Optical nanoscopy

AbstractThis article deals with the developments of optical microscopy towards nanoscopy. Basic concepts of the methods implemented to obtain spatial super-resolution are described, along with concepts related to the study of biological systems at the molecular level. Fluorescence as a mechanism of contrast and spatial resolution will be the starting point to developing a multi-messenger optical microscope tunable down to the nanoscale in living systems. Moreover, the integration of optical nanoscopy with scanning probe microscopy and the charming possibility of using artificial intelligence approaches will be shortly outlined

Cell biomechanics of the central nervous system

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011.Cataloged from PDF version of thesis.Includes bibliographical references (p. 133-153).Traumatic brain injury (TBI) is a significant cause of death and morbidity in both the civilian and military populations. The major causes of TBI, such as motor vehicle accidents, falls, sports concussions, and ballistic and explosive blast threats for military personnel, are well established and extensively characterized; however, there remains much to be learned about the specific mechanisms of damage leading to brain injury, especially at the cellular level. In order to understand how cells of the central nervous system (CNS) respond to mechanical insults and stimuli, a combined modeling/experimental approach was adopted. A computational framework was developed to accurately model how cells deform under various macroscopically imposed loading conditions. In addition, in vitro (cell culture) models were established to investigate damage responses to biologically relevant mechanical insults. In order to develop computational models of cell response to mechanical loading, it is essential to have accurate material properties for all cells of interest. In this work, the mechanical responses of neurons and astrocytes were quantified using atomic force microscopy (AFM) at three different loading rates and under relaxation to enable characterization of both the elastic and viscous components of the cell response. AFM data were used to calibrate an eight-parameter rheological model implemented in the framework of a commercial finite element package (Abaqus). Model parameters fit to the measured responses of neurons and astrocytes provide a quantitative measure of homogenized nonlinear viscoelastic properties for each cell type. In order to ensure that the measured responses could be considered representative of cell populations in their physiological environment, cells were also grown and tested on substrates of various stiffness, with the softest substrate mimicking the stiffness of brain tissue. Results of this study showed both the morphology and measured force response of astrocytes to be significantly affected by the stiffness of their substrate, with cells becoming increasingly rounded on soft substrates. Results of simulations suggested that changes in cell morphology were able to account for the observed changes in AFM force response, without significant changes to the cell material properties. In contrast, no significant changes in cell morphology were observed for neurons. These results highlight the importance of growing cells in a biologically relevant environment when studying mechanically mediated responses, such as TBI. To address this requirement, we developed two model systems with CNS cells grown in soft, 3D gels to investigate damage arising from dynamic compressive loading and from a shock pressure wave. These damage protocols, coupled with the single cell computational models, provide a new tool set for characterizing damage mechanisms in CNS cells and for studying TBI in highly controllable in vitro conditions.by Kristin Briana Bernick.Ph.D

Gradient light interference microscopy for imaging strongly scattering samples

A growing interest in three-dimensional cellular systems has raised new challenges for light microscopy. The fundamental difficulty is the tendency for the optical field to scramble when interacting with turbid media, leading to contrast images. In this work, we outline the development of an instrument that uses broadband optical fields in conjunction with phase-shifting interferometry to extract high-resolution and high-contrast structures from otherwise cloudy images. We construct our system from a differential interference contrast microscope, demonstrating our new modality in transmission and reflection geometries. We call this modality Gradient Light Interference Microscopy (GLIM) as the image measures the gradient of the object’s scattering potential. To facilitate complex experiments, we develop a high-throughput acquisition software and propose several ways to analyze this new kind of data using deep convolutional neural networks. This new proposal, termed phase imaging with computational specificity (PICS), allows for non-destructive yet chemically motivated annotation of microscopy images. The results presented in this dissertation provide templates that are readily extendible to other quantitative phase imaging modalities

