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.

No description available.

The purpose of this study was to assess the utility of confocal microscopy to examine nuclear organization and chromosome pairing for intact Arabidopsis male meiocytes. The efficiency of the confocal technique was evaluated by analyzing wild-type nuclei throughout meiosis. Early-mid leptotene meiocytes demonstrated the presence of several propidium iodide stained signals within the nucleolus prior to the onset of chromosome pairing in zygotene. Pachytene chromosomes were completely paired and were traced to confirm the Arabidopsis karyotype. Additionally, the confocal technique was employed on meiotic mutants, ahp2 and spo11-2-2, to characterize their meiotic defects. Leptotene ahp2 meiocytes and zygotene meiocytes in both meiotic mutants appeared normal. In contrast, pachytene meiocytes in ahp2 and spo11-2-2 mutants demonstrated a wide-spread lack of paired chromosomes. Despite this general lack of pairing, a small amount of chromosome pairing was detected on the short arms of NOR-bearing chromosomes 2 and 4 in ahp2 and spo11-2-2 mutants.

The purpose of this study was to assess the utility of confocal microscopy to examine nuclear organization and chromosome pairing for intact Arabidopsis male meiocytes. The efficiency of the confocal technique was evaluated by analyzing wild-type nuclei throughout meiosis. Early-mid leptotene meiocytes demonstrated the presence of several propidium iodide stained signals within the nucleolus prior to the onset of chromosome pairing in zygotene. Pachytene chromosomes were completely paired and were traced to confirm the Arabidopsis karyotype. Additionally, the confocal technique was employed on meiotic mutants, ahp2 and spo11-2-2, to characterize their meiotic defects. Leptotene ahp2 meiocytes and zygotene meiocytes in both meiotic mutants appeared normal. In contrast, pachytene meiocytes in ahp2 and spo11-2-2 mutants demonstrated a wide-spread lack of paired chromosomes. Despite this general lack of pairing, a small amount of chromosome pairing was detected on the short arms of NOR-bearing chromosomes 2 and 4 in ahp2 and spo11-2-2 mutants.

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.

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.

Thesis (Ph. D.)--University of Hong Kong, 1991.

Le travail de thèse présenté dans ce manuscrit est consacré à l’étude de la statis-tique et de la dynamique d’ondes partiellement cohérentes se propageant dans un milieunon linéaire, la fibre optique. Les études effectuées durant ce travail de thèse se placentprincipalement dans le champ de la turbulence intégrable qui examine la propagationnon linéaire d’ondes partiellement cohérentes dans des systèmes physiques décrits par deséquations intégrables telles que l’équation de Schrödinger non linéaire à une dimension.Nous avons reproduit en optique une expérience déjà réalisée en hydrodynamique.Nous comparons ainsi les déviations de la statistique gaussienne résultant de la propaga-tion non linéaire d’ondes lumineuses dans une fibre optique et de vagues dans un canalunidirectionnel. Afin d’observer la dynamique des ondes partiellement cohérentes se propageant dans unefibre optique en régime de dispersion anormale, nous avons construit un microscope tem-porel qui a permis d’observer des structures cohérentes particulières présentant des pro-priétés de localisation dans l’espace et dans le temps similaires à celles des ondes scélérates.Finalement, nous avons étudié le régime de propagation très faiblement non linéaire. Lathéorie cinétique des ondes (appelée encore théorie de la Turbulence d’ondes) prédit quel’élargissement spectral ne dépend pas du signe de la dispersion et nous avons présentédans ce manuscrit la preuve expérimentale de cette hypothèse. / The work presented in this thesis is related to the statistical and dynamical propertiesof partially coherent waves propagating inside an optical fiber. Our work mainly enterswithin the field of Integrable Turbulence that deals with nonlinear partially coherentwaves described by integrable equations, such as the one-dimensional nonlinear Shcrödin-ger equation. We have reproduced an experiment in optics that has been done some years ago inhydrodynamics. We compare the statistics of optical waves propagating inside an opticalfiber to the the statistics of waves propagating inside a water tank. Moreover we have built a time microscope in order to observe the real-time evolution of partially coherent waves. The soliton-like structures that have been observed in our expe-riments have localization properties in space and time that are similar to those typifyingrogue waves found in the field of oceanography. We have also examined the weakly nonlinear regime that can be described by using the so-called wave turbulence (WT) theory. WT theory states that the spectral broadening insuch a weakly nonlinear regime does not depend on the sign of the second-order dispersioncoefficient. In this thesis, we presented an experimental result confirming this theoreticalprediction.

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.

A microscope was interfaced to a PDP-9 computer in order to develop techniques suitable for on-line classification of white blood cells. The computer visual-input system is composed of an image dissector optically coupled to a microscope used in a transmitted light mode. The position of the slide under the microscope and the fine focus control are controllable from the computer. A technique of auto-focusing was developed to efficiently focus the microscope under computer control. This algorithm is described in detail, followed by a discussion of the physical factors that affect the performance of this technique. A method of locating or isolating the leukocytes (white blood cells) is described next. A constant, referred to as the "contrast ratio' is used to extract the threshold for the nuclei of the leukocytes based on the average background intensity. Finally, contour tracing and curvature function extraction are used as a means of testing the system. A specific test is conducted to obtain a comparison of the system's efficiency as compared to that of "manual" techniques used by a technician. The system described here is not only suitable for automatic leukocyte classification, but could also be used for many other routine tests requiring the examination of microscopic cells. / Applied Science, Faculty of / Electrical and Computer Engineering, Department of / Graduate