Abstract: Nonlinear multicontrast microscopy is established in this study as an important tool for understanding biological structure and function of muscle cells. Second harmonic generation, third harmonic generation and multi-photon excitation fluorescence are acquired simultaneously in order to establish the origin of nonlinear signal generation in myocytes, and investigate myocyte structure and functionality during muscle contraction. Using structural cross-correlation image analysis, an algorithm developed specifically for this research, for the first time, third harmonic generation is shown to originate from the mitochondria in myocytes. The second harmonic, which is generated from the anisotropic bands of the sarcomeres, is further shown to be dependent on the crystalline order of the sarcomeres, thereby providing a potential diagnostic tool to evaluate disorder in muscle cells. The combination of the second and third harmonic provides complementary information that can be used to further elucidate the basic principles of muscle contraction. Time-lapse nonlinear microscopic imaging showed structural and functional dynamics in the myocytes. The second harmonic contrast revealed nonsynchronized nanocontractions of sarcomeres in relaxed, non-contracting, cardiomyocytes and Drosophila muscle samples, providing insight into the asynchronous behaviour of individual sarcomeres. Furthermore, macrocontracting samples were found to exhibit a synchronization of nanocontractions, providing new evidence for how muscles contract. Dynamic image correlation analysis, another algorithm developed specifically for this investigation, is used to reveal networks of mitochondria, which show fluctuations of multi-photon excitation fluorescence and third harmonic generation signals. The intensity fluctuations in the networks reveal both slow and fast dynamics; phase shifts of the slow dynamics between different networks are observed. Fast dynamics appear only in the inner networks, suggesting functional difference between interfibrillar and subsarcolemma mitochondria. The groundwork for studying bioenergetics of mitochondria in cardiomyocytes with nonlinear multimodal microscopy is fully developed in this work. The origin of the nonlinear signals and the development of the image analysis techniques provide a solid foundation to further study of muscle contractility and bioenergetics.

Abstract: Nonlinear multicontrast microscopy is established in this study as an important tool for understanding biological structure and function of muscle cells. Second harmonic generation, third harmonic generation and multi-photon excitation fluorescence are acquired simultaneously in order to establish the origin of nonlinear signal generation in myocytes, and investigate myocyte structure and functionality during muscle contraction. Using structural cross-correlation image analysis, an algorithm developed specifically for this research, for the first time, third harmonic generation is shown to originate from the mitochondria in myocytes. The second harmonic, which is generated from the anisotropic bands of the sarcomeres, is further shown to be dependent on the crystalline order of the sarcomeres, thereby providing a potential diagnostic tool to evaluate disorder in muscle cells. The combination of the second and third harmonic provides complementary information that can be used to further elucidate the basic principles of muscle contraction. Time-lapse nonlinear microscopic imaging showed structural and functional dynamics in the myocytes. The second harmonic contrast revealed nonsynchronized nanocontractions of sarcomeres in relaxed, non-contracting, cardiomyocytes and Drosophila muscle samples, providing insight into the asynchronous behaviour of individual sarcomeres. Furthermore, macrocontracting samples were found to exhibit a synchronization of nanocontractions, providing new evidence for how muscles contract. Dynamic image correlation analysis, another algorithm developed specifically for this investigation, is used to reveal networks of mitochondria, which show fluctuations of multi-photon excitation fluorescence and third harmonic generation signals. The intensity fluctuations in the networks reveal both slow and fast dynamics; phase shifts of the slow dynamics between different networks are observed. Fast dynamics appear only in the inner networks, suggesting functional difference between interfibrillar and subsarcolemma mitochondria. The groundwork for studying bioenergetics of mitochondria in cardiomyocytes with nonlinear multimodal microscopy is fully developed in this work. The origin of the nonlinear signals and the development of the image analysis techniques provide a solid foundation to further study of muscle contractility and bioenergetics.

Thesis (Ph. D.)--Materials science and engineering, Georgia Institute of Technology, 2005. / Stock, Stuart, Committee Chair ; Sanders, Thomas, Committee Member ; Snyder, Robert, Committee Member ; Johnson, Steven, Committee Member ; Wilkinson, Angus, Committee Member. Vita. Includes bibliographical references.

