Matching Items (69)

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Combined AFM and Fluorescence Measurements for the Investigation of Nanophotonic Effects on Single Fluorophores

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

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.

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  • 2013-05

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Correlating Confocal Microscopy and Atomic Force Indentation Reveals Metastatic Cancer Cells Stiffen During Invasion Into Collagen I Matrices

Description

Mechanical interactions between cells and their microenvironment dictate cell phenotype and behavior, calling for cell mechanics measurements in three-dimensional (3D) extracellular matrices (ECM). Here we describe a novel technique for

Mechanical interactions between cells and their microenvironment dictate cell phenotype and behavior, calling for cell mechanics measurements in three-dimensional (3D) extracellular matrices (ECM). Here we describe a novel technique for quantitative mechanical characterization of soft, heterogeneous samples in 3D. The technique is based on the integration of atomic force microscopy (AFM) based deep indentation, confocal fluorescence microscopy, finite element (FE) simulations and analytical modeling. With this method, the force response of a cell embedded in 3D ECM can be decoupled from that of its surroundings, enabling quantitative determination of the elastic properties of both the cell and the matrix. We applied the technique to the quantification of the elastic properties of metastatic breast adenocarcinoma cells invading into collagen hydrogels. We found that actively invading and fully embedded cells are significantly stiffer than cells remaining on top of the collagen, a clear example of phenotypical change in response to the 3D environment. Treatment with Rho-associated protein kinase (ROCK) inhibitor significantly reduces this stiffening, indicating that actomyosin contractility plays a major role in the initial steps of metastatic invasion.

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  • 2016-01-27

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Computational Study of Conformation Dynamics and Allostery in WW Protein Domains

Description

Proteins continually and naturally incur evolutionary selection through mutagenesis that optimizes their fitness, which is primarily determined by their function. It is known that allosteric regulation alters a protein's conformational

Proteins continually and naturally incur evolutionary selection through mutagenesis that optimizes their fitness, which is primarily determined by their function. It is known that allosteric regulation alters a protein's conformational dynamics leading to functional changes. We have computationally introduced a mutation at a predicted regulatory site of a short, 46 residue-long, protein interaction module composed of a WW domain and corresponding polyproline ligand (PDB id: 1k9r). The dynamic flexibility index (DFI) was computed for the binding site of the wild type and mutant WW domains to quantify the mutations effect on the rigidity of the binding pocket. DFI is used as a metric to quantify the resilience of a given position to perturbation along the chain. Using steered molecular dynamics (SMD), we also measure the effect of the point mutation on allosteric regulation by approximating the binding free energy of the system calculated using Jarzynski's Equality. Calculation of the DFI shows that the overall flexibility of the protein complex increases as a result of the distal point mutation. Total change in DFI percentile of the binding site showed a 0.067 increase suggesting an allosteric, loss of function mutation. Furthermore, we see that the change in the binding free energy is greater for that of the mutated complex supporting the idea that an increase in flexibility is correlated to a decrease in proteinlig and binding affinity. We show that sequence mutation of an allosteric site affects the mechanical stability and functionality of the binding pocket.

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  • 2018-05

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Atomic Force Microscopy Imaging of Chromatin in Cancerous and Non-Cancerous Esophageal Cells

Description

Atomic force microscopy (AFM) was used to study structural differences in the chromatin of cancerous (CP-D) and non-cancerous (EPC2) cell lines. Chromatin samples were extracted using a salt fractionation

Atomic force microscopy (AFM) was used to study structural differences in the chromatin of cancerous (CP-D) and non-cancerous (EPC2) cell lines. Chromatin samples were extracted using a salt fractionation protocol and subject to Mnase digestion for 2, 4, 8, and 16 minutes. Samples were then immobilized on APTES-functionalized mica sheets. Images were produced using the tapping mode capabilities of the AFM and structural differences between cell lines were quantified using image processing software. Vast differences in chromatin structure were observed between cancerous and non-cancerous cell lines and it was discovered that CP-D chromatin is present as scattered nucleosomes and nucleosome aggregates while EPC2 chromatin is present in intricate arrays. It was also observed that in both the CP-D and EPC2 cell lines, nucleosomes were more isolated and less apparent at longer Mnase digestion times. These findings lead to the conclusion that as the DNA becomes sufficiently digested, chromatin and nucleosomal arrays begin to deteriorate and lose their complex and elaborate structure.