Physical properties of nanostructures induced by irradiation in diamond

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in ful llment of the requirements for the degree of Doctor of Philosophy in Physics. Johannesburg, 10 August 2017.We investigate the interaction of slow highly charged ions (SHCIs) with insulating type-Ib diamond (111) surfaces. Bismuth and Xenon SHCI beams produced using an Electron Beam Ion Trap (EBIT) and an Electron Cyclotron Resonance source (ECR) respectively, are accelerated onto type Ib diamond (111) surfaces with impact velocities up to 0.4 Bohr. SHCIs with charge states corresponding to potential energies between 4.5 keV and 110 keV are produced for this purpose. Atomic Force Microscopy analysis (AFM) of the diamond surfaces following SHCI impact reveals surface morphological modi cations characterized as nanoscale craters (nano-craters). To interpret the results from Tapping Mode AFM analysis of the irradiated diamond surfaces we discuss the interplay between kinetic and potential energy in nanocrater formation using empirical data together with Stopping and Range of Ions in Matter (SRIM) Monte Carlo Simulations. In the case of irradiation induced magnetic e ects in diamond, we investigate the magnetic properties of ultra-pure type-IIa diamond following irradiation with proton beams of 1-2 MeV energy. SQUID magnetometry of proton irradiated non-annealed diamond indicates formation of Curie type paramagnetism according to the Curie law. Raman and Photoluminescence spectroscopy measurements show that the primary structural features created by proton irradiation are the centers: GR1, ND1, TR12 and 3H. The Stopping and Range of Ions in Matter (SRIM) Monte Carlo simulations together with ii iii SQUID observations show a strong correlation between vacancy production, proton uence and the paramagnetic factor. At an average surface vacancy spacing of 1-1.6 nm and bulk (peak) vacancy spacing of 0.3-0.5 nm Curie paramagnetism is induced by formation of ND1 centres with an e ective magnetic moment eff (0.1-0.2) B. Post annealing SQUID analysis of proton irradiated diamond shows formation temperature independent magnetism with magnetic moment 6-7 emu superimposed to Curie-type paramagnetism. The response of ultra-pure type-IIa single crystal CVD diamond following 2.2 MeV proton micro-irradiation is further investigated using Atomic Force, Magnetic Force and Electrostatic Force Microscopy (AFM, MFM and EFM) under ambient conditions. Analysis of the phase shift signals using probe polarization dependent magnetization measurements and comparison of the MFM and EFM signals at zero electrical bias, show that measured force gradients originate from a radiation induced magnetic response in the micro-irradiated regions in diamond.LG201

