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1
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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Cacovich, Stefania. "Electron microscopy studies of hybrid perovskite solar cells." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/276753.
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Over the last five years hybrid organic-inorganic metal halide perovskites have attracted strong interest in the solar cell community as a result of their high power conversion efficiency and the solid opportunity to realise a low-cost as well as industry-scalable technology. Nevertheless, several aspects of this novel class of materials still need to be explored and the level of our understanding is rapidly and constantly evolving, from month to month. This dissertation reports investigations of perovskite solar cells with a particular focus on their local chemical composition. The analytical characterisation of such devices is very challenging due to the intrinsic instability of the organic component in the nanostructured compounds building up the cell. STEM-EDX (Scanning Transmission Electron Microscopy - Energy Dispersive X-ray spectroscopy) was employed to resolve at the nanoscale the morphology and the elemental composition of the devices. Firstly, a powerful procedure, involving FIB (Focus Ion Beam) sample preparation, the acquisition of STEM-EDX maps and the application of cutting edge post-processing data techniques based on multivariate analysis was developed and tested. The application of this method has drastically improved the quality of the signal that can be extracted from perovskite thin films before the onset of beam-induced transformations. Morphology, composition and interfaces in devices deposited by using different methodologies and external conditions were then explored in detail by combining multiple complementary advanced characterisation tools. The observed variations in the nanostructure of the cells were related to different photovoltaic performance, providing instructive indications for the synthesis and fabrication routes of the devices. Finally, the main degradation processes that affect perovskite solar cells were probed. STEM-EDX was used in conjunction with the application of in situ heating, leading to the direct observation of elemental species migration within the device, reported here for the first time with nanometric spatial resolution. Further analyses, involving a set of experiments aimed to study the effects of air exposure and light soaking on the cells, were designed and performed, providing evidence of the main pathways leading to the drastic drop in the device performance.
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Wong, Tsz-wai Terence, and 黃子維. "Optical time-stretch microscopy: a new tool for ultrafast and high-throughput cell imaging." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hub.hku.hk/bib/B5066234X.
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The exponential expansion in the field of biophotonics over the past half-century has been leading to ubiquitous basic science investigations, ranging from single cell to brain networking analysis. There is also one biophotonics technology used in clinic, which is optical coherence tomography, mostly for high-speed and high-resolution endoscopy. To keep up such momentum, new biophotonics technologies should be aiming at improving either the spatial resolution or temporal resolution of optical imaging. To this end, this thesis will address a new imaging technique which has an ultra-high temporal resolution. The applications and its cost-effective implementations will also be encompassed.
In the first part, I will introduce an entirely new optical imaging modality coined as optical time-stretch microscopy. This technology allows ultra-fast real-time imaging capability with an unprecedented line-scan rate (~10 million frames per second). This ultrafast microscope is renowned as the world’s fastest camera. However, this imaging system is previously not specially designed for biophotonics applications. Through the endeavors of our group, we are able to demonstrate this optical time-stretch microscopy for biomedical applications with less biomolecules absorption and higher diffraction limited resolution (<2 μm). This ultrafast imaging technique is particularly useful for high-throughput and high-accuracy cells/drugs screening applications, such as imaging flow cytometry and emulsion encapsulated drugs imaging.
In the second part, two cost-effective approaches for implementing optical time-stretch confocal microscopy are discussed in details. We experimentally demonstrate that even if we employ the two cost-effective approaches simultaneously, the images share comparable image quality to that of captured by costly specialty 1μm fiber and high-speed ( >16 GHz bandwidth) digitizer. In other words, the cost is drastically reduced while we can preserve similar image quality.
At the end, I will be wrapping up my thesis by concluding all my work done and forecasting the future challenges concerning the development of optical time-stretch microscopy. In particular, three different research directions are discussed.published_or_final_version
Electrical and Electronic Engineering
Master
Master of Philosophy
4
Wätjen, Jörn Timo. "Microscopic Characterisation of Solar Cells : An Electron Microscopy Study of Cu(In,Ga)Se2 and Cu2ZnSn(S,Se)4 Solar Cells." Doctoral thesis, Uppsala universitet, Fasta tillståndets elektronik, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-199432.
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The sun provides us with a surplus of energy convertible to electricity using solar cells. This thesis focuses on solar cells based on chalcopyrite (CIGSe) as well as kesterite (CZTS(e)) absorber layers. These materials yield record efficiencies of 20.4 % and 11.1 %, respectively. Especially for CZTS(e), the absorber layers often do not consist of one single desired phase but can exhibit areas with deviating material properties, referred to as secondary phases. Furthermore, several material layers are required for a working solar cell, each exhibiting interfaces. Even though secondary phases and interfaces represent a very small fraction of the solar cell they can have a profound influence on the over-all electrical solar cell characteristics. As such, it is crucial to understand how secondary phases and interfaces influence the local electrical characteristics. Characterising secondary phases and interfaces is challenging due to their small sample volume and relatively small differences in composition amongst others. This is where electronmicroscopy, especially transmission electron microscopy, offers valuable insight to material properties on the microscopic scale. The main challenge is, however, to link these material properties to the corresponding electrical characteristics of a solar cell. This thesis uses electron beam induced current imaging and introduces a new method for JV characterisation of solar cells on the micron scale. Combining microscopic structural and electrical characterisation techniques allowed identifying and characterising local defects found in the absorber layer of CIGS solar cells after thermal treatment. Furthermore, CZTSe solar cells in this thesis exhibited a low photo-current density which is traced to the formation of a current blocking ZnSe secondary phase at the front contact interface. The electron microscopy work has contributed to an understanding of the chemical stability of CZTS and has shown the need for an optimised back contact interface in order to avoid chemical decomposition reactions and formation of detrimental secondary phases. With this additional knowledge, a comprehensive picture of the material properties from the macroscopic down to the microscopic level can be attained throughout all required material layers.
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Barnes, Clifford Alexander. "Supra-vital atomic force microscopy of living cultured cells." Thesis, University of Ulster, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.494333.
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Zeskind, Benjamin J. "Quantitative imaging of living cells by deep ultraviolet microscopy." Thesis, Massachusetts Institute of Technology, 2006. http://hdl.handle.net/1721.1/38693.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006.Includes bibliographical references (p. 139-145).
Developments in light microscopy over the past three centuries have opened new windows into cell structure and function, yet many questions remain unanswered by current imaging approaches. Deep ultraviolet microscopy received attention in the 1950s as a way to generate image contrast from the strong absorbance of proteins and nucleic acids at wavelengths shorter than 300 nm. However, the lethal effects of these wavelengths limited their usefulness in studies of cell function, separating the contributions of protein and nucleic acid proved difficult, and scattering artifacts were a significant concern. We have used short exposures of deep-ultraviolet light synchronized with an ultraviolet-sensitive camera to observe mitosis and motility in living cells without causing necrosis, and quantified absorbance at 280 nm and 260 nm together with tryptophan native fluorescence in order to calculate maps of nucleic acid mass, protein mass, and quantum yield in unlabeled cells. We have also developed a method using images acquired at 320nm and 340nm, and an equation for Mie scattering, to determine a scattering correction factor for each pixel at 260nm and 280nm. These developments overcome the three main obstacles to previous deep UV microscopy efforts, creating a new approach to imaging unlabeled living cells that acquires quantitative information about protein and nucleic acid as a function of position and time.
by Benjamin J. Zeskind.
Ph.D.
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Joensuu, Jenny. "Online Image Analysis of Jurkat T Cells using in situ Microscopy." Thesis, Linköpings universitet, Institutionen för fysik, kemi och biologi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-153313.
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Cell cultivation in bioreactors would benefit from developed monitoring systems with online real-time imaging to evaluate cell culture conditions and processes. This opportunity can be provided with the newly developed in situ Microscope also called ISM. The ISM probe is mounted into the wall of a bioreactor and consists of a measurement zone with an illuminating light source to obtain real-time images of moving cells in suspension. The instrument is linked to advanced imaging analysis software which can be specifically adapted for the objects in study. The aim of this project is to analyze the T lymphocyte cell line Jurkat T cells using the ISM equipment and identify specific features of the cells that can be obtained. The results show that the equipment and linked software are suitable for monitoring cell density, cell size distribution and cell surface analysis of the Jurkat cells during cultivation. The ISM could also detect induced changes in cell size caused by osmotic shifts and the course of an infection occurring in the cell suspension using a developed software for online real-time monitoring.
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Al-Rekabi, Zeinab. "Investigating Mechanotransduction and Mechanosensitivity in Mammalian Cells." Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/30256.
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Living organisms are made up of a multitude of individual cells that are surrounded by biomolecules and fluids. It is well known that cells are highly regulated by biochemical signals; however it is now becoming clear that cells are also influenced by the mechanical forces and mechanical properties of the local microenvironment. Extracellular forces causing cellular deformation can originate from many sources, such as fluid shear stresses arising from interstitial or blood flow, mechanical stretching during breathing or compression during muscle contraction. Cells are able to sense variations in the mechanical properties (elasticity) of their microenvironment by actively probing their surroundings by utilizing specialized proteins that are involved in sensing and transmitting mechanical information. The actin cytoskeleton and myosin-II motor proteins form a contractile (actomyosin) network inside the cell that is connected to the extracellular microenvironment through focal adhesion and integrin sites. The transmission of internal actomyosin strain to the microenvironment via focal adhesion sites generates mechanical traction forces. Importantly, cells generate traction forces in response to extracellular forces and also to actively probe the elasticity of the microenvironment. Many studies have demonstrated that extracellular forces can lead to rapid cytoskeletal remodeling, focal adhesion regulation, and intracellular signalling which can alter traction force dynamics. As well, cell migration, proliferation and stem cell fate are regulated by the ability of cells to sense the elasticity of their microenvironment through the generation of traction forces. In vitro studies have largely explored the influence of substrate elasticity and extracellular forces in isolation, however, in vivo cells are exposed to both mechanical cues simultaneously and their combined effect remains largely unexplored. Therefore, a series of experiments were performed in which cells were subjected to controlled extracellular forces as on substrates of increasing elasticity. The cellular response was quantified by measuring the resulting traction force magnitude dynamics. Two cell types were shown to increase their traction forces in response to extracellular forces only on substrates of specific elasticities. Therefore, cellular traction forces are regulated by an ability to sense and integrate at least two pieces of mechanical information - elasticity and deformation. Finally, this ability is shown to be dependent on the microtubule network and regulators of myosin-II activity.
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Muys, James Johan. "Cellular Analysis by Atomic Force Microscopy." Thesis, University of Canterbury. Electrical and Computer Engineering, 2006. http://hdl.handle.net/10092/1158.
