de processus neuronaux dynamiques dans des tranches de cerveau aigus et in vivo. Le nouveau microscope permet d'obtenir une résolution spatiale latérale de ~ 50 nm à des profondeurs d'imagerie de ~ 50 μm dans du tissu cérébral vivant. Il fonctionne avec des fluorophores verts, y compris les protéines fluorescentes communes telles que la GFP et YFP, offrant le contraste de deux couleurs basé sur la détection spectrale et linéaire ‘unmixing’. S’agissant d’un microscope droit, utilisant un objectif à immersion ayant une grande distance de travail, nous avons pu incorporer des techniques électrophysiologiques comme patch-clamp et ajouter une plateforme pour l'imagerie in vivo. J’ai utilise ce nouveau microscope pour imager des processus neuronaux fins et leur dynamique à l’échelle nanométrique dans différent types de préparations et des régions différentes du cerveau. J’ai pu révéler des nouvelles caractéristiques morphologique des dendrites et épines. En outre, j'ai exploré différentes stratégies de marquage pour pouvoir utiliser la microscopie STED pour imager le trafic des protéines et de leur dynamique à l'échelle nanométrique dans des tranches de cerveauThe advent of STED microscopy has created a lot of excitement in the field of neuroscience becausemany important neuronal structures, such as dendritic spines, axonal shafts or astroglial processes,cannot be properly resolved by regular light microscopy techniques. Two-photon fluorescence microscopy is a widely used imaging technique in neuroscience because it permits imaging dynamic events deep inside light-scattering brain tissue, providing high optical sectioning and depth penetration. However, the spatial resolution of this approach is limited to around half a micron, and hence is inadequate for revealing many morphological details of neurons and synapses. The aim of my PhD work was to A) develop a microscope that improves on two-photon imaging by combining it with STED microscopy and to B) demonstrate its potential for nanoscale imaging of dynamic neural processes in acute brain slices and in vivo. The new microscope achieves a lateral spatial resolution of ~50 nm at imaging depths of ~50 μm in living brain slices. It works with green fluorophores, including common fluorescent proteins like GFP and YFP, offering two-color contrast based on spectral detection and linear unmixing. Because of its upright design using a long working distance water-immersion objective, it was possible to incorporate electrophysiological techniques like patch-clamping or to add a stage for in vivo imaging. I have used the new microscope to image fine neural processes and their nanoscale dynamics in different experimental preparations and brain regions, revealing new and interesting morphological features of dendrites and spines. In addition, I have explored different labeling strategies to be able to use STED microscopy for visualizing protein trafficking and dynamics at the nanoscale in brain slices
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Canonge, Rafael. "Imagerie moléculaire 3D quantitative des tissus en utilisant la microscopie Raman cohérente sans marquage." Thesis, Ecole centrale de Marseille, 2017. http://www.theses.fr/2017ECDM0010/document.
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Cette thèse porte sur l'utilisation et le développement de techniques de microscopie multiphotonique pour l'imagerie d'échantillons biologiques humains. Une plateforme d'imagerie multiphotonique utilisant les contrastes non linéaires sans marquage tels que la fluorescence à deux photons, la génération de seconde harmonique, et les mécanismes Raman cohérent (CARS et SRS) a été conçue et développée au cours de cette thèse, et les travaux expérimentaux suivant deux axes de recherche principaux sont présentés.Dans une première partie , l'imagerie tridimensionnelle et sans marquage des muqueuses du système digestif humain est comparée aux images histologiques classiques avec marquages colorimétriques. Nous montrons que les techniques multiphotoniques utilisées permettent de reconstituer la structure et de discerner les différents éléments moléculaires présents dans les tissus dans le but d'obtenir une caractérisation des zones touchées par le développement de tumeurs cancéreusesThis thesis focuses on multiphotonic microscopy techniques development and use in order to image human biological samples. A multiphotonic imaging setup using label-free nonlinear contrasts mechanisms such as two-photons fluorescence, second harmonic generation, or stimulated Raman effect (CARS or SRS) has been designed and developped during this PhD, and I present the experimental work in two main research topics.In a first part, we compare label-free 3D imaging with classic histological imaging using colorimetric labels in human digestive system. We show that multiphotonic technics allow to reconstruct the organization and discern the molecular compounds inside the tissues, in order to get a caratérization of the cancerous tumors developpement.The second part is related to the application of our multimodal setup to the quantitative study of real active molecular compounds real time penetration into in vivo human skin. We show that multiphotonic microscopy make possible to mesure active molecules in depth 3D concentration in the skin in order to understand transcutaneous diffusion mechanisms in cosmetic and pharmacological applications
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Konda, Pavan Chandra. "Multi-Aperture Fourier Ptychographic Microscopy : development of a high-speed gigapixel coherent computational microscope." Thesis, University of Glasgow, 2018. http://theses.gla.ac.uk/9015/.