Biopolymeric microbeads as a 3D scaffold for soft tissue engineering

The increase of different types of cell cultures, which can be used for the in vitro studies of physiological and/or pathological processes, has introduced the need to improve culture techniques through the use of materials and culture media that promote growth, recreating a cellular micro-environment that can be asserted in in vivo condition. Therefore, it is important to design and develop new biologically sustainable methods, such as to contribute to the \u201ccloser-to-in vivo\u201d condition. In particular, the design of a 3D in vitro model of neuronal culture is an important step to better understand the mechanisms of cell-cell communication, synaptogenesis and neurophysiological circuits. In order to mimic the ECM environment, a granular, porous and soft structure is preferred in the design of an artificial neural network. The granular structure is preferred due to the fact that CNS tissue seems to be organized as a greater proportion of the microscale tissue, that can be thought of as granular. For this reason, the thesis is focused on the production and characterization of bipolymeric microbeads as a 3D scaffold for soft tissue engineering. The biopolymer Chitosan is presented as an alternative adhesion factor and support for 2D and 3D neuronal cell cultures. Chitosan is a copolymer of glucosamine and N-acetyl-glucosamine, obtained by the deacetylation of chitin; it is well known for its low-cost, biocompatibility, biodegradability, muco-adhesiveness, antibacterial activity as well as its bioaffinity. Chitosan backbone shows positive charges of primary ammines that favor the electrostatic interactions with the negatively charged cell membranes promoting cell adhesion and growth. The standard studies focoused on the development of nervous system, have been performed using traditional monolayer culture onto supports modified by extracellular matrix components or synthetic biopolymers such as poly-ornithine and poly-lysine which are expressed at stages critical for neuronal differentiation in situ and are functional in neurite outgrowth in vitro, acting as adhesion proteins. Morphological and functional characterization of 2D neuronal culture grew up onto chitosan susbtrates are carried out and compared with the gold standard reported in literature, in order to validate the ability of chitosan to support neuronal adhesion, networks development and the differentiation capacity. 3D cultured neurons on chitosan microbeads based-scaffold, showed a structural development of a functional network that are more representative of the in vivo environment. The studies reported in this thesis, successfully demonstrate the alternative use of the polysaccharide chitosan as adhesion factor and as a structural component for 2D/3D neuronal cultures. Definitely, thanks to its low cost and versatility, it could be easily functionalized for the fabrication of personalized of in vitro models. In this thesis, a new technology to converts monodisperse microbead hydrogels to fine powders, is reported. Microengineered emulsion-to-powder (MEtoP) technology generates microgels with all the molecular, colloidal, and bulk characteristics of fresh microbeas upon resuspension in aqueous media. GelMA microbeads are fabricated by microfluidic technique, that is one of the most effective techniques, and allows precise tuning of the compositions and geometrical characteristics of microbeads. Gelatin-methacryloyl (GelMA) is a semi-synthetic hydrogel which consists of gelatin derivatized with methacrylamide and methacrylate groups. These hydrogels provide cells with an optimal biological environment (e.g., RGD motifs for adhesion) and can be quickly photo-crosslinked, which provide shape fidelity and stability at physiological temperature. MEtoP technology is based on protecting the dispersed phase of an emulsion to preserve its physical and chemical cues during harsh freezing and lyophilization procedures. This technology avoids the persistent problems of colloids, including difficulty in sterilization, bacterial and viral contamination, impaired stability, high processing costs, and difficult packaging and transportation