This thesis proposed and demonstrated a new all-polymer based fabrication process for an all-polymer flexible and parallel optical interconnect cable having a vertical light coupler, which can not only cut down the cost by eliminating metallization process for alignment but also facilitate both in production and application. Throughout the process, polyimide was used as the substrate, coated by Epoclad as claddings, then AP2210B and WPR 5100 were used to fabricate waveguides and 45 degree mirror couplers, respectively. In addition, precisely aligned mirror couplers to waveguides are fabricated by using polymer-based, non-metallic, and transparent alignment marks. Conventional and metallic alignment marks are easy to be detected by camera, when a layer of high reflective material, generally Cr metal, is patterned. However, transparent polymer material is used in this process, as alignment marks made of it which are actually buried phase structures. Therefore, it is hardly to be observed by conventional microscopy system. Hence, to increase the contrast of the alignment marks, I proposed and tested a feature specific alignment camera system for which the shape and depth of the alignment marks are optimized for phase-based imaging, such as phase contrast and Schlieren imaging. The results showed a contrast enhancement of alignment marks image compared to that of a conventional microscopy system. By using the fabrication and alignment process, process for adding waveguides to the structure is identified by using the polymer based alignment marks on the WPR 5100 layer. Mask was made by etch down process using fused silica wafer plate, Cr and AZ 3312 photoresist. At last, the developed and proposed process provides means of all-polymer based fabrication process for a flexible and parallel optical interconnect.

This dissertation presents techniques of three-dimensional microscopy. First, an economical method of microscopic image formation that employs a raster-scanning laser beam focused on a sample, while non-imaging detector receives the scattered light is presented. The images produced by this method are analogous to the scanning electron microscopy with visible effects of shadowing and reflection. Compared to a conventional wide-field imaging system, the system allows for a greater flexibility, as the variety of optical detectors, such as PMT and position-sensitive quadrant photodiode can be used to acquire images. The system demonstrates a simple, low-cost method of achieving the resolution on the order of a micron. A further gain in terms of resolution and the depth of focus by using Bessel rather than Gaussian beams is discussed. Then, a phase-imaging technique to quantitatively study the three-dimensional structure of reflective and transmissive microscopic samples is presented. The method, based on the simultaneous dual-wavelength digital holography, allows for higher axial range at which the unambiguous phase imaging can be performed. The technique is capable of nanometer axial resolution. The noise level, which increases as a result of using two wavelengths, is then reduced to the level of a single wavelength. The method compares favorably to software unwrapping, as the technique does not produce non-existent phase steps. Curvature mismatch between the reference and object beams is numerically compensated. The 3D images of porous coal samples and SKOV-3 ovarian cancer cells are presented.

This dissertation presents a quantitative phase imaging microscopy technique that combines phase-shifting interferometry with multi-wavelength optical phase unwrapping. The technique consists of a Michelson-type interferometer illuminated with any of three types of light sources; light emitting diodes, laser diodes and a ring dye laser. Interference images are obtained by using a 4-frame phase shifting method, and are combined to calculate the phase of the object surface. The 2π ambiguities are removed by repeating the experiment combining two and three different wavelengths, which yields phase images of effective wavelength much longer than the original. The resulting image is a profile of the object surface with a height resolution of several nanometers and range of several microns. To our knowledge, this is the first time that a three wavelength optical phase unwrapping method with no amplified phase noise has been presented for fullframe phase images. The results presented here are divided into three main categories based on the source of illumination; light emitting diodes, laser diodes and a ring dye laser. Results for both two-wavelength optical unwrapping and three-wavelength optical unwrapping techniques are demonstrated. The interferographic images using broadband sources such as light emitting diodes are significantly less affected by coherent noise compared to images obtained using lasers. Our results show that the three wavelength optical phase unwrapping can also be effectively applied to unwrap phase images obtained using coherent light sources such as lasers and laser diodes, without amplifying phase noise in the final phase image. We have successfully shown that our multi-wavelength phase-shifting technique extends the range free of 2π ambiguities in the phase map without using conventional computation intensive phase unwrapping methods. This phase imaging technique can be used to measure physical thickness or height of both biological and other microscopic samples, with nanometer axial resolution. An added advantage of the multi-wavelength optical phase unwrapping technique is that the beat wavelength can be tailored to match height variations of specific samples.