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Exocytosis is a fundamental cellular process where membrane-bound secretory granules from within the cell fuse with the plasma membrane to form fusion pore openings through which they expel their contents. This mechanism occurs constitutively in all eukaryotic cells and is responsible for the regulation of numerous bodily functions. Despite intensive study on exocytosis the fusion pore is poorly understood. In this research micro-fabrication techniques were integrated with biology to facilitate the study of fusion pores from cells in the anterior pituitary using the atomic force microscope (AFM). In one method cells were chemically fixed to reveal a diverse range of pore morphologies, which were characterised according to generic descriptions and compared to those in literature. The various pore topographies potentially illustrates different fusion mechanisms or artifacts caused from the impact of chemicals and solvents in distorting dynamic cellular events. Studies were performed to investigate changes in fusion pores in response to stimuli along with techniques designed to image membrane topography with nanometre resolution. To circumvent some deficiencies in traditional chemical fixation methodologies, a Bioimprint replication process was designed to create molecular imprints of cells using imprinting and soft moulding techniques with photo and thermal activated elastomers. Motivation for the transfer of cellular ultrastructure was to enable the non-destructive analysis of cells using the AFM while avoiding the need for chemical fixation. Cell replicas produced accurate images of membrane topology and contained certain fusion pore types similar to those in chemically fixed cells. However, replicas were often dehydrated and overall experiments testing stimuli responses were inconclusive. In a preliminary investigation, a soft replication moulding technique using a PDMS-elastomer was tested on human endometrial cancer cells with the aim of highlighting malignant mutations. Finally, a Biochip comprised of a series of interdigitated microelectrodes was used to position single-cells within an array of cavities using positive and negative dielectrophoresis (DEP). Selective sites either between or on the electrode were exposed as cavities designed to trap and incubate pituitary and cancer cells for analysis by atomic force microscopy (AFMy). Results achieved trapping of pituitary and cancer cells within cavities and demonstrated that positive DEP could be used as a force to effectively position living cells. AFM images of replicas created from cells trapped within cavities illustrated the advantage of integrating the Biochip with Bioimprint for cellular analysis.
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Chhun, Bryant B. "Super-resolution video microscopy of live cells by structured illumination." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:1473623.
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Owen, Rachel Jane. "Dynamic Scanning probe microscopy of cells and chromosomes in liquid." Thesis, University of Bristol, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.409431.
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Wassie, Asmamaw T. "Nanoscale biomolecular mapping in cells and tissues with expansion microscopy." Thesis, Massachusetts Institute of Technology, 2019. https://hdl.handle.net/1721.1/123069.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019Cataloged from PDF version of thesis. "June 2019."
Includes bibliographical references.
The ability to map the molecular organization of cells and tissues with nanoscale precision would open the door to understanding their biological functions as well as the mechanisms that lead to pathologies. Though recent technological advances have expanded the repertoire of biological tools, this crucial ability remains an unmet need. Expansion Microscopy (ExM) enables the 3D, nanoscale imaging of biological structures by physically magnifying cells and tissues. Specimens, embedded in a swellable hydrogel, undergo uniform expansion as covalently anchored labels and tags are isotropically separated. ExM thereby allows for the inexpensive nanoscale imaging of biological samples on conventional light microscopes. In this thesis, I describe the development of a method called Expansion FISH (ExFISH) that uses ExM to enable the nanoscale imaging of RNA throughout cells and tissues. A novel chemical approach covalently retains endogenous RNA molecules in the ExM hydrogel. After expansion, RNA molecules can be interrogated with in situ hybridization. ExFISH opens the door for the investigation of the nanoscale organization of RNA molecules in various contexts. Applied to the brain, ExFISH allows for the precise localization of RNA in nanoscale neuronal compartments such as dendrites and spines. Furthermore, the optical homogeneity of expanded samples enables the imaging of RNA in thick tissue-sections. ExFISH also supports multiplexed imaging of RNA as well as signal amplification techniques. Finally, this thesis describes strategies for the multiplexed characterization of biological specimens. Taken together, these approaches will find applications in developing an integrative understanding of cellular and tissue biology.
by Asmamaw T. Wassie.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering
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Naidoo, Thegaran. "Digital holographic microscopy with automated detection of red blood cells." Diss., University of Pretoria, 2017. http://hdl.handle.net/2263/61032.
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The digital in-line holographic configuration is motivated by the goal of developing a portable, cost effective sensor system for pre-screening patient blood samples. The theory of holography is explained from the foundational concepts in scalar diffraction theory all the way through to the implementation of reconstruction algorithms. Methods for the enhancement of holographic reconstructions are described. The algorithms that perform an automated count of the reconstructed objects are described and demonstrated. Simulated and experimental results are provided. Together, the lens-free holographic microscopy of micro-sized particles along with the application of image processing techniques for the automated detection and counting of objects of interest, provide a component towards realising a sensor system that can be used for pre-screening patient blood samples.Dissertation (MSc)--University of Pretoria, 2017.
CSIR
Computer Science
MSc
Unrestricted
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Narchi, Paul. "Investigation of crystalline silicon solar cells at the nano-scale using scanning probe microscopy techniques." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLX085/document.
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Cette thèse s’intéresse à l’analyse de cellules silicium cristallin à l’échelle nanométrique, à l’aide de techniques de microscopie à sonde locale (SPM). En particulier, nous avons choisi d’analyser les propriétés électriques à l’échelle locale, grâce à deux techniques SPM : la microcopie à sonde de Kelvin (KPFM) et la microscopie à force atomique à sonde conductrice (CP-AFM).Tout d’abord, nous présentons les forces et faiblesses de ces deux techniques, comparées à la microscopie électronique, qui permet également d’analyser les propriétés électrique à l’échelle nanométrique. Cette comparaison approfondie nous permet d’identifier des mesures où le KPFM et le CP-AFM sont particulièrement adéquat et peuvent apporter de la valeur. Ces mesures sont divisées en deux catégories : les analyses matériaux et les analyses dispositifs.Ensuite, nous nous focalisons sur les analyses matériaux à l’échelle nanométrique. Nous présentons d’abord des mesures de dopage à l’échelle nanométrique, à l’aide d’une technique avancée de CP-AFM, appelée Resiscope. Nous montrons que cette technique peut détecter des changements de dopage dans la gamme 1015 à 1020 atomes.cm-3, avec une résolution nanométrique et un bon ratio signal/bruit. Puis, nous présentons des mesures de durée de décroissance sur des wafers silicium cristallin passivés. Les mesures sont réalisées sur la tranche non-passivée des échantillons. Nous montrons que, même si la tranche n’est pas passivée, les durées de décroissance obtenue par KPFM ont une bonne corrélation avec les temps de vie des wafers mesurées par décroissance de la photoconductivité détectée par micro-ondes.Par la suite, nous nous concentrons sur les analyses dispositif. A l’aide du KPFM, nous analysons deux types de cellules solaires silicium cristallin : les cellules solaires silicium épitaxié (epi-Si) et les cellules solaires hétérojonctions à contact arrière (IBC). En particulier, nous nous focalisons sur l’analyse de dispositifs en condition d’opération. Nous étudions d’abord l’influence de la tension électrique appliquée et nous montrons que les effets de résistance et de diode peuvent être détectés à l’échelle nanométrique. Les mesures de KPFM sont comparées aux mesures de microscopie électronique à balayage (SEM) dans les mêmes conditions, puisque le SEM est aussi sensible au potentiel de surface. Nous montrons que les mesures KPFM sur la tranche de cellules solaires epi-Si peuvent permettre d’étudier les changements de champ électrique avec la tension électrique appliquée. De plus, si la tension électrique est modulée en fréquence, nous montrons que des mesures de temps de vie peuvent être effectuées à l’échelle locale sur la tranche de cellules solaires epi-Si, ce qui peut permettre de détecter les interfaces limitantes. Puis, nous étudions l’influence de l’illumination sur les mesures KPFM et CP-AFM. Nous effectuons des mesures sur la tranche de cellules epi-Si sous différentes valeurs d’intensité et longueurs d’onde d’illumination. Nous montrons une bonne sensibilité des mesures KPFM à l’illumination. Cependant, nous montrons que pour différentes longueurs d’onde, à tension de circuit ouvert fixé, nos mesures ne sont pas corrélées avec les mesures de rendement quantique interne, comme nous le pensions.Enfin, nous résumons notre travail dans un tableau qui représente les forces et faiblesses des techniques pour les différentes mesures d’intérêt exposées précédemment. A partir de ce tableau, nous imaginons un setup de microscopie « idéal » qui permette d’analyser les cellules solaires de manière fiable, versatile et précise. Pour finir, nous proposons des mesures d’intérêt qui pourraient être réalisées avec ce setup « idéal »This thesis focuses on the investigation of crystalline silicon solar cells at the nano-scale using scanning probe microscopy (SPM) techniques. In particular, we chose to investigate electrical properties at the nano-scale using two SPM techniques: Kelvin Probe Force Microscopy (KPFM) and Conducting Probe Atomic Force Microscopy (CP-AFM).First, we highlight the strengths and weaknesses of both these techniques compared to electron microscopy techniques, which can also help investigate electrical properties at the nano-scale. This comprehensive comparison enables to identify measurements where KPFM and CP-AFM are particularly adequate. These measurements are divided in two categories: material investigation and devices investigation.Then, we focus on materials investigation at the nano-scale using SPM techniques. We first present doping measurements at the nano-scale using an advanced CP-AFM technique called Resiscope. We prove that this technique could detect doping changes in the range 1015 and 1020 atoms.cm-3 with a nano-scale resolution and a high signal/noise ratio. Then, we highlight decay time measurements on passivated crystalline silicon wafers using KPFM. Measurements are performed on the unpassivated cross-section. We show that, even though the cross-section is not passivated, decay times measurements obtained with KPFM are in good agreement with lifetimes measured by microwave photoconductivity decay.Subsequently, we focus on device measurements. Using KPFM, we investigate two different crystalline silicon solar cell architectures: epitaxial silicon (epi-Si) solar cells and interdigitated back contact (IBC) heterojunction solar cells. In particular, we focus on measurements on devices under operating conditions. We first study the influence of the applied electrical bias. We study the sensitivity of surface potential to electrical bias and we show that diode and resistance effects can be detected at the nano-scale. KPFM measurements are compared to scanning electron microscopy (SEM) measurements in the same conditions since SEM is also sensitive to surface potential. We show that KPFM measurements on the cross-section of epi-Si solar cells can help detect electric field changes with electrical bias. Besides, if the electrical bias is frequency modulated, we show that lifetime measurements can be performed on the cross-section of epi-Si solar cells and can help detect limiting interfaces and layers. Then, we study the influence of illumination on KPFM and CP-AFM measurements. We perform photovoltage and photocurrent measurements on the cross-section of epi-Si solar under different values of illumination intensity and illumination wavelength. We show a good sensitivity of KPFM measurements to illumination. However, we show that measurements for different wavelengths at a given open circuit voltage, are not correlated with the internal quantum efficiency, as we could have expected.Finally, we summarize our work in a table showing the impact of strengths and weaknesses of the techniques for the different measurements highlighted. From this table, we imagine an “ideal” microscopy setup to investigate crystalline silicon solar cells in a reliable, versatile and accurate way. We propose investigations of interest that could be carried out using this “ideal” setup
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Kosmacek, Elizabeth Anne Ianzini Fiorenza Mackey Michael A. "Live cell imaging technology development for cancer research." [Iowa City, Iowa] : University of Iowa, 2009. http://ir.uiowa.edu/etd/388.
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Neils, Christopher Martin. "Laser scanning microscopy of broad freezing interfaces with applications to biological cells /." Full text (PDF) from UMI/Dissertation Abstracts International, 2000. http://wwwlib.umi.com/cr/utexas/fullcit?p3004349.
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Donno, Roberto. "Nanomechanical characterisation of cells and biocompatible substrates." Thesis, University of Manchester, 2014. https://www.research.manchester.ac.uk/portal/en/theses/nanomechanical-characterisation-of-cells-and-biocompatible-substrates(f4d8bf94-035f-4798-ad64-02d319756974).html.