Application of Parylene C thin films in cardiac cell culturing

There are two main challenges when producing in vitro cell systems: first, to reconstitute the in situ cellular microenvironment, thus delivering more representative and reliable cell models for drug screening and disease modelling studies. Second, to record and quantify the electrical and chemical gradients across the culture. Ideally, both challenges should be accomplished within a single platform towards a lab-on-chip implementation. This research work investigates the application of Parylene C in cardiac cell scaffolding and its integrability with electrochemical monitoring technologies for measuring extracellular action potentials and pH. The surface properties of Parylene C in terms of water affinity, chemical composition and nanotopography were characterised before and after modifying the material's inherent hydrophobicity through oxygen plasma. A technology was developed to selectively alter the surface hydrophobicity of Parylene C through standard lithography and oxygen plasma, which is characterised by μm-resolution and long-term pattern stability, and can accurately control the extent of induced hydrophilicity, the pattern layout and 3-D geometry. The micro-engineered Parylene C films were employed as scaffolds for cardiac cells with immature physiological properties, such as neonatal rat ventricular myocytes (NRVM). The scaffolds promoted a more in situ cellular structure and organisation, while they improved important calcium (Ca2+) cycling parameters such as fluorescent amplitude, time to peak (Tp), time to 50% (T50) and 90% (T90) decay at 0.5-2 Hz field stimulation. The thickness of the patterned Parylene C films was found to regulate the shape of the cells by controlling their adhesion area on the Parylene substrate through a thickness-dependent hydrophobicity. NRVM on thin (2 μm) membranes tended to bridge across the hydrophobic areas and adopt a spread-out shape (average contact angle at the level of the nucleus was 64.51o). On the other hand, cells on thick (10 μm) films were mostly constrained on the hydrophilic areas and demonstrated a more elongated, cylindrical (in vivo-like) shape (average contact angle was 84.73o). The cylindrical shape and a significantly (p <0.05) denser microtubule structure in cells on thick films possibly suggest a more mature cardiomyocyte. However, there was no significant effect on the Ca2+ physiology between the two groups. The micro-patterning technology was able to deliver free-standing Parylene C thin films (2-10 μm) to study the effect of substrate elasticity and flexibility on the Ca2+ physiology of NRVM. Preliminary results showed that fluorescent amplitude and time to peak were improved in structured NRVM cultures on stand-alone Parylene films compared to rigid Parylene-coated glass surfaces. However, no such trend was present in Ca2+ release parameters (T50, T90). The flexibility of the culture substrate was also manipulated by employing free-standing micro-patterned Parylene C films of distinct thicknesses (2-10 μm), but did not affect the cellular Ca2+ physiology. Further biological validation is needed with a larger sample size to draw a certain conclusion. The cell patterning technology was transferred to commercially available planar Multi-Electrode arrays (MEAs) to demonstrate integrability of this method with existing monitoring tools. The micro-patterned MEAs induced anisotropic cardiomyocyte cultures, as they substantially increased the longitudinal-to-transverse velocity anisotropy ratios (1.09, n=4 to 1.69, n=2), promoting action potential propagation profiles that closer resembled native cardiac tissue. Furthermore, the micro-engineered MEAs were proven to be reusable, yielding a versatile and low-cost approach that is compatible with state-of-art recording equipment and can be employed as a more reliable, off-the-shelf tool for drug screening studies. Selective hydrophilic modification of Parylene C was also employed to activate locally the H+ sensing capacity of such films, implementing extended-gate pH sensors. The ability of Parylene C to act in a dual way - as an encapsulation material and as an active pH sensing membrane - was demonstrated. The material exhibited a distinguishable sensitivity dependent on the oxygen plasma recipe, relatively low drift rates and excellent encapsulation quality. Based on these principles, flexible Parylene-based high-density miniaturised electrode arrays were fabricated, employing Parylene as a flexible structure material and as a H+ sensing membrane for local detection of pH. The presented Parylene-based technology has the potential to deliver integrated lab-on-chip implementations for growing cells in vitro with controlled microtopography while monitoring the extracellular electrical and pH gradients across the culture in a non-invasive way, with application in drug screening and disease modelling.Open Acces

Novel miniaturised and highly versatile biomechatronic platforms for the characterisation of melanoma cancer cells

There has been an increasing demand to acquire highly sensitive devices that are able to detect and characterize cancer at a single cell level. Despite the moderate progress in this field, the majority of approaches failed to reach cell characterization with optimal sensitivity and specificity. Accordingly, in this study highly sensitive, miniaturized-biomechatronic platforms have been modeled, designed, optimized, microfabricated, and characterized, which can be used to detect and differentiate various stages of melanoma cancer cells. The melanoma cell has been chosen as a legitimate cancer model, where electrophysiological and analytical expression of cell-membrane potential have been derived, and cellular contractile force has been obtained through a correlation with micromechanical deflections of a miniaturized cantilever beam. The main objectives of this study are in fourfold: (1) to quantify cell-membrane potential, (2) correlate cellular biophysics to respective contractile force of a cell in association with various stages of the melanoma disease, (3) examine the morphology of each stage of melanoma, and (4) arrive at a relation that would interrelate stage of the disease, cellular contractile force, and cellular electrophysiology based on conducted in vitro experimental findings. Various well-characterized melanoma cancer cell lines, with varying degrees of genetic complexities have been utilized. In this study, two-miniaturized-versatile-biomechatronic platforms have been developed to extract the electrophysiology of cells, and cellular mechanics (mechanobiology). The former platform consists of a microfluidic module, and stimulating and recording array of electrodes patterned on a glass substrate, forming multi-electrode arrays (MEAs), whereas the latter system consists of a microcantilever-based biosensor with an embedded Wheatstone bridge, and a microfluidic module. Furthermore, in support of this work main objectives, dedicated microelectronics together with customized software have been attained to functionalize, and empower the two-biomechatronic platforms. The bio-mechatronic system performance has been tested throughout a sufficient number of in vitro experiments.Open Acces

