Adaption and application of morphological pseudoconvolutions to scanning tunneling and atomic force microscopy

A recently developed class of digital filters known as morphological pseudoconvolutions are adapted and applied to Scanning Tunneling Microscopy (STM) and Atomic Force Microscopy (AFM) images. These filters are shown to outperform, both visually and in the mean square error sense, previously introduced Wiener filtering techniques. The filters are compared on typical STM/AFM images, using both modeled and actual data. The technique is general, and is shown to perform very well on many types of STM and AFM images

Deep Learning Enables Large Depth-of-Field Images for Sub-Diffraction-Limit Scanning Superlens Microscopy

Scanning electron microscopy (SEM) is indispensable in diverse applications ranging from microelectronics to food processing because it provides large depth-of-field images with a resolution beyond the optical diffraction limit. However, the technology requires coating conductive films on insulator samples and a vacuum environment. We use deep learning to obtain the mapping relationship between optical super-resolution (OSR) images and SEM domain images, which enables the transformation of OSR images into SEM-like large depth-of-field images. Our custom-built scanning superlens microscopy (SSUM) system, which requires neither coating samples by conductive films nor a vacuum environment, is used to acquire the OSR images with features down to ~80 nm. The peak signal-to-noise ratio (PSNR) and structural similarity index measure values indicate that the deep learning method performs excellently in image-to-image translation, with a PSNR improvement of about 0.74 dB over the optical super-resolution images. The proposed method provides a high level of detail in the reconstructed results, indicating that it has broad applicability to chip-level defect detection, biological sample analysis, forensics, and various other fields.Comment: 13 pages,7 figure

CHARACTERIZATION OF VASCULAR SMOOTH MUSCLE CELL MECHANICAL AND FRICTIONAL PROPERTIES USING ATOMIC FORCE MICROSCOPY

A working hypothesis within the Laboratory of Vascular Research is that mechanical loading on vascular smooth muscle cells (VSMCs), especially due to solid contact from endovascular devices, contributes to the development of restenosis. In order to better understand the role of mechanical loading on VSMCs in vascular disease development, it is imperative to understand the mechanical properties of VSMCs themselves. To measure the viscoelastic and frictional properties of living VSMCs in an in vitro setting, an atomic force microscope (AFM) was utilized, thereby allowing for mechanical testing of living cells in a fluid environment. In the first phase of research, it was found that proliferative VSMCs, similar to those commonly found in atherosclerotic lesions, had lower stiffness and higher hysteresis values than quiescent VSMCs. Furthermore, measured stiffness values did not appear to deviate greatly within the central region of adherent cells. As VSMCs are viscoelastic, rather than purely elastic in their mechanical behavior, phase two involved the development of an AFM-based stress relaxation technique, in order to quantify VSMC viscoelastic behavior. Suitable mechanical models, including the QLV reduced relaxation function and a simple power-law model, were identified and applied to accurately describe VSMC stress relaxation. In addition, the roles of two major cytoskeletal components, actin and microtubules, in governing stress relaxation behavior, were quantified via the aforementioned mechanical models. In phase three, the surface frictional properties of VSMCs were focused upon, and a novel method to quantify surface shear forces on VSMCs using lateral force microscopy was developed. It was determined that VSMC frictional properties are greatly influenced by cell stiffness, and elastohydrodynamic lubrication was proposed as a possible cellular lubricating mechanism. During research phase four, each of the techniques developed during the preceding phases was employed to test the effects of a clinically relevant biomolecule, oxidized low-density lipoprotein (oxLDL) on VSMC mechanical properties. It was concluded that oxLDL is associated with decreased cell stiffness, and decreased viscosity, as measured by stress relaxation and indentation tests. Furthermore, frictional coefficients were found to correlate positively with more fluid-like cells. This research project has led to a better understanding of VSMC mechanical behavior, as well as the development of AFM-based techniques and models that will be useful in determining cellular mechanical and frictional effects of various stimuli in an in vitro environment

Design and fabrication of biocompatible scaffolds for the regeneration of tissues

Regenerative medicine and tissue engineering attempt to repair or improve the biological functions of tissues that have been damaged or have ceased to perform their role through three main components: a biocompatible scaffold, cellular component and bioactive molecules. Nanotechnology provide a toolbox of innovative scaffold fabrication procedures in regenerative medicine. In fact, nanotechnology, using manufacturing techniques such as conventional and unconventional lithography, allows fabricating supports with different geometries and sizes as well as displaying physical chemical properties tunable over different length scales. Soft lithography techniques allow to functionalize the support by specific molecules that promote adhesion and control the growth of cells. Understanding cell response to scaffold, and viceversa, is a key issue; here we show our investigation of the essential features required for improving the cell-surface interaction over different scale lengths. The main goal of this thesis has been to devise a nanotechnology-based strategy for the fabrication of scaffolds for tissue regeneration. We made four types of scaffolds, which are able to accurately control cell adhesion and proliferation. For each scaffold, we chose properly designed materials, fabrication and characterization techniques

The combined AFM manipulation and fluorescence imaging of single DNA molecules

A combined fluorescence microscope/AFM set-up was constructed, which enabled the real-time manipulation of single DNA molecules. Fluorescence images of these TO-PRO-3 intercalated strands could be taken, while they were stretched with an AFM tip on silanised or polylysine covered glass surfaces. Characteristic AFM force spectra of single DNA molecules were achieved and a statistical analysis of the rupture forces, plateau heights and rupture lengths was compiled. The wide-field fluorescence images indicated that the DNA underwent condensation on polylysine to form aggregated rods and globular structures. Due to the strong unspecific adhesion of the DNA to the polylysine surface, AFM tip manipulation frequently led to strand scission. In addition, it was possible to “write” nm-sized letters of fluorescent DNA by unraveling agglomerated strands from the tip onto the surface. In contrast, DNA strands on silane showed far less condensation. Extended single chains were bound to the surface by the termini or at specific sites along the double helix. These fixed and straightened strands could be overstretched laterally to ca. 1.6 times the original contour length. Chain rupture occurred at the tip position, but occasionally mid-strand rupture was also observed. An analysis of the chain fluorescence intensity for different stretching lengths revealed that the dyes remain intercalated up to the end of the DNA B-S overstretching transition