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Atomic Force Microscopy (AFM) is a powerful technique that has evolved from being a purely imaging tool to a one capable of providing multifunctional information, offering exciting new possibilities for nano-biotechnology. The project focuses on the use of the AFM in order to morphologically and mechanically characterise cells and biomaterials demonstrating how versatile this instrument can be. The project is divided in the following parts:Part 1: establishment of AFM protocols for the nano-scale morphological and mechanical characterisation of soft and hard macroscopic substrates and of objects such as adsorbed nanoparticles. In particular, these techniques were tested on:Hyaluronic acid (HA)/poly(ethylene glycol) (PEG)-based hydrogels, which provide an artificial model for the mechanical behaviour of some biological tissues and organs. The elastic modulus, measured via AFM nanoindentation, of these hydrogels increased by decreasing the concentration and the molecular weight (MW) of HA in the hydrogels. We have then verified a clear relation between the mechanical properties of the hydrogels and the proliferation of cells cultured on them. Chitosan nanoparticle (popular carriers for the delivery of negatively charged macromolecular payloads, e.g. nucleic acids) cross-linked with triphosphate (TPP) and then coated with HA. We focussed on the influence of chitosan molecular weight (Mw) on nanoparticle properties. HA was able to penetrate into the more porous nanoparticles (high MW chitosan), whereas it formed a corona around the more cross-linked ones (low MW chitosan). AFM imaging was used to highlight the presence of this corona and also to estimate its apparent thickness to about 20-30 nm (in dry state).Silicone substrates modified with amphiphilic triblock copolymer (Sil-GMMA) layers. Extensive AFM (imaging and nanoindentation) provided evidence that silicone substrates are prevalently coated with Sil-GMMA thin layers that exhibit negligible hydrophobic recovery during drying and change the surface from more to less cell-adhesive. Part 2: AFM mechanical characterisation of fibroblast-to-myofibroblast differentiation process. Fibroblasts were stimulated to differentiate into myofibroblasts by Transforming Grow Factor β1 (TGFβ1) on hard substrate. AFM force maps performed both on fibroblasts (untreated cells) and myofibroblasts (TGFβ1-treated cells) revealed a significant increase in the elastic modulus in treated cells. Part 3: preparation and AFM characterisation of poly(ethylene glycol) diacrylate/acrylate (PEGDA/A) hydrogels. Since the mechanical properties of the substrate plays a pivotal role in fibroblast-to-myofibroblast differentiation process, hydrogels were prepared and characterised at the macro/nanoscale with AFM indentation, providing us with cell-adhesive substrates that cover a wide range of elastic modulus. These substrates are optimal candidates for future investigations to better understand and possibly control the differentiation process.
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Parshall, Daniel. "Phase imaging digital holography for biological microscopy." [Tampa, Fla.] : University of South Florida, 2004. http://purl.fcla.edu/fcla/etd/SFE0000285.
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Friedrichs, Jens. "Analyzing Interactions Between Cells And Extracellular Matrix By Atomic Force Microscopy." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2009. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-25093.
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Interactions of cells with the extracellular matrix (ECM) have important roles in various physiological and pathological processes, including tissue morphogenesis during embryonic development, wound healing and tumor invasion. Although most of the proteins involved in cell-ECM interactions have been identified, the underlying mechanisms and involved signaling pathways are incompletely understood. Here, atomic force microscope-based imaging and single-cell force measurements were used to characterize the interactions of different cell types with ECM proteins.
The interplay between cells and ECM is complex. However, two interaction types, protein-protein and protein-carbohydrate, predominate. Integrins, adhesion receptors for ECM, mediate the former, galectins, a family of animal lectins, the latter. In the second chapter of this thesis, the contributions of both receptor families to the interactions of epithelial MDCK cells with ECM proteins are presented. It was found that galectins-3 and 9 are highly expressed in MDCK cells and required for optimal long-term adhesion (90 minutes) to ECM proteins collagen-I and laminin-111. Interestingly, early adhesion (< 2 minutes) to laminin-111, was integrin-independent and instead mediated by carbohydrate interactions and galectins. In contrast, early adhesion to collagen-I was exclusively mediated by integrins. Moreover, cells frequently entered an enhanced adhesion state, marked by a significant increase in the force required for cell detachment. Although adhesion was mediated by integrins, adhesion enhancement was especially observed in cells depleted for galectin-3. It was proposed that galectin-3 influences integrin-mediated adhesion complex formation by altering receptor clustering.
To control their attachment to ECM proteins, cells regulate integrin receptors. One regulatory process is integrin crosstalk, where the binding of one type of integrin influences the activity of another type. In the third chapter, the implementation of a single-cell force spectroscopy assay to identify such crosstalks and gain insight into their mechanisms is described. In this assay the interactions of integrin receptors being specifically attached to one ligand are characterized in dependence of another ligand-bond receptor pair. With this assay a crosstalk between collagen-binding integrin α1β1 and fibronectin-binding integrin α5β1 was identified in HeLa cells. This crosstalk was directional from integrin α1β1 to integrin α5β1 and appeared to regulate integrin α5β1 by inducing its endocytosis.
In the fourth and final chapter, mechanisms of matrix-induced cell alignment were studied by imaging cells on two-dimensional matrices assembled of highly aligned collagen fibrils. Integrin α2β1 was identified as the predominant receptor mediating cell polarization. Time-lapse AFM demonstrated that during alignment cells deform the matrix by reorienting individual collagen fibrils. Cells deformed the collagen matrix asymmetrically, revealing an anisotropy in matrix rigidity. When matrix rigidity was rendered uniform by chemical cross-linking or when the matrix was formed from collagen fibrils of reduced tensile strength, cell polarization did not occur. This suggested that both the high tensile strength and pliability of collagen fibrils contribute to the anisotropic rigidity of the matrix and lead to directional cellular traction and cell polarization. During alignment, cellular protrusions contacted the collagen matrix from below and above. This complex entanglement of cellular protrusions and collagen fibrils may further promote cell alignment by maximizing cellular traction.
The work presented here adds to the understanding of cell-ECM interactions. Atomic force microscopy imaging allowed characterizing the behavior of cells on nanopatterned collagen matrices whereas single-cell force spectroscopy revealed insights into the regulation of cell adhesion by galectins. Furthermore, methodological advances in the single-cell force spectroscopy assay allowed the intracellular regulation of receptor molecules to be studied. The work demonstrates that atomic force microscopy is a versatile tool to study cell-ECM interactions.
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Michiels, Rebecca [Verfasser], and Alexander [Akademischer Betreuer] Rohrbach. "Investigation of filopodia dynamics in macrophage cells by photonic force microscopy." Freiburg : Universität, 2019. http://d-nb.info/1185977295/34.
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Oh, Seung-eun. "Quantitative phase microscopy for the study of electromotility in living cells." Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/62650.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Physics, 2010.Cataloged from PDF version of thesis.
Includes bibliographical references (p. 118-123).
The electric activity of living cells is accompanied with changes in their optical and mechanical properties, which arise from the intrinsic biophysics of the cell membrane. These intrinsic changes can be used as an indicator for cell electric activity, but, to our knowledge, the intrinsic signal of electric activity has never been detected in single vertebrate cells. We describe here our development of a quantitative phase microscopy technique that is capable of detecting the intrinsic changes induced by electric activity in a human cell line. Chapter 1 provides introductory material regarding cellular electrophysiology and a review of the literature on the intrinsic signal of cell electric activity. This chapter also briefly introduces the quantitative phase microscope. In Chapter 2, we discuss our pilot studies and introduce the electromotility of prestinexpressing HEK293 cells as a test system. We describe our design of an effective optical detection scheme based on quantitative phase imaging and frequency domain detection which provides full-field, high resolution, high sensitivity, quantitative detection of electrically induced optical signals in cells. In Chapter 3, we demonstrate an improved quantitative phase microscope based on low-coherence interferometry with enhanced sensitivity and lower noise. We successfully acquired images of the intrinsic optical signal from electrically stimulated single HEK293 cells. In Chapter 4, we characterized the electrochemical properties and dynamic properties of the intrinsic optical signal. We argue that the signal is generated through the electromechanical coupling mechanism called membrane electromotility (MEM). Using the MEM signal as an indicator of membrane electric activity, we imaged the propagation of an applied potential in a network of cells in Chapter 5. Our research shows that high resolution quantitative phase imaging is a powerful tool that can provide significant insight into the underlying mechanism of cellular intrinsic optical signal of electric activity. Membrane electromotility imaging provides a novel opportunity for the visualization of the electrical connectivity of cultured cells.
by Seungeun Oh.
Ph.D.
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Magnusson, Klas E. G. "Segmentation and tracking of cells and particles in time-lapse microscopy." Doctoral thesis, KTH, Signalbehandling, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-196911.
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In biology, many different kinds of microscopy are used to study cells. There are many different kinds of transmission microscopy, where light is passed through the cells, that can be used without staining or other treatments that can harm the cells. There is also fluorescence microscopy, where fluorescent proteins or dyes are placed in the cells or in parts of the cells, so that they emit light of a specific wavelength when they are illuminated with light of a different wavelength. Many fluorescence microscopes can take images on many different depths in a sample and thereby build a three-dimensional image of the sample. Fluorescence microscopy can also be used to study particles, for example viruses, inside cells. Modern microscopes often have digital cameras or other equipment to take images or record time-lapse video. When biologists perform experiments on cells, they often record image sequences or sequences of three-dimensional volumes to see how the cells behave when they are subjected to different drugs, culture substrates, or other external factors. Previously, the analysis of recorded data has often been done manually, but that is very time-consuming and the results often become subjective and hard to reproduce. Therefore there is a great need for technology for automated analysis of image sequences with cells and particles inside cells. Such technology is needed especially in biological research and drug development. But the technology could also be used clinically, for example to tailor a cancer treatment to an individual patient by evaluating different treatments on cells from a biopsy. This thesis presents algorithms to find cells and particles in images, and to calculate tracks that show how they have moved during an experiment. We have developed a complete system that can find and track cells in all commonly used imaging modalities. We selected and extended a number of existing segmentation algorithms, and thereby created a complete tool to find cell outlines. To link the segmented objects into tracks, we developed a new track linking algorithm. The algorithm adds tracks one by one using dynamic programming, and has many advantages over prior algorithms. Among other things, it is fast, it calculates tracks which are optimal for the entire image sequence, and it can handle situations where multiple cells have been segmented incorrectly as one object. To make it possible to use information about the velocities of the objects in the linking, we developed a method where the positions of the objects are preprocessed using a filter before the linking is performed. This is important for tracking of some particles inside cells and for tracking of cell nuclei in some embryos. We have developed an open source software which contains all tools that are necessary to analyze image sequences with cells or particles. It has tools for segmentation and tracking of objects, optimization of settings, manual correction, and analysis of outlines and tracks. We developed the software together with biologists who used it in their research. The software has already been used for data analysis in a number of biology publications. Our system has also achieved outstanding performance in three international objective comparisons of systems for tracking of cells.Inom biologi används många olika typer av mikroskopi för att studera celler. Det finns många typer av genomlysningsmikroskopi, där ljus passerar genom cellerna, som kan användas utan färgning eller andra åtgärder som riskerar att skada cellerna. Det finns också fluorescensmikroskopi där fluorescerande proteiner eller färger förs in i cellerna eller i delar av cellerna, så att de emitterar ljus av en viss våglängd då de belyses med ljus av en annan våglängd. Många fluorescensmikroskop kan ta bilder på flera olika djup i ett prov och på så sätt bygga upp en tre-dimensionell bild av provet. Fluorescensmikroskopi kan även användas för att studera partiklar, som exempelvis virus, inuti celler. Moderna mikroskop har ofta digitala kameror eller liknande utrustning för att ta bilder och spela in bildsekvenser. När biologer gör experiment på celler spelar de ofta in bildsekvenser eller sekvenser av tre-dimensionella volymer för att se hur cellerna beter sig när de utsätts för olika läkemedel, odlingssubstrat, eller andra yttre faktorer. Tidigare har analysen av inspelad data ofta gjorts manuellt, men detta är mycket tidskrävande och resultaten blir ofta subjektiva och svåra att reproducera. Därför finns det ett stort behov av teknik för automatiserad analys av bildsekvenser med celler och partiklar inuti celler. Sådan teknik behövs framförallt inom biologisk forskning och utveckling av läkemedel. Men tekniken skulle också kunna användas kliniskt, exempelvis för att skräddarsy en cancerbehandling till en enskild patient genom att utvärdera olika behandlingar på celler från en biopsi. I denna avhandling presenteras algoritmer för att hitta celler och partiklar i bilder, och för att beräkna trajektorier som visar hur de har förflyttat sig under ett experiment. Vi har utvecklat ett komplett system som kan hitta och följa celler i alla vanligt förekommande typer av mikroskopi. Vi valde ut och vidareutvecklade ett antal existerande segmenteringsalgoritmer, och skapade på så sätt ett heltäckande verktyg för att hitta cellkonturer. För att länka ihop de segmenterade objekten till trajektorier utvecklade vi en ny länkningsalgoritm. Algoritmen lägger till trajektorier en och en med hjälp av dynamisk programmering, och har många fördelar jämfört med tidigare algoritmer. Bland annat är den snabb, den beräknar trajektorier som är optimala över hela bildsekvensen, och den kan hantera fall då flera celler felaktigt segmenterats som ett objekt. För att kunna använda information om objektens hastighet vid länkningen utvecklade vi en metod där objektens positioner förbehandlas med hjälp av ett filter innan länkningen utförs. Detta är betydelsefullt för följning av vissa partiklar inuti celler och för följning av cellkärnor i vissa embryon. Vi har utvecklat en mjukvara med öppen källkod, som innehåller alla verktyg som krävs för att analysera bildsekvenser med celler eller partiklar. Den har verktyg för segmentering och följning av objekt, optimering av inställningar, manuell korrektion, och analys av konturer och trajektorier. Vi utvecklade mjukvaran i samarbete med biologer som använde den i sin forskning. Mjukvaran har redan använts för dataanalys i ett antal biologiska publikationer. Vårt system har även uppnått enastående resultat i tre internationella objektiva jämförelser av system för följning av celler.