Viscoelasticity

This book contains a wealth of useful information on current research on viscoelasticity. By covering a broad variety of rheology, non-Newtonian fluid mechanics and viscoelasticity-related topics, this book is addressed to a wide spectrum of academic and applied researchers and scientists but it could also prove useful to industry specialists. The subject areas include, theory, simulations, biological materials and food products among others

DEVELOPMENT OF FUNCTIONAL NANOCOMPOSITE MATERIALS TOWARDS BIODEGRADABLE SOFT ROBOTICS AND FLEXIBLE ELECTRONICS

World population is continuously growing, as well as the influence we have on the ecosystem\u2019s natural equilibrium. Moreover, such growth is not homogeneous and it results in an overall increase of older people. Humanity\u2019s activity, growth and aging leads to many challenging issues to address: among them, there are the spread of suddenly and/or chronic diseases, malnutrition, resource pressure and environmental pollution. Research in the novel field of biodegradable soft robotics and electronics can help dealing with these issues. In fact, to face the aging of the population, it is necessary an improvement in rehabilitation technologies, physiological and continuous monitoring, as well as personalized care and therapy. Also in the agricultural sector, an accurate and efficient direct measure of the plants health conditions would be of help especially in the less-developed countries. But since living beings, such as humans and plants, are constituted by soft tissues that continuously change their size and shapes, today\u2019s traditional technologies, based on rigid materials, may not be able to provide an efficient interaction necessary to satisfy these needs: the mechanical mismatch is too prohibitive. Instead, soft robotic systems and devices can be designed to combine active functionalities with soft mechanical properties that can allow them to efficiently and safely interact with soft living tissues. Soft implantable biomedical devices, smart rehabilitation devices and compliant sensors for plants are all applications that can be achieved with soft technologies. The development of sophisticated autonomous soft systems needs the integration on a unique soft body or platform of many functionalities (such as mechanical actuation, energy harvesting, storage and delivery, sensing capabilities). A great research interest is recently arising on this topic, but yet not so many groups are focusing their efforts in the use of natural-derived and biodegradable raw materials. In fact, resource pressure and environmental pollution are becoming more and more critical problems. It should be completely avoided the use of in exhaustion, pollutant, toxic and non-degradable resources, such as lithium, petroleum derivatives, halogenated compounds and organic solvents. So-obtained biodegradable soft systems and devices could then be manufactured in high number and deployed in the environment to fulfil their duties without the need to recover them, since they can safely degrade in the environment. The aim of the current Ph.D. project is the use of natural-derived and biodegradable polymers and substances as building blocks for the development of smart composite materials that could operate as functional elements in a soft robotic system or device. Soft mechanical properties and electronic/ionic conductive properties are here combined together within smart nanocomposite materials. The use of supersonic cluster beam deposition (SCBD) technique enabled the fabrication of cluster-assembled Au electrodes that can partially penetrate into the surface of soft materials, providing an efficient solution to the challenge of coupling conductive metallic layers and soft deformable polymeric substrates. In this work, cellulose derivatives and poly(3-hydroxybutyrate) bioplastic are used as building blocks for the development of both underwater and in-air soft electromechanical actuators that are characterized and tested. A cellulosic matrix is blended with natural-derived ionic liquids to design and manufacture completely biodegradable supercapacitors, extremely interesting energy storage devices. Lastly, ultrathin Au electrodes are here deposited on biodegradable cellulose acetate sheets, in order to develop transparent flexible electronics as well as bidirectional resistive-type strain sensors. The results obtained in this work can be regarded as a preliminary study towards the realization of full natural-derived and biodegradable soft robotic and electronic systems and devices