Browsing by Subject "Atomic force microscopy"

Now showing 1 - 7 of 7
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    Developing an approach to improve beta-phase properties in ferroelectric pvd-hfp thin films
    (2020-05) Dale, Ashley S.; Cheng, Ruihua; Petrache, Horia; Wassall, Stephen
    Improved fabrication of poly(vinylindenefluoride)-hexafluoropropylene (PVDF-HFP) thin films is of particular interest due to the high electric coercivity found in the beta-phase structure of the thin film. We show that it is possible to obtain high-quality, beta-phase dominant PVDF-HFP thin films using a direct approach to Langmuir-Blodgett deposition without the use of annealing or additives. To improve sample quality, an automated Langmuir-Blodgett thin film deposition system was developed; a custom dipping trough was fabricated, a sample dipping mechanism was designed and constructed, and the system was automated using custom LabVIEW software. Samples were fabricated in the form of ferroelectric capacitors on substrates of glass and silicon, and implement a unique step design with a bottom electrode of copper with an aluminum wetting layer and a top electrode of gold with an aluminum wetting layer. Samples were then characterized using a custom ferroelectric measurement program implemented in LabVIEW with a Keithley picoammeter/voltage supply to confirm electric coercivity properties. Further characterization using scanning electron microscopy and atomic force microscopy confirmed the improvement in thin film fabrication over previous methods.
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    Multi-scale analysis of morphology, mechanics, and composition of collagen in murine osteogenesis imperfecta
    (2013-11-06) Bart, Zachary Ryan; Wallace, Joseph; Na, Sungsoo; Yokota, Hiroki, 1955-; Schild, John H.
    Osteogenesis imperfecta is a rare congenital disease commonly characterized by brittle bones caused by mutations in the genes encoding Type I collagen, the single most abundant protein produced by the body. The murine model (oim) exists as a natural mutation of this protein, converting its heterotrimeric structure of two Col1a1 molecules and a single Col1a2 molecule into homotrimers composed of only the former. This defect impacts bone mechanical integrity, greatly weakening their structure. Femurs from male wild type (WT), heterozygous (oim/+), and homozygous (oim/oim) mice, all at 12 weeks of age, were assessed using assays at multiple length scales with minimal sample processing to ensure a near-physiological state. Atomic force microscopy (AFM) demonstrated detectable differences in the organization of collagen at the nanometer scale that may partially attribute to alterations in material and structural behavior obtained through mechanical testing and reference point indentation (RPI). Changes in geometric and chemical structure through the use of µ-Computed Tomography and Raman spectroscopy respectively indicate a smaller, brittle phenotype caused by oim. Changes within the periodic D-spacing of collagen point towards a reduced mineral nucleation site, supported by reduced mineral crystallinity, resulting in altered material and structural behavior in oim/oim mice. Multi-scale analyses of this nature offer much in assessing how molecular changes can compound to create a degraded, brittle phenotype.
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    Multi-scale measurement of stiffness in the developing ferret brain
    (Springer Nature, 2023-11-23) Walter, Christopher; Balouchzadeh, Ramin; Garcia, Kara E.; Kroenke, Christopher D.; Pathak, Amit; Bayly, Philip V.; Radiology and Imaging Sciences, School of Medicine
    Cortical folding is an important process during brain development, and aberrant folding is linked to disorders such as autism and schizophrenia. Changes in cell numbers, size, and morphology have been proposed to exert forces that control the folding process, but these changes may also influence the mechanical properties of developing brain tissue. Currently, the changes in tissue stiffness during brain folding are unknown. Here, we report stiffness in the developing ferret brain across multiple length scales, emphasizing changes in folding cortical tissue. Using rheometry to measure the bulk properties of brain tissue, we found that overall brain stiffness increases with age over the period of cortical folding. Using atomic force microscopy to target the cortical plate, we found that the occipital cortex increases in stiffness as well as stiffness heterogeneity over the course of development and folding. These findings can help to elucidate the mechanics of the cortical folding process by clarifying the concurrent evolution of tissue properties.