QC 20161125
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Taleb. "Raman Microscopy and ComputationalTechniques for the Chemometric Analysis of Tumor Cells." Thesis, Queen's University Belfast, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.501411.
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Yu, Xiao. "Study of the Motility of Biological Cells by Digital Holographic Microscopy." Scholar Commons, 2014. https://scholarcommons.usf.edu/etd/5159.
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In this dissertation, I utilize digital holographic microscopy (DHM) to study the motility of biological cells. As an important feature of DHM, quantitative phase microscopy by digital holography (DH-QPM) is applied to study the cell-substrate interactions and migratory behavior of adhesive cells. The traction force exerted by biological cells is visualized as distortions in flexible substrata. Motile fibroblasts produce wrinkles when attached to a silicone rubber film. For the non-wrinkling elastic substrate polyacrylamide (PAA), surface deformation due to fibroblast adhesion and motility is visualized as tangential and vertical displacement. This surface deformation and the associated cellular traction forces are measured from phase profiles based on the degree of distortion. Intracellular fluctuations in amoeba cells are also analyzed statistically by DH-QPM. With the capacity of yielding quantitative measures directly, DH-QPM provides efficient and versatile means for quantitative analysis of cellular or intracellular motility.
Three-dimensional profiling and tracking by DHM enable label-free and quantitative analysis of the characteristics and dynamic processes of objects, since DHM can record real-time data for micro-scale objects and produce a single hologram containing all the information about their three-dimensional structure. Here, I utilize DHM to visualize suspended microspheres and microfibers in three dimensions, and record the four-dimensional trajectories of free-swimming cells in the absence of mechanical focus adjustment. The displacement of microfibers due to interactions with cells in three spatial dimensions is measured as a function of time at sub-second and micrometer levels in a direct and straightforward manner. It has thus been shown that DHM is a highly efficient and versatile means for quantitative tracking and analysis of cell motility.
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Streetley, J. W. A. "Cryo-electron microscopy of Weibel-Palade Bodies in human endothelial cells." Thesis, University College London (University of London), 2014. http://discovery.ucl.ac.uk/1458642/.
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Weibel-Palade bodies (WPBs) are a secretory storage organelle found in endothelial cells. Their primary cargo is the large haemostatic protein von Willebrand factor (VWF), however there are many other minor components, both in the membrane and lumen of the WPB. VWF is stored in long helical tubules which define the ‘rod-like’ shape of the WPBs. WPBs are released in response to raised intracellular Ca²⁺ or cAMP levels, secreting the VWF content. Upon exocytosis, the environment of the VWF changes from acidic to neutral pH, causing the tubules of VWF to unfurl into strings. The expansion of the VWF tubules into strings exposes binding and cleavage sites, turning VWF into its haemostatically active form. As tubules transform into strings, the overall shape of the WPB changes, first becoming round before releasing its contents. Imaging at the periphery of frozen-hydrated, whole human umbilical vein endothelial cells (HUVECs) by cryo-electron microscopy (cryo-EM) and cryo-electron tomography has been used to study WPBs within their native environment. The structure of WPBs within HUVECs both unstimulated and stimulated to release WPBs with raised intracellular Ca²⁺ has been examined. This has enabled imaging of both rod-shaped and rounded WPBs. Round WPBs in HUVECs exposed to hypotonic shock and within human heart microvasculature endothelial cells (HHMECs) have also been imaged. A subset of the WPBs imaged within endothelial cells contain an internal vesicle within them. Light microscopy studies have shown a vesicle with corresponding characteristics is enriched for the membrane protein CD63, a known constituent of WPBs. Finally, a technique for chemically fixing cells for light microscopy studies prior to plunge-freezing is shown, with subsequent correlative light and electron microscopy experiments looking at CD63-enriched vesicles within WPBs. The quality of preservation for cryo-EM experiments, including tomography and image processing for correlative microscopy is discussed.
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Zheng, Tao. "Investigation of plant tissue by environmental scanning electron microscopy." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609068.
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Pérez, Rodríguez Ana. "Nanoscale interpretation of performances in organic solar cells and field effect transistors." Doctoral thesis, Universitat Autònoma de Barcelona, 2018. http://hdl.handle.net/10803/565824.
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Dos de los principales retos a superar en el campo de la electrónica orgánica son el control la morfología de la capa semiconductora y las propiedades de las interfases. En particular, las interfases formadas por la capa orgánica semiconductora y los electrodos metálicos influyen fuertemente en el comportamiento de los dispositivos. Hasta el momento se ha dedicado un gran esfuerzo a la mejora de estas interfases siguiendo para ello diferentes estrategias como son el uso de monocapas autoensambladas (SAMs), capas de óxidos metálicos o mediante el dopaje de los contactos. Con respecto a la morfología, se ha probado que ésta juega un papel fundamental en la disociación de los excitones, en la extracción de la carga y en la recombinación en células solares orgánicas (OSCs), así como en las propiedades de trasporte en transistores orgánicos de efecto campo (OFETs). En este trabajo se utiliza principalmente el microscopio de fuerza atómica (AFM) y, en menor medida, otras técnicas de caracterización de superficies, y en la investigación de interfases que forman parte de estos dispositivos. En particular, se ha empleado el microscopio de fricción atómica (FFM), el microscopio de conducción de fuerza atómica (AFM) y el microscopio de Kelvin de fuerza atómica (KPFM) en dispositivos OSC y OFETs, con el objetivo de correlacionar la caracterización a escala nanométrica con el rendimiento macroscópico de los dispositivos. Este documento está organizado de la siguiente forma. Las motivaciones del trabajo se presentan en el Capítulo 1. En el Capítulo 2 se incluye una introducción teórica a los semiconductores orgánicos y a los conceptos de autoensamblado y nanoestructuración. Las técnicas y metodologías empleadas en la tesis se describen en el Capítulo 3. Los resultados se presentan en los capítulos 4, 5, 6 y 7.
En el Capítulo 4 se detalla el fundamento físico del Transverse Shear Microscopy (TSM). La combinación de datos experimentales con resultados de simulaciones numéricas nos ha permitido probar que la señal de TSM tiene naturaleza disipativa.
El Capítulo 5 se centra en el efecto de las capas transportadoras de huecos (HTLs) y su impacto en el rendimiento de células solares fabricados con materiales orgánicos y con perovskitas. Respecto a las células solares, hemos demostrado que el uso de SAMs de ácidos fosfónicos cambia favorablemente la función de trabajo del cátodo de ITO, pero también induce una acumulación de carga en la interfase con efectos negativos en el rendimiento del dispositivo. En las células solares de perovskitas, a pesar de utilizar un material recientemente sintetizado como HTL con una posición del HOMO más favorable, el alineamiento energético en la interfase con el TiO2 resulta menos favorable, dando lugar a peores propiedades de las células solares.
El Capítulo 6 está dedicado al efecto del solvent vapor annealing (SVA) en la cristalinidad y la separación vertical de fase en células solares de oligotiofenos comprobando que, al aplicar el método SVA, los dominios del oligómero muestran mejor cristalinidad, mientras que los dominios del fullereno aumentan en tamaño, mejorando las propiedades fotovoltaicas de los dispositivos.
En el Capítulo 7 hemos realizado una caracterización a escala nanométrica de OFETs fabricados con C8-BTBT:PS mediante FFM de OFETs fabricados con C8-BTBT:PS, que ha permitido resolver la separación vertical de fase del PS y el C8-BTBT. Mediante KPFM, se obtuvieron mapas del potencial de superficie de los OFETs, a partir de los que se han obtenido valores de la resistencia de contacto y de la movilidad de carga para diferentes electrodos y concluyendo que la resistencia de contacto es un factor crítico que limita el rendimiento de estos dispositivos.