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  • 2019-05

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Collective behavior of swimming bimetallic motors in chemical concentration gradients

Description

Locomotion of microorganisms is commonly observed in nature. Although microorganism locomotion is commonly attributed to mechanical deformation of solid appendages, in 1956 Nobel Laureate Peter Mitchell proposed that an asymmetric

Locomotion of microorganisms is commonly observed in nature. Although microorganism locomotion is commonly attributed to mechanical deformation of solid appendages, in 1956 Nobel Laureate Peter Mitchell proposed that an asymmetric ion flux on a bacterium's surface could generate electric fields that drive locomotion via self-electrophoresis. Recent advances in nanofabrication have enabled the engineering of synthetic analogues, bimetallic colloidal particles, that swim due to asymmetric ion flux originally proposed by Mitchell. Bimetallic colloidal particles swim through aqueous solutions by converting chemical fuel to fluid motion through asymmetric electrochemical reactions. This dissertation presents novel bimetallic motor fabrication strategies, motor functionality, and a study of the motor collective behavior in chemical concentration gradients. Brownian dynamics simulations and experiments show that the motors exhibit chemokinesis, a motile response to chemical gradients that results in net migration and concentration of particles. Chemokinesis is typically observed in living organisms and distinct from chemotaxis in that there is no particle directional sensing. The synthetic motor chemokinesis observed in this work is due to variation in the motor's velocity and effective diffusivity as a function of the fuel and salt concentration. Static concentration fields are generated in microfluidic devices fabricated with porous walls. The development of nanoscale particles that swim autonomously and collectively in chemical concentration gradients can be leveraged for a wide range of applications such as directed drug delivery, self-healing materials, and environmental remediation.

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Date Created
  • 2011

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Recognition tunneling: approaches towards next generation DNA sequencing

Description

This thesis describes several approaches to next generation DNA sequencing via tunneling current method based on a Scanning Tunneling Microscope system. In chapters 5 and 6, preliminary results have shown

This thesis describes several approaches to next generation DNA sequencing via tunneling current method based on a Scanning Tunneling Microscope system. In chapters 5 and 6, preliminary results have shown that DNA bases could be identified by their characteristic tunneling signals. Measurements taken in aqueous buffered solution showed that single base resolution could be achieved with economic setups. In chapter 7, it is illustrated that some ongoing measurements are indicating the sequence readout by making linear scan on a piece of short DNA oligomer. However, to overcome the difficulties of controlling DNA especially ssDNA movement, it is much better to have the tunneling measurement incorporated onto a robust nanopore device to realize sequential reading of the DNA sequence while it is being translocated.

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Date Created
  • 2011

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Ferroelectric lithium niobate surfaces for depositions of metallic nanostructure and ZnO semiconducting thin film

Description

A distinct characteristic of ferroelectric materials is the existence of a reversible spontaneous polarization with the application of an electric field. The relevant properties ferroelectric lithium niobate surfaces include a

A distinct characteristic of ferroelectric materials is the existence of a reversible spontaneous polarization with the application of an electric field. The relevant properties ferroelectric lithium niobate surfaces include a low density of defects and external screening of the bound polarization charge. These properties result in unique surface electric field distribution with a strong electric field in the vicinity of domain boundaries, while away from the boundaries, the field decreases rapidly. In this work, ferroelectric lithium niobate (LN) is used as a template to direct the assembly of metallic nanostructures via photo-induced reduction and a substrate for deposition of ZnO semiconducting thin films via plasma enhanced atomic layer deposition (PE-ALD). To understand the mechanism the photo-induced deposition process the following effects were considered: the illumination photon energy and intensity, the polarization screening mechanism of the lithium niobate template and the chemical concentration. Depending on the UV wavelength, variation of Ag deposition rate and boundary nanowire formation are observed and attributed to the unique surface electric field distribution of the polarity patterned template and the penetration depth of UV light. Oxygen implantation is employed to transition the surface from external screening to internal screening, which results in depressed boundary nanowire formation. The ratio of the photon flux and Ag ion flux to the surface determine the deposition pattern. Domain boundary deposition is enhanced with a high photon/Ag ion flux ratio while domain boundary deposition is depressed with a low photon/Ag ion flux ratio. These results also support the photo-induced deposition model where the process is limited by carrier generation, and the cation reduction occurs at the surface. These findings will provide a foundational understanding to employ ferroelectric templates for assembly and patterning of inorganic, organic, biological, and integrated structures. ZnO films deposited on positive and negative domain surfaces of LN demonstrate different I-V curve behavior at different temperatures. At room temperature, ZnO deposited on positive domains exhibits almost two orders of magnitude greater conductance than on negative domains. The conductance of ZnO on positive domains decreases with increasing temperature while the conductance of ZnO on negative domains increases with increasing temperature. The observations are interpreted in terms of the downward or upward band bending at the ZnO/LN interface which is induced by the ferroelectric polarization charge. Possible application of this effect in non-volatile memory devices is proposed for future work.