Observation of Single Atom Defects and Sub-molecular Resolution with Atomic Force Microscopy in Ambient Environments

Improvements in microscopy enable the imaging of new phenomena, driving scientific advancement and the technological change it brings. Imaging in ambient conditions in particular can create challenges which limit the resolution. If phenomena can be imaged in an ambient environment the images can be collected more quickly, allowing for faster iteration, and enabling the user to work with samples which cannot enter ultra-high vacuum equipment. In this thesis, I demonstrate the imaging of transition metal dichalcogenides with atomic resolution conductive atomic force microscopy in ambient conditions and a comparative study alongside optical spectroscopy. Such resolution enables the differentiation of defect categories and the imaging of new types of defects created in samples via exposure to nitrogen plasma. The introduction of defects also induces changes in optical spectroscopy which can be identified in cryogenic measurements. This work builds a strong foundation for future work to establish correlations between the population of defects and features of photoluminescence spectra. Further work on simulations explores atomic force microscopy techniques for imaging non-planar molecules, demonstrating that constant force approaches could yield high-resolution images of molecules and allow the user to extract quantitative information such as the angle of molecular moieties. Comparisons between experimental and simulated off-resonance atomic force microscope images of a network of Zn tetra-phenyl porphyrin on a Au (111) substrate show promising progress. Such images are created using atomic coordinates from density functional theory simulations and could enable the determination of the adsorption geometry of the molecules in the network in ambient conditions

Cell mechanics and cell-cell interactions of fibroblasts from Dupuytren's Patient : Atomic Force Microscopy Investigation

Cells as a biological entity of tissue, itself made of biomolecules such as mostly proteins, lipids and carbohydrates, creates its own meshwork of biopolymers named extracellular matrix (ECM) particularly fibroblasts. With the advanced light and force microscopies, inter-cellular, cell-ECM and intracellular signaling pathways are deeply explored either by tagging the biomolecule of interest with fluorophores or by applying certain forces(in the order of pN to nN). In the field of mechanobiology, interplay between cell function and physical forces are studied using biophysical tools thatprobe their diverse mechanisms. Cells exert forces( inside-out signalling)and also respond to physical forces from their micro-environment( outside-in signalling) through participation of chain of varying protein signaling molecules. Actin molecules from cytoskeleton family form filaments in the cytoplasmic side of the cell and myosin walk on these filaments generates contractile tension. These traction forces get transmitted to the extracellular matrixof the cell or to the neighboring cells through protein complexes such as integrin and cadherins, respectively. Fibroblasts,from the mesenchymal family, are the abundant cells found in the connective tissue. Basically, fibroblasts synthesize,degrade and maintain the extracellular matrix components of the tissue. Fibroblasts, by acquiring different phenotypes called protomyofibroblast/myofibroblast, play a huge participation in various connective tissue related diseases. Myofibroblast are large cells possessing large bundles of actin filaments of isomers named alpha smooth muscle actin (I /--SMA). On the other hand, protomyofibroblast share the similar characteristic appearance but shows I /--SMA negative large stress fibres. In Dupuytrena s disease, thesemyofibroblasts persists and deform the surrounding matrix environment thus results in tissue stiffening and further leads to tissue contracture. Existing various biophysical tools maps forces such as tractile force, cell-cell interaction force and cell-ECM interaction force. One among such tool is Atomic Force Microscopy, a multifunctional toolbox in cellular biology to observe various cell types mechanics. Observing cell viscoelastic properties by application of controlled force (nanonewton) to the adherent cell become more common in the biomedical community. This thesis demonstrates the measurement of viscoelastic properties of fibroblast of different phenotypes extracted from a Dupuytrena s diseased patient and ECM derived from various tissues.The bio-mechanical interplay between cell and ECM has been studied with careful design of the AFM experiments. Fibroblasts extracted from the cords and nodular region of the palmar fascia exhibits myofibroblast phenotype and migrate slower than the fibroblast extracted from dermal and scar region. Normal and scar fibroblasts migrate faster in the wound healing assay.On the decellularized matrices, scar fibroblasts exhibitprotomyofibroblast phenotype by expressing large stress fibres. Whereas, normal fibroblasts derived from the dermal region express the healthy phenotypic appearance. From AFM based Single-cell force spectroscopy (SCFS), cell-cell interaction force measurements evaluatethe homophilic and heterophilic cadherinpairs mechanical bond strength expressed in homo-cellular (fibroblast of similar phenotype) and hetero-cellular (fibroblast-epithelial cell) arrangements. SCFS measurements also illustrate the significant role of actomyosin contractile apparatus in cadherin extracellular iidomain binding dynamics. With this evidence, SCFS setup has become an excellent spectroscopic tool to study the intracellular signalingcascades that are linked to the extracellular domain consisting transmembrane proteins such as cadherins. Therefore, an understanding of the unique fibroblasts mechanobiology is necessary to study the healthy and diseased tissue dynamics. The cell-cell and cell-ECM bio-chemical and bio-mechanical cues are strongly interdependent. Finally, the current thesis opens the basic understanding of the fibroblasts biophysical properties using AFM nano-mechanical tool and unravels the fibroblasts biomechanical function in sub-tissue level biology