Las principales conclusiones de la tesis se incluyen en el Capítulo 8Two of the main challenges in organic electronic devices are the semiconducting layer morphology and the interface properties. Particularly, the interfaces formed by the semiconducting organic layer and the metallic electrodes strongly influence the performance of the devices. Thus, a strong effort has been devoted to improve these interfaces by different approaches such as self assembled monolayers (SAMs), layer of metalic oxides or contact doping. Concerning the morphology, it has been proven that it plays a fundamental role in exciton dissociation, charge collection and recombination in organic solar cells (OSC), as well as in the transport properties in organic field effect transistors (OFETs). In this work we make use of atomic force microscopy (AFM) and, in a lesser extent, of other surface characterization techniques for the study of surfaces and interfaces that conform organic electronic devices. In particular, we focus on the use of friction force microscopy (FFM), conductive atomic force microscopy (C-AFM) and Kelvin probe force microscopy (KPFM) operating modes on OSC and OFETs devices with the goal of correlating the nanoscale characterization with the macroscopic performance of the devices. This thesis is organized in the following way: the motivations of this work are presented in Chapter 1. In Chapter 2 a brief theoretical introduction on organic semiconductors and the concept of self assembly and nanostructuration is given, while in Chapter 3 the techniques employed during this thesis as well as the used methodologies are described. The results are presented in the Chapters 4, 5, 6 and 7. In Chapter 4 we study in detail the physical origin behind Transverse Shear Microscopy (TSM). By combining experimental data with simulations, we prove that the TSM signal has a dissipative origin and we use the technique to obtain the crystalline orientation of tip-induced grown PTCDI-C8 islands. In Chapter 5 we focus on the effect of hole transport layers (HTLs) for both organic and perovskites solar cells. For bulk-heterojunction solar cells (BHJ) we prove that the use of phosphonic acid self assembled monolayers (SAMs) changes the workfunction of the ITO cathode in a favourable way, but also induces a charge accumulation density at the interface with detrimental effects for the cell performance. In perovskite solar cells, despite using newly synthesized HTL with more favourable HOMO position, the energy level alignment at the interface with the TiO2 results less favourable leading to worse photovoltaic device properties. Chapter 6 is devoted to the solvent vapor annealing (SVA) effect on the crystallinity and vertical phase separation on oligothiophene bulkheterojunction solar cells. We prove that, upon SVA, the oligomer domains present better crystallinity while the fullerene domains increase in size, enhancing the photovoltaic performance of the devices. In Chapter 7, a nanoscale characterization by means of FFM was correlated with the device performance for C8-BTBT:PS OFETs, providing with a picture at the nanoscale of the organic films vertical phase separation. By means of KPFM, maps of the surface potential of the OFETs were obtained, allowing us to extract contact resistance and charge mobility values for different electrodes, concluding that the contact resistance is the critical factor limiting the devices performance. Finally, in Chapter 8, the main conclusions of this thesis will be collected.
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Andersson, Schönn Mikael. "Promoter regulation : designing cells for biotechnological applications." Thesis, Uppsala universitet, Molekylär biomimetik, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-297502.
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The filamentous cyanobacteria Nostoc punctiforme ATCC 29133 is a model species fordevelopment of sustainable production methods of numerous compounds. One of its uniquefeatures is the anaerobic environment of the strains nitrogen fixing heterocyst cells. To be ableto properly utilize this environment, more knowledge regarding what regulates cell specificexpression is required. In this study, three motifs of the NsiR I promoter of Anabaena sp.PCC 7120 was studied in this system utilizing YFP-fluorescence as a reporter to determinetheir impact on spatial expression pattern. Investigations were performed on immobilizedcells with the use of confocal microscopy and results point towards sigma factor regulation.
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Kroeger, Benjamin Robert. "The genetic regulation and subcellular dynamics of secretory and endolysosomal organelles of Drosophila secondary cells." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:dce9ae14-b03d-4fca-8429-de839cc40d6a.
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Secretory processes underpin the emergence of cellular diversity in complex multicellular organisms. However, our understanding of the basic mechanisms controlling the different secretory and endosomal compartments involved remains surprisingly incomplete. During my DPhil I have studied a specialised epithelial cell type in the male Drosophila accessory glands, the secondary cell, which contains unusually large intracellular compartments that are accessible to detailed morphological study. I characterise the organisation, ultrastructure and molecular composition of this cell's secretory and endosomal compartments, and I employ specific Rab GTPases, conserved coordinators of membrane trafficking and identity, to define multiple compartmental subtypes. By developing super-resolution and time-lapse microscopy approaches in these cells, I show that numerous intraluminal vesicles (ILVs) are formed within Rab11-labelled secretory compartments and released into the accessory gland lumen as exosomes, the first clear demonstration in eukaryotic cells of exosome biogenesis within a non-late endosomal compartment. Biogenesis of these ILVs is dependent on evolutionarily conserved Endosomal Sorting Complexes Required for Transport (ESCRT) 0-III genes and involves loading of compartment-specific cargoes. Work by others, some in collaboration with me, has shown that these novel mechanisms are conserved in human cells. I show that dense-core granules, the structures employed to package proteins and other molecules destined for regulated secretion, form within large non-cored Rab6- positive compartments, in a process that seems to involve inputs from both the Golgi and recycling endosomal pathways. Further analysis has revealed roles for specific Rabs, for ILVs, and for the conserved fibrillar protein Mfas/TGFBI in different aspects of DCG formation. I also show that DCGs are not only secreted, but can also be degraded by fusion to acidic endosomal compartments. Remarkably, there is evidence that mammalian cells may employ all of these mechanisms and defects in these processes may be linked to diseases like cancer, diabetes and neurodegenerative disorders. Hence my work has established a new system to study complex secretory mechanisms, which can now be developed to model specific disease processes in the future. In summary, I have discovered several novel cell biological mechanisms controlling exosome biology, dense-core granule biogenesis, regulated secretion, and endolysosomal trafficking. Some of these already appear relevant to human health and disease, suggesting that the secondary cell system has considerable further potential for unravelling the fundamental processes underlying eukaryotic secretion in the future.
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Lähdesmäki, Ilkka Johannes. "Flow injection methods for drug-receptor interaction studies, based on probing cell metabolism /." Thesis, Connect to this title online; UW restricted, 1999. http://hdl.handle.net/1773/8590.
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Al-Roubaie, Sarah. "Time-lapse microscopy of endothelial cells and macrophages during embryonic vascular development." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=107779.
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Traditional in vivo time-lapse microscopy relies on transgenic animals in which a fluorescent protein is expressed in a subset of cells. A recent development of Tg(tie1:H2B-eYFP) quail embryos have made it possible to image endothelial cells (ECs) in vivo using fluorescence microscopy. The transgenic quails highlighted not only ECs in vessel walls, but also ECs in circulation. The role of ECs in circulation during vascular development is unknown. We time-lapsed transgenic embryos during early vascular development and observed that TIE1+ ECs either remained in free circulation or were seen rolling along the endothelium. ECs in circulation previously observed in areas of vascular damage and tumor growth have been identified as circulating endothelial cells (CECs), a sign of vascular damage, or endothelial progenitor cells (EPCs), a sign of neovascularization. We believe that the cells in circulation we observed in the embryonic vasculature are CECs and that the high rate of vessel reorganization that occurs during early embryonic vascular remodeling results in the sloughing off of endothelial cells from the vessel wall.Though transgenics are a powerful tool, they are incredibly difficult, expensive, and time consuming to produce. We have developed a technique to concurrently label both ECs and macrophages and time-lapse them in vivo. We used intra-vascular injection of fluorescently conjugated acetylated low-density-lipoprotein (AcLDL) to label ECs and macrophages. We then injected PKH26-PCL, a phagocyte specific dye to double label macrophages. Leukocytes are known to be present before the onset of blood flow but little is known about their function in vascular development. We found many double-labeled cells circulating, rolling along the endothelium, as well as a subset that extravasate. We quantified the expression of genes that are active in macrophage recruitment during vascular development and saw an upregulation of expression during vascular remodelling. We looked at expression of vcam1 by in situ hybridization and found that regions undergoing remodeling expressed vcam1 in a punctate manner, however regions in which morphologically distinct arteries and veins were present (i.e. regions that had remodeled) had very little vcam1 expression.Traditionnellement, l'imagerie à lapse de temps en biologie s'appuie sur des animaux transgéniques dans lesquels une protéine fluorescente est exprimée par une population spécifique de cellules. Un collaborateur a récemment produit une caille transgénique qui exprime du YFP, pour « Yellow Fluorescent Protein », sous le contrôle d'un promoteur pour les cellules endothéliales (TIE1). Dans cet animal, non seulement est-il possible de visualiser les cellules endothéliales de la paroi des vaisseaux sanguins, mais le gène fluorescent est aussi exprimé dans des cellules endothéliales en circulation. Le rôle des cellules endothéliales en circulation au cours du développement vasculaire est inconnu. Nous avons imagé en temps réel les embryons transgéniques et nous avons observé que les cellules endothéliales TIE1+ roulent et interagissent avec l'endothélium des vaisseaux sanguins mais ne s'intègre jamais. Dans l'adulte, deux sortes de cellules endothéliales circulantes sont connues. La première population consiste de cellules matures, dépouillées après des lésions vasculaires. Celle-ci sont nommé des CECs (« Circulating Endothelial Cells ») et sont signe de pathologies. La deuxième population de cellules endothéliales circulantes représente des cellules progénitrices, qui retienne la capacité d'induire de la néovascularisation. Elle sont nommée EPCs (« Endothelial Progenitor Cells »). Nous croyons que les cellules en circulation dans l'embryon sont des cellules endothéliales circulantes (CECs) et que le remodelage vasculaire embryonnaire représente une insulte sur la paroi vasculaire qui est produit par l'initiation des flux sanguins lors du développement. Bien que les animaux transgéniques soient un outil puissant, ils sont coûteux et long à produire. Nous avons développé une technique pour marquer les cellules endothéliales et les macrophages en même temps pour l'imagerie à lapse de temps. La méthode consiste d'une injection intravasculaire de lipoprotéines acétylée conjuguées à un colorant fluorescent (AcLDL). Les lipoprotéines acétylée sont absorbées par les cellules endothéliales et les macrophages. Nous avons ensuite injecté un deuxième colorant, PKH26-PCL, qui est spécifique pour les phagocytes. La présence de macrophage avant l'apparition du flux sanguin était connue, mais leur fonction dans le développement vasculaire est très peu étudiée. Nous avons trouvé de nombreuses cellules doublement marquées en circulation, qui roulent le long de l'endothélium, ainsi qu'un sous-ensemble qui s'extravaser. Nous avons quantifié l'expression des gènes lier au recrutement de macrophage lors du développement vasculaire et avons vu augmentation. Nous avons examiné l'expression de vcam1 par hybridation in situ. Nous trouvons que les régions qui sont en train de remodeler expriment vcam1 de manière ponctuée, toutefois des régions dans lesquelles les artères et les veines sont visibles (ce qui représentent des régions qui ont déjà remodelé) avait peu d'expression de vcam1.
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Rodríguez, Fajardo Valeria. "Novel methods for plasmonic nanoparticles imaging inside cells using dark-field microscopy." Doctoral thesis, Universitat Politècnica de Catalunya, 2018. http://hdl.handle.net/10803/462174.
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The potential impact of noble metal nanoparticles (NPs) in diverse fields, particularly medicine, is tremendous. However, before NPs can be routinely used in clinical practices, it is fundamental to comprehensively study their interaction with cells. This study is non-trivial because such interaction is an extremely complex process that depends on multiple factors.
In order to use plasmonic NPs as biosensing probes or to investigate their interaction with biological specimens, it is necessary to detect them. The most frequently used methods are electron microscopy (SEM/TEM), two-photon luminescence (TPL) and dark-field (DF) microscopy. While the first one outperforms in terms of resolution, it requires intensive sample preparation and is highly invasive. Although TPL is capable of identifying NPs with good accuracy and contrast, measurements can be affected by high peak powers of pulsed illumination. Moreover, they require specialized equipment, and are limited by insufficient temporal resolution to follow the NP-cell interaction dynamics or track cells in flow. In this context, DF stands out as a great option. However, since the cell’s scattering can be very high, DF alone is not reliable for detecting metal NPs immersed in cells.
In this thesis we present two alternative methods for imaging of plasmonic NPs embedded in cells, both based on DF microscopy. Scattering is very attractive for several reasons: acquisition time is not fundamentally limited; it is harmless for the cells or plasmonic NPs; it does not suffer from blinking or bleaching; and its implementation is simple and does not require specialized elements. The key idea is removing the cell's contribution to the total scattering by using the distinct optical properties of cells and plasmonic NPs, thus overcoming the reliability issue of standard DF.
