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- Creators: Ros, Robert
Description
In this project, we introduce a type of microscopy which produces correlated topography and fluorescence lifetime images with nanometer resolution. This technique combines atomic force microscopy (AFM) and time resolved confocal fluorescence microscopy to conduct biological and materials research. This method is used to investigate nanophotonic effects on single fluorophores, including quantum dots and fluorescent molecules. For single fluorescent molecules, we investigate the effects of quenching of fluorescence with the probe of an atomic force microscope which is combined and synchronized with a confocal fluorescence lifetime microscope. For quantum dots, we investigate the correlation between the topographic and fluorescence data. With this method of combining an atomic force microscope with a confocal microscope, it is anticipated that there will be applications in nanomaterial characterization and life sciences; such as the determination of the structure of small molecular systems on surfaces, molecular interactions, as well as the structure and properties of fluorescent nanomaterials.
ContributorsWard, Alex Mark (Author) / Ros, Robert (Thesis director) / Shumway, John (Committee member) / Schulz, Olaf (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Department of Physics (Contributor)
Created2013-05
Description
Cell adhesion is an important aspect of many biological processes. The atomic force microscope (AFM) has made it possible to quantify the forces involved in cellular adhesion using a technique called single cell force spectroscopy (SCFS). AFM based SCFS offers versatile control over experimental conditions for probing directly the interaction between specific cell types and specific proteins, surfaces, or other cells. Transmembrane integrins are the primary proteins involved in cellular adhesion to the extra cellular matix (ECM). One of the chief integrins involved in the adhesion of leukocyte cells is αMβ2 (Mac-1). The experiments in this dissertation quantify the adhesion of Mac-1 expressing human embryonic kidney (HEK Mac-1), platelets, and neutrophils cells on substrates with different concentrations of fibrinogen and on fibrin gels and multi-layered fibrinogen coated fibrin gels. It was shown that multi-layered fibrinogen reduces the adhesion force of these cells considerably. A novel method was developed as part of this research combining total internal reflection microscopy (TIRFM) with SCFS allowing for optical microscopy of HEK Mac-1 cells interacting with bovine serum albumin (BSA) coated glass after interacting with multi-layered fibrinogen. HEK Mac-1 cells are able to remove fibrinogen molecules from the multi-layered fibrinogen matrix. An analysis methodology for quantifying the kinetic parameters of integrin-ligand interactions from SCFS experiments is proposed, and the kinetic parameters of the Mac-1 fibrinogen bond are quantified. Additional SCFS experiments quantify the adhesion of macrophages and HEK Mac-1 cells on functionalized glass surfaces and normal glass surfaces. Both cell types show highest adhesion on a novel functionalized glass surface that was prepared to induce macrophage fusion. These experiments demonstrate the versatility of AFM based SCFS, and how it can be applied to address many questions in cellular biology offering quantitative insights.
ContributorsChristenson, Wayne B (Author) / Ros, Robert (Thesis advisor) / Beckstein, Oliver (Committee member) / Lindsay, Stuart (Committee member) / Ugarova, Tatiana (Committee member) / Arizona State University (Publisher)
Created2016