This thesis quantitatively investigates gaseous electron-ion recombination in an environmental scanning electron microscope (ESEM) at a transient level by utilizing the dark shadows/streaks seen in gaseous secondary electron detector (GSED) images immediately after a region of enhanced secondary electron (SE) emission is encountered by a scanning electron beam. The investigation firstly derives a theoretical model of gaseous electron-ion recombination that takes into consideration transients caused by the time constant of the GSED electronics and external circuitry used to generate images. Experimental data of pixel intensity versus time of the streaks is then simulated using the model enabling the relative magnitudes of (i) ionization and recombination rates, (ii) recombination coefficients, and (iii) electron drift velocities, as well as absolute values of the total time constant of the detection system, to be determined as a function of microscope operating parameters. Results reveal the exact dependence that the effects of SE-ion recombination on signal formation have on reduced electric field intensity and time in ESEM. Furthermore, the model implicitly demonstrates that signal loss as a consequence of field retardation due to ion space charges, although obviously present, is not the foremost phenomenon causing streaking in images, as previously thought. Following that the generation and detection of gaseous scintillation and electro- luminescence produced via electron-gas molecule excitation reactions in ESEM is investigated. Here a novel gaseous scintillation detection (GSD) system is developed to efficiently detect photons produced. Images acquired using GSD are compared to those obtained using conventional GSED detection, and demonstrate that images rich in SE contrast can be achieved using such systems. A theoretical model is developed that describes the generation of photon signals by cascading SEs, high energy backscattered electrons (BSEs) and primary beam electrons (PEs). Photon amplification, or the total number of photons produced per sample emissive electron, is then investigated, and compared to conventional electronic amplification, over a wide range of microscope operating parameters, imaging gases and photon collection geometries. The main findings of the investigation revealed that detected electroluminescent signals exhibit larger SE signal-to-background levels than that of conventional electronic signals detected via GSED. Also, dragging the electron cascade towards the light pipe assemblage of GSD systems, or electrostatic focusing, dramatically increases photon collection efficiencies. The attainment of such an improvement being a direct consequence of increasing the `effective' solid angle for photon collection. Finally, in attempt to characterize the scintillating wavelengths arising from sample emissive SEs, PEs, BSEs, and their respective cascaded electrons, such that future photon filtering techniques can be employed to extract nominated GSD imaging signals, the emission spectra of commonly utilized electroluminescent gases in ESEM, such as argon (Ar) and nitrogen (N2), were collected and investigated. Spectra of Ar and N2 reveal several major emission lines that occur in the ultraviolet (UV) to near infrared (NIR) regions of the electromagnetic spectrum. The major photon emissions discovered in Ar are attributed to occur via atomic de-excitation transitions of neutral Ar (Ar I), whilst for N2, major emissions are attributed to be a consequence of second positive band vibrational de-excitation reactions. Major wavelength intensity versus gas pressure data, for both Ar and N2, illustrate that wavelength intensities increase with decreasing pressure. This phenomenon strongly suggesting that quenching effects and reductions in excitation mean free paths increase with imaging gas pressure.

It is becoming clear that mechanical stimuli are critical in regulating cell biology; however, the short-term structural response of a cell to mechanical forces remains relatively poorly understood. We mechanically stimulated cells expressing actin-EGFP with controlled forces (0-20nN) in order to investigate the cell’s structural response. Two clear force dependent responses were observed: a short-term local deformation of actin stress fibres and a long-term force-induced remodelling of stress fibres at cell edges, far from the point of contact. We were also able to quantify strain dynamics occurring along stress fibres. The cell exhibits complex heterogeneous negative and positive strain fluctuations along stress fibres, indicating localized dynamic contraction and expansion. A ~50% increase in myosin contractile activity is apparent following application of 20nN force. Directly visualizing force-propagation and stress fibre strain dynamics has revealed new information about the pathways involved in mechanotransduction which ultimately govern the downstream response of a cell.

It is becoming clear that mechanical stimuli are critical in regulating cell biology; however, the short-term structural response of a cell to mechanical forces remains relatively poorly understood. We mechanically stimulated cells expressing actin-EGFP with controlled forces (0-20nN) in order to investigate the cell’s structural response. Two clear force dependent responses were observed: a short-term local deformation of actin stress fibres and a long-term force-induced remodelling of stress fibres at cell edges, far from the point of contact. We were also able to quantify strain dynamics occurring along stress fibres. The cell exhibits complex heterogeneous negative and positive strain fluctuations along stress fibres, indicating localized dynamic contraction and expansion. A ~50% increase in myosin contractile activity is apparent following application of 20nN force. Directly visualizing force-propagation and stress fibre strain dynamics has revealed new information about the pathways involved in mechanotransduction which ultimately govern the downstream response of a cell.