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    Physisorbed Polymer-Tethered Lipid Bilayer with Lipopolymer Gradient
    (MDPI, 2012-11) Lin, Yu-Hung; Minner, Daniel E.; Herring, Vincent L.; Naumann, Christoph A.; Chemistry and Chemical Biology, School of Science
    Physisorbed polymer-tethered lipid bilayers consisting of phospholipids and lipopolymers represent an attractive planar model membrane platform, in which bilayer fluidity and membrane elastic properties can be regulated through lipopolymer molar concentration. Herein we report a method for the fabrication of such a planar model membrane system with a lateral gradient of lipopolymer density. In addition, a procedure is described, which leads to a sharp boundary between regions of low and high lipopolymer molar concentrations. Resulting gradients and sharp boundaries are visualized on the basis of membrane buckling structures at elevated lipopolymer concentrations using epifluorescence microscopy and atomic force microscopy. Furthermore, results from spot photobleaching experiments are presented, which provide insight into the lipid lateral fluidity in these model membrane architectures. The presented experimental data highlight a planar, solid-supported membrane characterized by fascinating length scale-dependent dynamics and elastic properties with remarkable parallels to those observed in cellular membranes.
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    Regulating Lipid Organization and Investigating Membrane Protein Properties in Physisorbed Polymer-tethered Membranes
    (2012-08-07) Siegel, Amanda P.; Naumann, Christoph A.; Minto, Robert; Thompson, David H. Pai; Ritchie, Kenneth
    Cell membranes have remarkable properties both at the microscopic level and the molecular level. The current research describes the use of physisorbed polymer-grafted lipids in model membranes to investigate some of these properties on both of these length scales. On the microscopic scale, plasma membranes can be thought of as heterogenous thin films. Cell membranes adhered to elastic substrates are capable of sensing substrate/film mismatches and modulating their membrane stiffness to more closely match the substrate. Membrane/substrate mismatch can be modeled by constructing lipopolymer-enriched lipid monolayers with different bending stiffnesses and physisorbing them to rigid substrates which causes buckling. This report describes the use of atomic force microscopy and epimicroscopy to characterize these buckled structures and to illustrate the use of the buckled structures as diffusion barriers in lipid bilayers. In addition, a series of monolayers with varying bending stiffnesses and thicknesses are constructed on rigid substrates to analyze changes in buckling patterns and relate the experimental results to thin film buckling theory. On the molecular scale, plasma membranes can also be thought of as heterogeneous mixtures of lipids where the specific lipid environment is a crucial factor affecting membrane protein function. Unfortunately, heterogeneities involving cholesterol, labeled lipid rafts, are small and transient in live cells. To address this difficulty, the present work describes a model platform based on polymer-supported lipid bilayers containing stable raft-mimicking domains into which transmembrane proteins are incorporated (αvβ3, and α5β1integrins). This flexible platform enables the use of confocal fluorescence fluctuation spectroscopy to quantitatively probe the effect of cholesterol concentrations and the binding of native ligands (vitronectin and fibronectin for αvβ3, and α5β1) on protein oligomerization state and on domain-specific protein sequestration. In particular, the report shows significant ligand-induced integrin sequestration with a low level of dimerization. Cholesterol concentration increases rate of dimerization, but only moderately. Ligand addition does not affect rate of dimerization in either system. The combined results strongly suggest that ligands induce changes to integrin conformation and/or dynamics without inducing changes in integrin oligomerization state, and in fact these ligand-induce conformational changes impact protein-lipid interactions.