Polarization difference dark-field (PDDF) microscopy exploits the different responses of cells and gold nanorods (GNRs) to light's polarization orientation. While scattering intensity of GNRs is highly dependent on it, cell's is not. Therefore, by subtracting two images, one for each polarization direction, cells’ scattering is eliminated. We validated the concept using a phantom sample, and proved PDDF's ability to discriminate GNRs-loaded cells from bare ones is higher than DFs. However, its applicability is limited by two factors: it is only useful for asymmetric NPs, and it is not possible to carry out quantitative measurements.
Two-color dark-field (TCDF) microscopy uses the distinct optical properties of cells and plasmonic NPs on illumination wavelength. While NP's scattering strongly changes with wavelength, cell's does not. Hence, the subtraction of two images, one for each color, will cancel out cell's scattering. Using phantom samples we first proved standard DF is not suitable for plasmonic NPs detection in environments with non-negligible scattering, whereas TCDF is capable of doing so. We carried out experiments on mammalian cells that show TCDF performs better than standard DF in terms of both specificity and sensitivity. Finally we demonstrated the potential of TCDF for long-term tracking of NPs in cells and cell screening in mixed populations under both static and flowing conditions.
Thanks to its robustness, fewer limitations and better performance, the use of TCDF is more convenient than PDDFs. We demonstrated TCDF is efficient and versatile: It works for adherent and suspension cells, under static and flowing conditions, and for diverse applications. Importantly, TCDF's performance could be further improved by optimizing the optical setup and using more sophisticated calibration methods. TCDF is presented as a complement to existing methods (TPL and SEM/TEM), since it is more suitable for live-cell studies and performs better in terms of speed, although it is not as sensitive. TCDF could be applied to other resonant NPs and systems, as long as their scattering properties significantly differ.El impacto potencial de las nanopartículas (NPs) de metales nobles en diversos campos, en particular medicina,es inmenso. Sin embargo, antes de que puedan ser usadas rutinariamente en procedimientos clínicos es fundamental estudiar concienzudamente su interacción con las células, estudio que no es trivial porque es un proceso extremadamente complicado que depende de muchos factores. A fin de usar las NPs plasmónicas como sonda para biosensado o investigar su interacci ón con especímenes biológicos es necesario detectarlas. Los métodos más usados son la microscopia de electrones (SEM/TEM), luminiscencia de dos fotones (TPL) y campo oscuro (DF). Aunque la resoluci ón de la primera sobresale, requiere preparaci ón de muestras compleja y es altamente invasiva. Si bien TPL es capaz de identificar NPs con buena precisi ón y contraste, las mediciones pueden ser afectadas por las altas potencias pico de la iluminaci ón pulsada. Más aún, estas requieren equipos especializados y su resolución temporal es insuficiente para seguir la dinámica de la interacción entre las NPs y las células o rastrear células en flujo. En este contexto, DF sobresale como una excelente opci ón, sin embargo, dado que el esparcimiento de la célula misma puede ser bastante alto, no es confiable. En esta tesis presentamos dos métodos basados en microscopia DF para detectar NPs plasmónicas embebidas en células. El esparcimiento es bastante atractivo por varias razones: el tiempo de adquisici ón no está fundamentalmente limitado; no es dañino para las células o NPs; no sufre de parpadeo o fotoblanqueado; y su implementaci ón es simple y no requiere elementos especializados. La idea clave es remover la contribuci ón de la célula al esparcimiento total, y con ello superar el problema de confiabilidad del DF convencional. La microscopia de campo oscuro de diferencia de polarizaci ón (PDDF) explota el hecho que mientras que la intensidad del esparcimiento producido por nanocilindros de oro (GNRs) depende de la orientaci ón de la polarización de la luz, la de las células no lo hace. De esta manera, al substraer dos imágenes, una por cada dirección, el esparcimiento de la célula es eliminado. Validamos el concepto usando una muestra substituta y probamos que la habilidad de PDDF para discriminar células con GNRs de células sin ellos es mayor que la de DF. Sin embargo, dos factores limitan su aplicabilidad: solo es útil para NPs asimétricas y no es posible realizar medidas cuantitativas.La microscopia de campo oscuro de dos colores (TCDF) toma ventaja de que el esparcimiento producido por las NPs cambia drásticamente con la longitud de onda, mientras que el de las células no lo hace. Así, la substracción de dos imágenes, una por cada color, cancelará el esparcimiento de las células. Usando una muestra substituta probamos que el DF convencional no es fiable para detectar NPs plasmónicas en medios con esparcimiento no despreciable, mientras que TCDF sí lo es. Realizamos experimentos en células que demostraron que TCDF se desempeña mejor que DF en términos especificidad y sensibilidad. Mostramos tambi én el potencial de TCDF para el rastreo a largo plazo de NPs en células y la identificación de células en poblaciones mixtas en condiciones estáticas y en flujo. El uso de TCDF es más conveniente que el de PDDF gracias a su robustez, menos limitaciones y mejor desempe ño. TCDF es eficiente y vers átil: funciona para células adherentes y en suspensi ón, diversas aplicaciones, y en condiciones estáticas y de flujo. Además, su desempeño podría ser mejorado optimizando el montaje óptico y usando métodos de calibración más sofisticados. TCDF se presenta como un complemento a técnicas ya existentes (TPL and SEM/TEM), dado que es más adecuado para estudios con células vivas y se desempeña mejor en términos de velocidad, aunque no sea tan sensible. TCDF podría ser aplicado a otros sistemas y otras NPs con resonancia, siempre que sus propiedades de esparcimiento difieran lo suficiente.
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Agustin, Ramses Martinez. "Automated detection and classification of circulating cancer cells via high-throughput microscopy." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2007. http://wwwlib.umi.com/cr/ucsd/fullcit?p3258983.
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Thesis (Ph. D.)--University of California, San Diego, 2007.Title from first page of PDF file (viewed June 13, 2007). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 156-164).
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Byrne, Gerard. "Total internal reflection microscopy studies on colloidal particle endocytosis by living cells." Thesis, University of Nottingham, 2009. http://eprints.nottingham.ac.uk/10979/.
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The purpose of this study was to develop novel optical microscopy techniques in order to investigate colloidal drug particle endocytosis by mammalian cells. A total internal reflection microscope (TIRM) was initially developed for high resolution cellular imaging. TIRM is a non-fluorescent imaging technique based on the principle of ‘scattering’ of the evanescent field created when a light beam undergoes total internal reflection at an interface between two media with different refractive indices, such as glass and air. The key design considerations with respect to development of a TIRM instrument are discussed. The technique is also compared and contrasted to the more commonly known non-fluorescent RICM (Reflection Interference Contrast Microscopy) technique using computer simulations. Time-lapse video TIRM is applied to imaging the interaction between A549 and 3T3 cells, and a polylysine coated substrate. Real-time label-free visualisation of 0.5 and 1 m polystyrene particle endocytosis by living cells is then demonstrated. Modifications to the TIRM system to include a dual-colour fluorescent TIRF (Total Internal Reflection Fluorescence) microscope are described in detail. Results are shown which demonstrate the ability of a combined TIRM/TIRF instrument to selectively image the basal cell membrane both label-free and fluorescently. 3T3 fibroblast cells were genetically modified using standard molecular biology protocols to express the fluorescent fusion protein EGFP-Clathrin LCa (enhanced green fluorescent protein clathrin light chain a). Finally, colloidal particle endocytosis by the genetically modified cell was imaged using the TIRM/TIRF microscope. Direct visualisation of the internalisation of 500 nm particles via clathrin coated pits in 3T3 cells was shown for the first time.
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Lindqvist, Eliza. "An investigation of performing the proteinretention expansion microscopy protocol on neuronal cells." Thesis, KTH, Tillämpad fysik, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-233225.
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Tsikritsis, Dimitrios. "Vibrational spectroscopy and microscopy in colorectal cancer." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/33049.
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This project set out to examine the possibility that by acquiring Raman spectra and performing multi-photon imaging we can get better diagnosis and understanding of the biochemistry of an individual cancerous tumour and distinguish it from the healthy tissue. Within the frame of this study, colorectal primary and secondary cancer cells are examined with Raman spectroscopy in order to (i) study and distinguish them according to their chemical composition by applying multivariate methods and (ii) determine whether Raman spectroscopy can identify the cells which are the link between primary and secondary colorectal cancer cells, the so-called Cancer Stem Cells. The second part of this thesis is based on tissue studies. Human colorectal tissue sections are examined in a label-free manner with the use of multi-photon imaging modes (i) Two photon excitation fluorescence, (ii) stimulated Raman scattering and (iii) second harmonic generation, in order to determine whether these can provide fast and accurate diagnosis of colorectal cancer. These techniques were able to distinguish between healthy and cancerous tissue regions, based on the chemically-specific images of the tissue microenvironment and architecture. The hypothesis of Cancer stem cell is examined with the use of Raman spectroscopy shown that the CSCs have some small differences according to their tissue origin.
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Stayton, Isaac Alexander. "Investigation of the interactions between selected nanoparticles and human lung carcinoma cells at the single cell and single particle level." Diss., Rolla, Mo. : Missouri University of Science and Technology, 2009. http://scholarsmine.mst.edu/thesis/pdf/Stayton_09007dcc8065344d.pdf.
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Thesis (Ph. D.)--Missouri University of Science and Technology, 2009.Vita. The entire thesis text is included in file. Title from title screen of thesis/dissertation PDF file (viewed April 29, 2009) Includes bibliographical references.
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Msallam, Rasha. "Intravital imaging and immuno-regulatory functions of mast cells in cutaneous immune responses." Thesis, Sorbonne Paris Cité, 2015. http://www.theses.fr/2015PA05T019.