There are several reasons for imaging a single, developing embryo from multiple view points. The embryo is a complex biomechanical system and morphogenesis movements in one region typically produce motions in adjacent areas. Multi-view imaging can be used to observe morphogenesis and gain a better understanding of normal and abnormal embryo development. The system would allow the embryo to be rotated to a specific vantage point so that a particular morphogenetic process may be observed clearly. Moreover, a multi-view system can be used to gather images to create an accurate three-dimensional reconstruction of the embryo for computer simulations. The scope of this thesis was to construct an apparatus that could capture multi-view images for these applications. A multi-view system for imaging live Drosophila melanogaster embryos, the first of its kind, is presented. Embryos for imaging are collected from genetically modified Drosophila stocks that contain a green fluorescing protein (GFP), which highlights only specific cell components. The embryos are mounted on a wire that is rotated under computer control to desired viewpoints in front of the objective of a custom-built confocal microscope. The optical components for the orizontallyaligned microscope were researched, selected and installed specifically for this multi-viewing apparatus. The multiple images of the stacks from each viewpoint are deconvolved and collaged so as to show all of the cells visible from that view. The process of rotating and capturing images can be repeated for many angles over the course of one hour. Experiments were conducted to verify the repeatability of the rotation mechanism and to determine the number of image slices required to produce a satisfactory image collage from each viewpoint. Additional testing was conducted to establish that the system could capture a complete 360° view of the embryo, and a time-lapse study was done to verify that a developing embryo could be imaged repeatedly from two separate angles during ventral furrow formation. An analysis of the effects of the imaging system on embryos in terms of photo-bleaching and viability is presented.

There are several reasons for imaging a single, developing embryo from multiple view points. The embryo is a complex biomechanical system and morphogenesis movements in one region typically produce motions in adjacent areas. Multi-view imaging can be used to observe morphogenesis and gain a better understanding of normal and abnormal embryo development. The system would allow the embryo to be rotated to a specific vantage point so that a particular morphogenetic process may be observed clearly. Moreover, a multi-view system can be used to gather images to create an accurate three-dimensional reconstruction of the embryo for computer simulations. The scope of this thesis was to construct an apparatus that could capture multi-view images for these applications. A multi-view system for imaging live Drosophila melanogaster embryos, the first of its kind, is presented. Embryos for imaging are collected from genetically modified Drosophila stocks that contain a green fluorescing protein (GFP), which highlights only specific cell components. The embryos are mounted on a wire that is rotated under computer control to desired viewpoints in front of the objective of a custom-built confocal microscope. The optical components for the orizontallyaligned microscope were researched, selected and installed specifically for this multi-viewing apparatus. The multiple images of the stacks from each viewpoint are deconvolved and collaged so as to show all of the cells visible from that view. The process of rotating and capturing images can be repeated for many angles over the course of one hour. Experiments were conducted to verify the repeatability of the rotation mechanism and to determine the number of image slices required to produce a satisfactory image collage from each viewpoint. Additional testing was conducted to establish that the system could capture a complete 360° view of the embryo, and a time-lapse study was done to verify that a developing embryo could be imaged repeatedly from two separate angles during ventral furrow formation. An analysis of the effects of the imaging system on embryos in terms of photo-bleaching and viability is presented.

<p>This research presents new calibration techniques for the characterization of atomic force microscopy cantilevers. Atomic force microscopy cantilevers are sensors that detect forces on the order of pico- to nanonewtons and displacements on the order of nano- to micrometers. Several calibration techniques exist with a variety of strengths and weaknesses. This research presents techniques that enable the noncontact calibration of the output sensor voltage-to-displacement sensitivity and the cantilever stiffness through the analysis of the unscaled thermal vibration of a cantilever in a liquid environment.</p><p>A noncontact stiffness calibration method is presented that identifies cantilever characteristics by fitting a dynamic model of the cantilever reaction to a thermal bath according to the fluctuation-dissipation theorem. The fitting algorithm incorporates an assumption of heavy fluid loading, which is present in liquid environments.</p><p>The use of the Lorentzian line function and a variable-slope noise model as an alternate approach to the thermal noise method was found to reduce the difference between calibrations preformed on the same cantilever in air and in water relative to existing techniques. This alternate approach was used in combination with the new stiffness calibration technique to determine the voltage-to-displacement sensitivity without requiring contact loading of the cantilever.</p><p>Additionally, computational techniques are presented in the investigation of alternate cantilever geometries, including V-shaped cantilevers and warped cantilevers. These techniques offer opportunities for future research to further reduce the uncertainty of atomic force microscopy calibration.</p> / Dissertation