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Medical research and clinical diagnostics require imaging of large sample areas with sub-cellular resolution. Conventional imaging techniques can provide either high-resolution or wide field-of-view (FoV) but not both. This compromise is conventionally defeated by using a high NA objective with a small FoV and then mechanically scan the sample in order to acquire separate images of its different regions. By stitching these images together, a larger effective FoV is then obtained. This procedure, however, requires precise and expensive scanning stages and prolongs the acquisition time, thus rendering the observation of fast processes/phenomena impossible. A novel imaging configuration termed Multi-Aperture Fourier Ptychographic Microscopy (MA-FPM) is proposed here based on Fourier ptychography (FP), a technique to achieve wide-FoV and high-resolution using time-sequential synthesis of a high-NA coherent illumination. MA-FPM configuration utilises an array of objective lenses coupled with detectors to increase the bandwidth of the object spatial-frequencies captured in a single snapshot. This provides high-speed data-acquisition with wide FoV, high-resolution, long working distance and extended depth-of-field. In this work, a new reconstruction method based on Fresnel diffraction forward model was developed to extend FP reconstruction to the proposed MA-FPM technique. MA-FPM was validated experimentally by synthesis of a 3x3 lens array system from a translating objective-detector system. Additionally, a calibration procedure was also developed to register dissimilar images from multiple cameras and successfully implemented on the experimental data. A nine-fold improvement in captured data-bandwidth was demonstrated. Another experimental configuration was proposed using the Scheimpflug condition to correct for the aberrations present in the off-axis imaging systems. An experimental setup was built for this new configuration using 3D printed parts to minimise the cost. The design of this setup is discussed along with robustness analysis of the low-cost detectors used in this setup. A reconstruction model for the Scheimpflug configuration FP was developed and applied to the experimental data. Preliminary experimental results were found to be in agreement with this reconstruction model. Some artefacts were observed in these results due to the calibration errors in the experiment. These can be corrected by using the self-calibration algorithm proposed in the literature, which is left as a future work. Extensions to this work can include implementing multiplexed illumination for further increasing the data acquisition speed and diffraction tomography for imaging thick samples.
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于恩華 and Enhua Yu. "Crossed and uncrossed retinal fibres in normal and monocular hamsters: light and electron microscopic studies." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1990. http://hub.hku.hk/bib/B31232449.
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Bridger, Paul M. McGill T. C. McGill T. C. "Development of apertureless microscopy and force microscopy of GaN and CeO₂2 /." Diss., Pasadena, Calif. : California Institute of Technology, 1999. http://resolver.caltech.edu/CaltechETD:etd-10122007-132811.
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Schönenberger, Christian. "Understanding magnetic force microscopy /." Zürich : [ETH], 1990. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=9151.
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Diss. ETH no 9151, 1990.A dissertation submitted to the Swiss Federal Institute of Technology Zurich for the degree of Natural Sciences, accepted for publication in "Zeitschrift für Physik B" Bibliogr.: p. 40-43.
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Almqvist, Nils. "Scanning probe microscopy : Applications." Licentiate thesis, Luleå tekniska universitet, Materialvetenskap, 1994. http://urn.kb.se/resolve?urn=urn:nbn:se:ltu:diva-17980.
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Liu, Yanzhang. "Magnetic dissipation force microscopy." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape11/PQDD_0016/NQ44497.pdf.
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Donnermeyer, Achim. "Scanning ion-conductance microscopy." [S.l.] : [s.n.], 2007. http://nbn-resolving.de/urn/resolver.pl?urn=urn:nbn:de:hbz:361-11593.
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Liu, Yanzhang 1963. "Magnetic dissipation force microscopy." Thesis, McGill University, 1997. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=35002.
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This thesis concentrates on Magnetic Dissipation Force Microscopy---instrumentation, experiments and theory of the origin of magnetic dissipation.A home-built vacuum magnetic force microscope (MFM) was debugged. The electronic noise in the system was reduced to below the thermal cantilever noise and the microscope now operates at its theoretical maximum (thermally limited) sensitivity. Then, a new technique, magnetic dissipation imaging, was developed. It allows the imaging of variations of 10-17 W in dissipation with sub-100 nm resolution. A normal MFM image and a magnetic dissipation image can be acquired simultaneously on the same area of a sample.
A theory was developed which correlates the dissipation with micromagnetic structures in domain walls. We consider the energy dissipation through coherent generation of phonons via magnetostriction induced by domain wall width oscillations. A quantitative agreement of theory with experiments for a 110 nm thick Co/Ni multi-layer and a 4 nm thick Co film samples was obtained. This theory predicts two new phenomena: a minimum drive force needed to cause wall width oscillations and wall width resonances.
With the above mentioned microscope, magnetic domain structure, micromagnetic domain wall structure and the associated dissipation have been studied on several samples, including a 30 nm thick Ni80Fe20 patterned into 20 mum squares and a CoPtCr recording medium. The dissipation results show strong correlations with magnetic domain structure. In the Ni80 Fe20 sample, the dissipation signal shows pronounced maxima correlated with the domain wall positions. We suggest magnetoelastic losses and eddy current losses due to wall jumps are the origins of the dissipation. With an in-situ magnetizing stage, we also studied magnetization reversal processes and dissipation hysteresis in the Ni80Fe20 sample. Besides the nucleation and growth of reverse domains, the formation of a 360º wall was observed. The CoPtCr sample shows different dissipation properties with both larger and smaller than average dissipation value observed in the transition regions.
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Davies, D. G. "Scanning electron acoustic microscopy." Thesis, University of Cambridge, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.304042.
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Duncan, James Lyon. "Electron microscopy of photosystems." Thesis, Imperial College London, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.412477.
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Whitmore, Lawrence Charles. "Microscopy of nanomachined silicon." Thesis, University of Surrey, 1994. http://epubs.surrey.ac.uk/771883/.
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McJury, Mark. "NMR microscopy at 500MHz." Thesis, University of Nottingham, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.293694.
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Sutton, Simon Jonathan. "Microscopy of polypyrrole films." Thesis, University of Reading, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.317153.
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