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Date Created
  • 2011

Surface stress during electro-oxidation of carbon monoxide and bulk stress evolution during electrochemical intercalation of lithium

Description

This work investigates in-situ stress evolution of interfacial and bulk processes in electrochemical systems, and is divided into two projects. The first project examines the electrocapillarity of clean and CO-covered

This work investigates in-situ stress evolution of interfacial and bulk processes in electrochemical systems, and is divided into two projects. The first project examines the electrocapillarity of clean and CO-covered electrodes. It also investigates surface stress evolution during electro-oxidation of CO at Pt{111}, Ru/Pt{111} and Ru{0001} electrodes. The second project explores the evolution of bulk stress that occurs during intercalation (extraction) of lithium (Li) and formation of a solid electrolyte interphase during electrochemical reduction (oxidation) of Li at graphitic electrodes. Electrocapillarity measurements have shown that hydrogen and hydroxide adsorption are compressive on Pt{111}, Ru/Pt{111}, and Ru{0001}. The adsorption-induced surface stresses correlate strongly with adsorption charge. Electrocatalytic oxidation of CO on Pt{111} and Ru/Pt{111} gives a tensile surface stress. A numerical method was developed to separate both current and stress into background and active components. Applying this model to the CO oxidation signal on Ru{0001} gives a tensile surface stress and elucidates the rate limiting steps on all three electrodes. The enhanced catalysis of Ru/Pt{111} is confirmed to be bi-functional in nature: Ru provides adsorbed hydroxide to Pt allowing for rapid CO oxidation. The majority of Li-ion batteries have anodes consisting of graphite particles with polyvinylidene fluoride (PVDF) as binder. Intercalation of Li into graphite occurs in stages and produces anisotropic strains. As batteries have a fixed size and shape these strains are converted into mechanical stresses. Conventionally staging phenomena has been observed with X-ray diffraction and collaborated electrochemically with the potential. Work herein shows that staging is also clearly observed in stress. The Li staging potentials as measured by differential chronopotentiometry and stress are nearly identical. Relative peak heights of Li staging, as measured by these two techniques, are similar during reduction, but differ during oxidation due to non-linear stress relaxation phenomena. This stress relaxation appears to be due to homogenization of Li within graphite particles rather than viscous flow of the binder. The first Li reduction wave occurs simultaneously with formation of a passivating layer known as the solid electrolyte interphase (SEI). Preliminary experiments have shown the stress of SEI formation to be tensile (~+1.5 MPa).

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  • 2011

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Reconstruction methods In free electron laser X-ray diffraction

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One of the most important issues in femtosecond free electron laser X-ray diraction is to reconstruct the 3D charge density of molecule from a mass of diraction snapshots. In order