In recent years, nanowires have become increasingly important due to their unique properties and applications. Thus, processes in the fabrication to nanostructures has come a focal point in research. In this research, a new method to fabricate nanowires has been developed. The new technique is called the Scanning Probe Alloying Nanolithography (SPAN). The SPAN was processed using an Atomic Force Microscope (AFM) in ambient environment. Firstly, an AFM probe was coated with gold (Au), and then slid on a silicon (Si) substrate. The contact-sliding motion generated a nanostructure on the substrate, instead of wear. Subsequently, careful examination was carried out at the scale relevant to an AFM probe, in terms of physical dimension and electrical conductivity. The measured conductivity value of the generated microstructures was found to be between the conductivity values of pure silicon and gold. Simple analysis indicated that the microstructures were formed due to frictional energy dispersed in the interface forming a bond to sustain mechanical wear. This research proves the feasibilities of tip-based nanomanufacturing. The SPAN process was developed to increase efficiency of the technique. This study also explored the possibility of the applications as a biosensor and a flexible device. This dissertation contains nine sections. The first section introduces backgrounds necessary to understand the subject matter. It reviews current status of the nanofabrication technologies. The basic concepts of AFM are also provided. The second section discusses the motivation and goals in detail. The third section covers the new technology, scanning probe alloying nanolithography (SPAN) to fabricate nanostructures. The fourth talks about characterization of nanostructures. Subsequently, the characterized nanostructures and their mechanical, chemical, and electrical properties are discussed in the fifth section. In the sixth section, the new process to form a nanostructure is evaluated and its mechanism is discussed. The seventh section discusses the feasibility of the nanostructures to be used in biosensors and flexible devices. The conclusion of the research is summarized in the seventh section.

Recent advances in using microscopes in ultracold atom experiment have allowed experimenters for the first time to directly observe and manipulate individual atoms in individual lattice sites. This technique enhances our capability to simulate strongly correlated systems such as Mott insulator and high temperature superconductivity. Currently, all ultracold atom experiments with high resolution imaging capability use bosonic atoms. In this thesis, I present our progress towards creating the fermionic version of the microscope experiment which is more suitable for simulating real condensed matter systems. Lithium is ideal due to the existence of both fermionic and bosonic isotopes, its light mass, which means faster experiment time scales that suppresses many sources of technical noise, and also due to the existence of a broad Feshbach resonance, which can be used to tune the inter-particle interaction strength over a wide range from attractive, non-interacting, and repulsive interactions. A high numerical aperture objective will be used to image and manipulate the atoms with single lattice site resolution. This setup should allow us to implement the Hubbard hamiltonian which could describe interesting quantum phases such as antiferromagnetism, d-wave superfluidity, and high temperature superconductivity. I will also discuss the feasibility of the Raman sideband cooling method for cooling the atoms during the imaging process. We have also developed a new electronic control system to control the sequence of the experiment. This electronic system is very scalable in order to keep up with the increasing complexity of atomic physics experiments. Furthermore, the system is also designed to be more precise in order to keep up with the faster time scale of lithium experiment. / Physics

This thesis seeks to characterize the size of the interaction volume in a sample subject to electric force microscope (EFM) probing. It discusses the historical relevance of the EFM and the experimental method used. It then discusses the modeling of the fields surrounding the grating sample with the equivalent charge model (ECM) where a tip or other rotationally symmetric conducting element is replaced by a series of point charges on the vertical axis that mimic the original fields. The results of the model were then compared to the experimental data as well as a model simulated using COMSOL, a finite element analysis package. The electrostatic model was found to have good agreement with the simulated and experimental results and was then used to estimate the volume of interaction and the lateral resolution of this technique. The volume of interaction was estimated at 6000 μm3 and the lateral resolution was estimated at 10 μm.

This master’s project report deals with the process development for patterning thesub-micron features using Deep-UV photolithography. Patterning of the sub-micronstructures in the resists UV26 and ZEP520A-7 has been demonstrated successfully. Using theKarl Süss-MJB4 DUV mask aligner, trenches of width down to 535 nm have been obtained.Good results have been obtained in these experiments considering the development time andthe exposure time, which are found to be shorter compared to previously published results.This provides a faster process and higher throughput. Experimental steps along with thefurther improvement areas are discussed.Equipment used include a Karl Süss-MJB4 DUV mask aligner, an optical microscope anda Scanning Electron Microscope (SEM).