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    Removing or truncating connexin 43 in murine osteocytes alters cortical geometry, nanoscale morphology, and tissue mechanics in the tibia
    (Elsevier, 2016-07) Hammond, Max A.; Berman, Alycia G.; Pacheco-Costa, Rafael; Davis, Hannah M.; Plotkin, Lilian I.; Wallace, Joseph M.; Biomedical Engineering, School of Engineering and Technology
    Gap junctions are formed from ubiquitously expressed proteins called connexins that allow the transfer of small signaling molecules between adjacent cells. Gap junctions are especially important for signaling between osteocytes and other bone cell types. The most abundant type of connexin in bone is connexin 43 (Cx43). The C-terminal domain of Cx43 is thought to be an important modulator of gap junction function but the role that this domain plays in regulating tissue-level mechanics is largely unknown. We hypothesized that the lack of the C-terminal domain of Cx43 would cause morphological and compositional changes as well as differences in how bone responds to reference point indentation (RPI) and fracture toughness testing. The effects of the C-terminal domain of Cx43 in osteocytes and other cell types were assessed in a murine model (C57BL/6 background). Mice with endogenous Cx43 in their osteocytes removed via a Cre-loxP system were crossed with knock-in mice which expressed Cx43 that lacked the C-terminal domain in all cell types due to the insertion of a truncated allele to produce the four groups used in the study. The main effect of removing the C-terminal domain from osteocytic Cx43 increased cortical mineral crystallinity (p=0.036) and decreased fracture toughness (p=0.017). The main effect of the presence of the C-terminal domain in other cell types increased trabecular thickness (p<0.001), cortical thickness (p=0.008), and average RPI unloading slope (p=0.004). Collagen morphology was altered when either osteocytes lacked Cx43 (p=0.008) or some truncated Cx43 was expressed in all cell types (p<0.001) compared to controls but not when only the truncated form of Cx43 was expressed in osteocytes (p=0.641). In conclusion, the presence of the C-terminal domain of Cx43 in osteocytes and other cell types is important to maintain normal structure and mechanical integrity of bone.
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    β-Aminopropionitrile-Induced Reduction in Enzymatic Crosslinking Causes In Vitro Changes in Collagen Morphology and Molecular Composition
    (PLOS, 2016-11-09) Canelón, Silvia P.; Wallace, Joseph M.; Department of Biomedical Engineering, School of Engineering & Technology
    Type I collagen morphology can be characterized using fibril D-spacing, a metric which describes the periodicity of repeating bands of gap and overlap regions of collagen molecules arranged into collagen fibrils. This fibrillar structure is stabilized by enzymatic crosslinks initiated by lysyl oxidase (LOX), a step which can be disrupted using β-aminopropionitrile (BAPN). Murine in vivo studies have confirmed effects of BAPN on collagen nanostructure and the objective of this study was to evaluate the mechanism of these effects in vitro by measuring D-spacing, evaluating the ratio of mature to immature crosslinks, and quantifying gene expression of type I collagen and LOX. Osteoblasts were cultured in complete media, and differentiated using ascorbic acid, in the presence or absence of 0.25mM BAPN-fumarate. The matrix produced was imaged using atomic force microscopy (AFM) and 2D Fast Fourier transforms were performed to extract D-spacing from individual fibrils. The experiment was repeated for quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Fourier Transform infrared spectroscopy (FTIR) analyses. The D-spacing distribution of collagen produced in the presence of BAPN was shifted toward higher D-spacing values, indicating BAPN affects the morphology of collagen produced in vitro, supporting aforementioned in vivo experiments. In contrast, no difference in gene expression was found for any target gene, suggesting LOX inhibition does not upregulate the LOX gene to compensate for the reduction in aldehyde formation, or regulate expression of genes encoding type I collagen. Finally, the mature to immature crosslink ratio decreased with BAPN treatment and was linked to a reduction in peak percent area of mature crosslink hydroxylysylpyridinoline (HP). In conclusion, in vitro treatment of osteoblasts with low levels of BAPN did not induce changes in genes encoding LOX or type I collagen, but led to an increase in collagen D-spacing as well as a decrease in mature crosslinks.