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La peau est un « avant poste » fascinant du système immunitaire. Elle forme une barrière entre l'environnement extérieur et l’organisme. Elle est aussi le point d'entrée pour les agents pathogènes, contre lesquels le système immunitaire organise des réponses adaptatives. Les acteurs de l'immunité innée de la peau contrôlent l'invasion des pathogènes et perçoivent également des changements environnementaux physiques et chimiques directs. Plusieurs composants du système immunitaire, tels que des cellules dendritiques (DCs), les macrophages (MΦ) et les mastocytes (MCs), participent à l'éradication des pathogènes et à l'initiation des réponses mémoires adaptatives. Ce qui permet une mobilisation rapide des cellules T effectrices ainsi que la sécrétion des anticorps par les cellules B à la suite d’une seconde exposition aux agents pathogènes. Les MCs qui sont des cellules résidentes du derme, jouent un rôle déterminant dans la libération de signaux d’alertes et sont classiquement considérés comme des cellules effectrices de la réaction allergique cutanée liée à l'IgE. Plusieurs observations récentes indiquent que les MCs seraient aussi impliqués dans les processus immunorégulateurs lors de l'initiation des réponses immunitaires adaptatives, dans le maintien de la tolérance périphérique aux composants de la peau et dans la régénération de la peau au cours des processus de cicatrisation. Cependant, les interactions entre les MCs et d'autres cellules immunitaires innées et adaptatives recrutées dans des conditions inflammatoires cutanées n'ont pas été élucidées en détail. Dans ce travail, nous décrivons l'utilisation d'une nouvelle souris possédant des MCs fluorescents (RMB), dans laquelle nous avons marqué les MCs FcεRI+ avec un marqueur fluorescent rouge tomato (TdT) et avec un système d'ablation conditionnelle basé sur l'expression concurrente du récepteur de la toxine diphtérique (DTR). Avec ces souris RMB, nous avons visualisé la dynamique des MCs et nous avons suivi les interactions entre les MCs et les lymphocytes T régulateurs (Tregs) après l'activation des MCs par l'IgE, dans une réaction inflammatoire typique de l'anaphylaxie cutanée passive (PCA). Dans un second volet d’étude, nous avons évalué le rôle des MCs lors d'un modèle expérimental de la greffe de peau de l'oreille, afin de révéler leur influence dans la cinétique de rejet ou prise de greffe du transplant. Nous avons constaté que 1) l'activation et la dégranulation des MCs induites par le pontage du récepteur FcεRI via des IgE couplées à un antigène multivalent sont les seules responsables de la réaction de PCA, et induisent le recrutement de Tregs ayant une grande motilité sur le site de l'inflammation. Nous avons constaté dans ces conditions, que les MCs restent immobiles, et que les Tregs établissent des contacts dynamiques avec les MCs dans le derme. 2) En outre, nous avons mis en place un modèle pour identifier les paramètres moléculaires de l'interaction MC-Treg et avons constaté que le complexe de l'antigène avec l'IgE peut être présenté aux Tregs en association avec les molécules du complexe majeur d'histocompatibilité de classe II, permettant la formation des contacts stables MC-Treg. 3) En utilisant un modèle de transplantation de la peau in vivo, nous avons montré que l'ablation conditionnelle des MCs conduit à une accélération du rejet du greffon dans le cas d'une transplantation en présence d’une disparité d’antigènes d’histocompatibilité mineurs depuis une souris mâle sur une souris femelle. Nous avons également constaté un impact inattendu de l'ablation des MCs dans la greffe de peau en l’absence de disparité antigénique d'une souris femelle sur une souris femelle, conduisant à un rejet rapide. Les MCs semblent donc être essentiels pour la cicatrisation et la régénération tissulaire après greffe. (...)The skin is a fascinating outpost of the immune system. It performs a barrier function between the outside environment and the inner body and is also a port of entry for pathogens against which the immune system mounts adapted responses. The skin innate immune defenses control pathogen invasion and perceive also direct physical and chemical environmental changes. Several component of the immune system such as dendritic cells (DC), macrophages (MΦ) and mast cells (MC) participate in initial pathogen clearance and in initiating adaptive memory responses, allowing rapid mobilization of effector T cells and secretion of B cellderived antibodies after secondary pathogen challenge. MCs residing in the dermis exert a determinant alert function through the liberation of various factors and are classically considered as effector cells in the IgE-mediated cutaneous allergic reaction. As emerging now, MC are also involved in immunoregulatory processes during the initiation of adaptive immune responses, the maintenance of peripheral tolerance to skin components and skin regeneration during wound healing. Yet, the crosstalks between MCs and other innate and adaptive immune cells recruited during cutaneous inflammatory conditions have not been elucidated in detail. Here, we report the use of a novel Mast cell fluorescent reporter mouse (RMB), in which we tagged FcεRI+ MCs, with red fluorescence marker tomato (Tdt) and with a conditional ablation system based on concurrent diphtheria toxin receptor (DTR) expression. Using these RMB mice, we visualized MC dynamics and monitored MC interactions with regulatory T lymphocytes (Tregs) after IgE-mediated activation of MCs, in a typical passive cutaneous anaphylaxis (PCA) inflammatory reaction. Using another setting, we further assessed the role of MC during experimental ear skin grafting to reveal their potential influence in skin grafting and rejection. We found that 1) the activation and degranulation of MCs induced by FcεRI crosslinking by multivalent IgE is solely responsible for the PCA reaction and induces the recruitment of highly motile regulatory T cells (Tregs) to the site of inflammation. In these conditions, we found that MC remain sessile and Tregs establish dynamic contacts with MC in the dermis. 2) Further we set up a model system to reveal the molecular requirement for MC-Treg interaction and found that antigen complexed with IgE were able to be presented to Treg in association with major histocompatibility complex class II molecules allowing the formation of stable MC-Treg contacts. 3) Using in vivo skin transplantation model, we showed that conditional ablation of MCs leads to an acceleration of skin transplant rejection in sex-mismatched model (male skin transplant to female). We also found an unexpected impact of MC conditional ablation in sex-matched skin graft (female skin transplant to female) leading to rapid rejection, implying that MCs are essential for the wound healing reaction and the regeneration of tissue continuity after grafting. The aforementioned results point out to an important immunoregulatory role of MC beyond their classically described activator functions in inflamed tissues. The fact that MC constantly interact with Treg during inflammatory processes suggest that MCs could participate in skin homeostasis by exerting tolerogenic functions. These functions remain to be elucidated at the molecular level as presented in the discussion
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Orth, Antony G. "Imaging Pressure, Cells and Light Fields." Thesis, Harvard University, 2014. http://dissertations.umi.com/gsas.harvard:11349.
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Imaging systems often make use of macroscopic lenses to manipulate light. Modern microfabrication techniques, however, have opened up a pathway to the development of novel arrayed imaging systems. In such systems, centimeter-scale areas can contain thousands to millions of micro-scale optical elements, presenting exciting opportunities for new imaging applications. We show two such applications in this thesis: pressure sensing in microfluidics and high throughput fluorescence microscopy for high content screening. Conversely, we show that arrayed elements are not always needed for three dimensional light field imaging.Engineering and Applied Sciences
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Van, Der Hofstadt Serrano Marc. "Hygroscopic properties of single bacterial cells and endospores studied by Electrostatic Force Microscopy." Doctoral thesis, Universitat de Barcelona, 2016. http://hdl.handle.net/10803/400567.
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The large abundance of bacterial growth niches provide a rich diversity of bacterial traits. These are usually characterized using traditional microbiology research tools, and newer characterization techniques (which focus on addressing physical and physicochemical properties). Most of these techniques are performed at the level of colonies, where millions of cells are analysed and hinder the heterogeneity of single cells. The Atomic Force Microscope (AFM) is emerging as a promising nanotechnology tool for single bacterial cell studies (Nanomicrobiology), since it is capable of characterizing the structure and simultaneously obtaining other physical properties of interest under physiological conditions.
To sustain harsh conditions, some bacterial species have the ability to produce endospores. This environmental resistance has been mainly attributed to the way endospores control its water content. A heterogeneous distribution of the water content plays a key role in the resistance. Despite the large existing literature in hydration properties of bacterial endospores, the hydration capabilities of endospores still present some open questions. In this work of thesis the hygroscopic properties of single bacterial cells and endospores are studied under different environmental conditions. To achieve these results, we have made use of the Electrostatic Force Microscopy (EFM), an adaptation of the AFM which can report changes in the dielectric properties of individual bacterial samples.
Firstly of all, biocompatible gelatine was used to weakly attach bacterial cells, and the dynamic jumping mode was used to drastically reduce the shear forces provoked on bacterial samples during conventional AFM imaging. This methodology allowed us to observe in situ bacterial cell division at the single cell and nanoscale resolution.
Due to the large morphology of bacterial samples, lift mode EFM had to be used. This electrical imaging mode hinders the intrinsic contribution of the sample under study due to topographical crosstalk contribution. A method was proposed to remove topographical crosstalk contribution, which revealed electrical homogeneity of inorganic calibration samples and of dried single bacterial cells. The use of a subsurface sample revealed the capabilities of the EFM as a tool for subsurface characterization. Such ability revealed the potential of the EFM to detect water distribution within the bacterial cell samples under study in this work of thesis.
The electrical characterization of bacterial vegetative cells and bacterial endospores under a range of different relative humidity allowed us to study the difference in hygroscopic properties between the two samples. At low relative humidity, 40% RH, the bacterial endospores hardly hydrate in comparison to the bacterial vegetative cells. At high relative humidity, 80% RH, the bacterial vegetative cells drastically hydrate in comparison to the bacterial endospores. In the latter case, it has been demonstrated that the external layers accommodate most of the moisture absorbed, leaving the core at low hydration levels. In the case of the vegetative cells, the cell wall is not able to accommodate such high levels of moisture, forcing the cytoplasmic region to become highly hydrated. This discrepancy in the hydration behaviour seems key for the persistence of the core region as the driest region of the bacterial endospores in atmospheric conditions.
Finally, electrical measurements performed under liquid conditions revealed the high hydration state of the living bacterial cells in contraposition to bacterial endospores. This lower hydration
of the endospores under liquid conditions could be attributable to the difference in structure. All together, these results obtained in this work of thesis have shown the lower hydration properties of single bacterial endospores in contraposition to its vegetative cell in all environmental conditions, from dry conditions up to liquid environments.El microscopi de forces atòmiques (AFM) s'està convertint en una eina prometedora per a la caracterització de bacteris individuals, ja que presenten un ampli ventall de característiques. (Nanomicrobiologia). En particular, alguns bacteris són capaç de produir endòspores que resisteixen condicions ambientals extremes. S'ha observat que aquesta resistència està lligada al contingut de l'aigua, i en particular en la capacitat de mantenir el nucli sec. L'objectiu d'aquest treball de tesis és l'estudi de les seves propietats higroscòpiques en diferents condicions ambientals. En primer lloc es van obtenir imatges de bacteris individuals dividint-se amb resolució nanomètrica. L'ús de gelatina i un mètode d'imatge poc agressiu (dynamic jumping mode) va permetre imitar condicions naturals. A causa de la gran morfologia de les mostres bacterianes, es va utilitzar un mètode d'imatge elèctrica que emmascarava la contribució intrínseca de la mostra. La quantificació del sistema va permetre revelar homogeneïtat elèctrica de cèl·lules bacterianes individuals seques. L'ús d'una mostra subsuperficial va revelar el potencial del EFM per detectar la distribució de l'aigua dins de les cèl·lules bacterianes. La caracterització elèctrica de les cèl·lules vegetatives bacterianes i les endòspores bacterianes va revelar una major hidratació de les cèl·lules vegetatives bacterianes en contraposició a les endòspores bacterianes. A elevada humitat relativa, les cèl·lules vegetatives s'hidraten dràsticament i causen la hidratació de la regió citoplasmàtica, mentre que les endòspores tenen la capacitat de deixar el nucli en nivells baixos d'hidratació. Aquesta discrepància en el comportament d'hidratació sembla clau per a la persistència de la latència de les endòspores en condicions atmosfèriques. Finalment, mesures elèctriques realitzades en líquid van revelar un estat d'alta hidratació de les cèl·lules bacterianes vives en contraposició a les endòspores bacterianes. Aquesta hidratació inferior de les endòspores en condicions de líquid podria ser atribuïble a la diferència en l'estructura. Tot junt, aquests resultats obtinguts en aquest treball de tesi han demostrat una menor propietat d'hidratació en les endòspores bacterianes en contraposició a la seva cèl·lula vegetativa en totes les condicions ambients, des de condicions seques fins a líquides.
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Coffey, David C. "Characterizing the local optoelectronic performance of organic solar cells with scanning-probe microscopy /." Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/9688.
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Emilson, Axel. "Analysis of human epidermal Langerhans' cells and allergens with confocal laser scanning microscopy /." Stockholm, 1997. http://diss.kib.ki.se/1997/91-628-2734-0.