One of the most important issues in femtosecond free electron laser X-ray diraction is to reconstruct the 3D charge density of molecule from a mass of diraction snapshots. In order to determine the orientation of single molecule from diraction patterns, we rst determine the moments and products of inertia of this from 2D experiment data (diraction patterns or EM images to obtain the elements of the inertia tensor. If diraction patterns from uniformly random orientations or some preferred orientations are collected, the principal axes of the molecule can be extracted, together with the Euler angles which relate the principal axes of the molecule to the laboratory frame axes. This is achieved by nding the maximum and minimum values for the measured moments from many single-molecule patterns. Simulations for GroEL protein indicates that the calculation of the autocorrelation help eliminate the Poisson noise in Cryo- EM images and can make correct orientation determination. The eect of water jacket surrounding the protein molecule is studied based on molecular dynamics simulation result. The intensities from water and interference is found to suppress those from protein itself. A method is proposed and applied to the simulation data to show the possibility for it to overcome the water background problem. The scattering between Bragg re ections from nanocrystals is used to aid solution of the phase problem. We describe a method for reconstructing the charge density of a typical molecule within a single unit cell, if suciently nely-sampled diraction data are available from many nanocrystals of dierent sizes lying in the same orientations without knowledge of the distribution of particle size or requiring atomic-resolution data. Triple correlation of the diraction patterns are made use of to reconiii

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Date Created
  • 2011

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Interactions driving the collapse of islet amyloid polypeptide: implications for amyloid aggregation

Description

Human islet amyloid polypeptide (hIAPP), also known as amylin, is a 37-residue intrinsically disordered hormone involved in glucose regulation and gastric emptying. The aggregation of hIAPP into amyloid fibrils is

Human islet amyloid polypeptide (hIAPP), also known as amylin, is a 37-residue intrinsically disordered hormone involved in glucose regulation and gastric emptying. The aggregation of hIAPP into amyloid fibrils is believed to play a causal role in type 2 diabetes. To date, not much is known about the monomeric state of hIAPP or how it undergoes an irreversible transformation from disordered peptide to insoluble aggregate. IAPP contains a highly conserved disulfide bond that restricts hIAPP(1-8) into a short ring-like structure: N_loop. Removal or chemical reduction of N_loop not only prevents cell response upon binding to the CGRP receptor, but also alters the mass per length distribution of hIAPP fibers and the kinetics of fibril formation. The mechanism by which N_loop affects hIAPP aggregation is not yet understood, but is important for rationalizing kinetics and developing potential inhibitors. By measuring end-to-end contact formation rates, Vaiana et al. showed that N_loop induces collapsed states in IAPP monomers, implying attractive interactions between N_loop and other regions of the disordered polypeptide chain . We show that in addition to being involved in intra-protein interactions, the N_loop is involved in inter-protein interactions, which lead to the formation of extremely long and stable β-turn fibers. These non-amyloid fibers are present in the 10 μM concentration range, under the same solution conditions in which hIAPP forms amyloid fibers. We discuss the effect of peptide cyclization on both intra- and inter-protein interactions, and its possible implications for aggregation. Our findings indicate a potential role of N_loop-N_loop interactions in hIAPP aggregation, which has not previously been explored. Though our findings suggest that N_loop plays an important role in the pathway of amyloid formation, other naturally occurring IAPP variants that contain this structural feature are incapable of forming amyloids. For example, hIAPP readily forms amyloid fibrils in vitro, whereas the rat variant (rIAPP), differing by six amino acids, does not. In addition to being highly soluble, rIAPP is an effective inhibitor of hIAPP fibril formation . Both of these properties have been attributed to rIAPP's three proline residues: A25P, S28P and S29P. Single proline mutants of hIAPP have also been shown to kinetically inhibit hIAPP fibril formation. Because of their intrinsic dihedral angle preferences, prolines are expected to affect conformational ensembles of intrinsically disordered proteins. The specific effect of proline substitutions on IAPP structure and dynamics has not yet been explored, as the detection of such properties is experimentally challenging due to the low molecular weight, fast reconfiguration times, and very low solubility of IAPP peptides. High-resolution techniques able to measure tertiary contact formations are needed to address this issue. We employ a nanosecond laser spectroscopy technique to measure end-to-end contact formation rates in IAPP mutants. We explore the proline substitutions in IAPP and quantify their effects in terms of intrinsic chain stiffness. We find that the three proline mutations found in rIAPP increase chain stiffness. Interestingly, we also find that residue R18 plays an important role in rIAPP's unique chain stiffness and, together with the proline residues, is a determinant for its non-amyloidogenic properties. We discuss the implications of our findings on the role of prolines in IDPs.

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Date Created
  • 2013