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Randall, Catherine Marie. "Investigating the Mechanical and Structural Properties of Human Cells by Atomic Force Microscopy." Thesis, Liverpool John Moores University, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.515351.
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Basu, Srinjan. "Study of Chromatin Structure Using Stimulated Raman Scattering Microscopy in Living Mammalian Cells." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10416.
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DNA is packaged into the nucleus of a mammalian cell as a nucleoprotein complex called chromatin. Changes in chromatin structure occur during processes that are critical to an understanding of mammalian cell biology such as cell division. Existing fixed-cell techniques have provided insight into chromatin organization in the mammalian nucleus. In addition, fluorescence microscopy techniques have allowed us to study changes in chromatin structure in living cells. However, most of these fluorescence techniques cannot be used for tissue imaging or long-term imaging due to photobleaching. In this thesis, we demonstrate that a label-free technique called Stimulated Raman Scattering (SRS) microscopy can be used to solve these problems and study chromatin structure in living mammalian cells both in culture and in tissue. SRS is a vibrational microscopy technique that takes advantage of intrinsic contrast arising from specific chemical bonds in a molecule. Nucleic acids have specifc phosphate and CH vibrations that can be used to determine their cellular distributions. Imaging at specific phosphate peaks using fingerprint SRS microscopy allows the detection of polytene chromosomes in Drosophila salivary gland cells and condensed chromatin in metaphase mammalian cells. In addition, we develop a technique called multicolor SRS microscopy, in which we image at several wavelengths across the CH vibrational band, and then use linear combination to simultaneously determine the nucleic acid, lipid and protein distributions in living mammalian cells. This technique achieves greater contrast than imaging at the phosphate vibrational peak due to the stronger SRS signal in the high wavenumber CH band and so allows us to determine chromatin structure in interphase mammalian cells. This technique also allows long-term imaging of living mammalian cells and the imaging of tissue such as mouse skin. The technique is used to monitor mammalian cell division in culture and paves the way for similar studies in living tissue. This technique will provide insight into cell division, differentiation and apoptosis during development and in disease models such as cancer.
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Xu, Yang. "Multimodal Spectral Microscopy and Imaging Mass Spectrometry of Biomolecules in Cells and Tissues." University of Toledo / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1333769758.
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Haase, Kristina M. "Mechanics and Mechanotransduction of Adherent Cells: A Compendium of Atomic Force Microscopy Studies." Thesis, Université d'Ottawa / University of Ottawa, 2014. http://hdl.handle.net/10393/31850.
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Mechanical cues have been recognized to be critically important in the regulation of cells. A myriad of cellular processes including differentiation, proliferation, and gene expression are all affected by physical forces from the extra- and intra-cellular microenvironments. Despite recent advances in nano-technologies, many questions still surround how cells sense and respond to forces. Through a series of studies, we demonstrate how both the structure and inherent mechanical properties of the cell affect their response to mechanical cues. We first develop a methodology to mechanically manipulate cells while simultaneously characterizing their deformations. Using combined atomic force and confocal microscopy techniques and through systematic examination we demonstrate the role of the cytoskeleton and nucleus in the deformability and shape change of epithelial cells. Mechanical properties have been used in recent years to identify diseased states, including cancer. With this in mind, we used HeLa cells as a model and characterized significant deformability of their plasma membrane and underlying cortex. Importantly, we demonstrate and characterize their ability to recover from large shape changes, which we also observed in other epithelial cells. Shape recovery is shown to be rapid and reliant upon the actin cytoskeleton and intracellular fluid flow. Although the nucleus does not contribute significantly to the deformation and recovery of HeLa cells, the importance of nuclear mechanics cannot be forgone. In vitro studies have shown that mechanical forces transmitted through the cell’s cytoskeleton critically affect nuclear mechanics and gene transcription processes. Many others have used simple models and isolated nuclei in an attempt to characterize nuclear properties. Thus, in a subsequent study, we examine the nucleus within intact cells. Nuclear shape change, in response to force, is shown to be complex and cannot be well-characterized by isotropic mechanical properties. Characterization of the mechanics of the cell, as demonstrated through our findings, is crucial in the field of biological physics. The aforementioned studies, written as scientific articles, are presented in the body of this thesis (Chapters 2-5). A review article that focuses on mechanotransduction and relevant examples using AFM as a tool for its examination acts as an introductory chapter.
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Scherer, K. M. "Two-Photon Microscopy of E-Combretastatin uptake and activation in live mammalian cells." Thesis, University of Salford, 2012. http://usir.salford.ac.uk/38101/.
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Cancer is one of the most common causes of death in the Western world. Hence, the development of novel effective treatment modalities to combat this disease is important. Combretastatins are powerful anticancer drugs that are being evaluated in phase I/II clinical trials. Combretastatins act by binding to p-tubulin, preventing microtubule assembly and inhibiting angiogenesis in developing tumours. Most widely studied is the natural product c/5(Z)-combretastatin A4 that was originally isolated from the African bush willow Combretum caffrum by Pettit and co-workers. Chemically Combretastatins are substituted stilbenes, exhibiting major activity in the cis-form, whereas the corresponding fr-am'-isomers are ~ 2 - 3 orders of magnitude less cytotoxic. Combretastatins undergo reversible photoisomerization upon irradiation with UV/visible light. Here a novel form of two-photon phototherapy is proposed, in which the "inactive" fnms(£)-combretastatins are used as anticancer pro-drugs that may be activated (converted to the cw-isomer) by localized irradiation with light at the tumour site. The use of near-infrared (NIR) light to trigger a nonlinear two-photon excited photoactivation process provides deeper light penetration into tissues and minimizes cellular autofluorescence compared with UV/visible light. The work described in this thesis is highly multidisciplinary. A major task was the synthesis of a range of combretastatin derivatives as target compounds for the novel form of two-photon excited anticancer phototherapy proposed herein. The cytotoxicity of the drug candidates was assessed on live mammalian cell lines. Photochemical properties such as fluorescence quantum yields, fluorescence lifetimes, photoisomerization quantum yields and two-photon absorption characteristics of the combretastatin derivatives were investigated spectroscopically. The fact that the trans-isomers fluoresce in most cases, whereas ds-isomers are virtually non-fluorescent has allowed multiphoton fluorescence lifetime imaging (FLIM) of the real-time uptake of the inactive pro-drugs into live mammalian cells, as well as monitoring their intracellular distribution and concentration. Two-photon excitation of ^-Combretastatins on monolayers of live cells led to effective conversion of the inactive pro-drugs to the highly cytotoxic Z-derivatives leading to apoptosis in the irradiated areas within 24 h. In conclusion, the two-photon excited activation of £-combretastatins on live cells was demonstrated, making £-combretastatins promising pro-drug candidates for two-photon excited anticancer phototherapy.
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Tomalik, Edyta. "Image-based Microscale Particle Velocimetry in Live Cell Microscopy." Thesis, Blekinge Tekniska Högskola, Institutionen för programvaruteknik, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:bth-2564.
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Background: Nowadays, one of the medical problem is rolling cell adhesion. Rolling cell adhesion is a complex process that requires the analysis of the challenging environment such as body fluid and is the process responsible for recruiting the cell to specific organs. In order to explore the rolling cell adhesion, mathematical model is proposed. Different image processing methods are created, such as optical flow - Lucas Kanade algorithm, and other type of methods related to mechanical fluid, namely PIV (Particle Image Velocimetry). Aim: The aim of this master thesis is the identification of challenges while using PIV in live cell images and propose the algorithm, which may analyze the rolling cell adhesion problem. Methods: In order to understand properly the rolling cell adhesion problem from biological site, literature review combined with the expert consultation is performed. According to gather information, mathematical model is proposed. Particle Image Velocimetry is explained according to literature review, where at the beginning the expert recommends some books as a primary research. As a result of this research, PIV challenges are identified and generally PIV idea is explained. Then two experiments are performed. The first experiment evaluates detection algorithms and the second one, analyses track algorithm vs. PIV. In order to evaluate the mentioned algorithms, some evaluation method are selected and some criteria are defined. Unfortunately the found methods are not perfect, therefore a new method related to performance evaluation using time series is proposed. Thesis result: The result of this thesis is a proposition of the algorithm, which can be used in the rolling cell adhesion. The algorithm is formed according to the detailed exploration of the rolling cell adhesion and analysis of the selected algorithms related to the image analysis during the theoretical research and experiments.
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Colom, diego Adai. "From eye lens cells to lens membrane proteins : Development and application of a hybrid high-speed atomic force microscopy/optical microscopy setup." Thesis, Aix-Marseille, 2013. http://www.theses.fr/2013AIXM4033.
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Je utilise le AFM et le HS-AFM pour étudier les caractéristiques mécaniques du cellule du cristallin et aussi des protéines de membrane de la cellule, AQP0 et Connexon. L’énergie d'interaction de la AQP0 est -2.7 kBT, très nécessaire pour former les microdomaines de jonctions (junctional microdomain). Aussi c' est la première fois qu il est possible de voir des protéines individuel et son mouvement en cellules vivants. La formation de microdomaines est important pour la transparence du cristallin, et le AQP1 ne le peux faireI used the AFM and HS-AFM for characterise the eye lens and the eye lens membrane protein, AQP0 and connexon.A QP0-AQP0 interaction energy is -2.7kBT, it is important for the formation of junctional microdomains, which keep the distance between the cells lens and lens transparency. this is the first report which is present time the visualization of unlabelled membrane proteins on living cells under physiological conditions. AQP1 can not maintain the lens transparency because it does not form junctional microdomains
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Curry, Nathan. "Development and application of correlative STED and AFM to investigate neuronal cells." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/274579.
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Over the past three decades in cellular neuroscience there has been a shift towards the view of the 'tripartite synapse', where, astrocytes -- as well as the pre-synapse and post-synapse -- are involved in synaptic signalling. The migration of astrocytes to form branched networks in the brain is, therefore, of great interest in understanding brain development and neuronal function. Migration is a complex interplay between cytoskeletal reorganisation and cell mechanical stiffness. In order to improve understanding of this process, correlative measurements of cytoskeletal organisation and mechanical stiffness are required. To investigate astrocyte migration a technique combining atomic force microscopy (AFM) with stimulated emission depletion (STED) microscopy was developed. First a custom STED microscope was developed. To facilitate the design of this system the theoretical performance of a range of STED techniques (cw-STED, time-gated STED, pulsed STED and RESOLFT) were compared, identifying that pulsed STED theoretically has the highest photon efficiency. A pulsed STED microscope, which uses adaptive optics, was then designed, developed and characterised. The microscope was found to achieve resolutions below 50 nm. The STED microscope was combined with a commercial AFM to study live cells. Using the recently developed SiR-actin and SiR-tubulin dyes and AFM probes optimised for live cell mechanical property studies, images of the actin and tubulin cytoskeleton were correlated with AFM topography and mechanical stiffness measurements. It was found that, in astrocytes, actin contributes significantly both to astrocyte stiffness and topography. Investigations of migrating cells showed differences in actin organisation and mechanical stiffness between the basis and leading edge of migration. A further study was performed, investigating the effects of the gap-junction protein connexin30, which is expressed during the early stages of brain development, on migration. This protein was found to inhibit the actin reorganisation and mechanical stiffness changes observed in basal conditions. Overall the combination of mechanosensitive AFM measurements with advanced microscopy, such as super-resolution, on live cells is a promising approach which will enable a range of investigations, for instance when studying cell structural remodeling during brain development or tumorigenesis.