Optical phase microscopy provides a view of objects that have minimal to no effect on the detected intensity of light that are unobservable by standard microscopy techniques. Since its inception just over 60 years ago that gave us a vision to an unseen world and earned Frits Zernike the Nobel prize in physics in 1953, phase microscopy has evolved to find various applications in biological cell imaging, crystallography, semiconductor failure analysis, and more. Two common and commercially available techniques are phase contrast and differential interference contrast (DIC). In phase contrast method, a large portion of the unscattered light that accounts for the majority of the light passing unaffected through a transparent medium is blocked to allow the scattered light due to the object to be observed with higher contrast. DIC is a self-referenced interferometer that transduces phase variation to intensity variation. While being established as fundamental tools in many scientific and engineering disciplines, the traditional implementation of these techniques lacks the ability to provide the means for quantitative and repeatable measurement without an extensive and cumbersome calibration. The rapidly growing fields in modern biology meteorology and nano-technology have emphasized the demand for a more robust and convenient quantitative phase microscopy. The recent emergence of modern optical devices such as high resolution programmable spatial light modulators (SLM) has enabled a multitude of research activities over the past decade to reinvent phase microscopy in unconventional ways. This work is concerned with an implementation of a DIC microscope containing a 4-f system at its core with a programmable SLM placed at the frequency plane of the imaging system that allows for employing Fourier pair transforms for wavefront manipulation. This configuration of microscope provides a convenient way to perform both wavefront shearing with quantifiable arbitrary shear amount and direction as well as phase stepping interferometry by programming the SLM with a series of numerically generated patterns and digitally capturing interferograms for each step which are then used to calculate the objects phase gradient map. Wavefront shearing is performed by generating a pattern for the SLM where two phase ramp patterns with opposite slopes are interleaved through a random selection process with uniform distribution in order to mimic the simultaneous presence of the ramps on the same plane. The theoretical treatment accompanied by simulations and experimental results and discussion are presented in this work.

The objective of this research project was to evaluate the mechanical, morphological, and permeability properties of electrospun blends of polydioxanone and poly(lactic acid) for application as vascular grafts. Mechanical analysis was performed by uniaxial tensile testing to examine the peak load, peak stress, elastic modulus, and strain at break of the fibrous materials. The morphological characteristics of the polymer blends were analyzed using phase contrast microscopy, scanning electron microscopy, and image analysis software. Scanning electron microscopy and image analysis software were used to assess fiber diameter and pore size of electrospun scaffolds. Scaffold permeability measurements were also used to calculate fiber diameter and pore size, and the values were compared to those obtained using image analysis. The material property results acquired from the research suggest that the electrospun polymer blends have potential for use in vascular graft applications.

In this dissertation, improved techniques in digital holography, that have produced high-resolution, high-fidelity images, are discussed. In particular, the angular spectrum method of calculating holographic optical field is noted to have several advantages over the more commonly used Fresnel transformation or Huygens convolution method. It is observed that spurious noise and interference components can be tightly controlled through the analysis and filtering of the angular spectrum. In the angular spectrum method, the reconstruction distance does not have a lower limit, and the off-axis angle between the object and reference waves can be lower than that of the Fresnel requirement, while still allowing the zero-order background to be cleanly separated. Holographic phase images are largely immune from the coherent noise commonly found in amplitude images. With the use of a miniature pulsed laser, the resulting images have 0.5um diffraction-limited lateral resolution and the phase profile is accurate to about several nanometers of optical path length. Samples such as ovarian cancer cells (SKOV-3) and mouse-embryo fibroblast cells have been imaged. These images display intra-cellular and intra-nuclear organelles with clarity and quantitative accuracy. This technique clearly exceeds currently available methods in phase-contrast opticalmicroscopy in both resolution and detail and provides a new modality for imaging morphology of cellular and intracellular structures that is not currently available. Furthermore, we also demonstrate that phase imaging digital holographic movies provide a novel method of non-invasive quantitative viewing of living cells and other objects. This technique is shown to have significant advantages over conventional microscopy.

Αμίαντος (asbestos) θεωρείται μια ομάδα ινωδών ορυκτών τα οποία παρουσιάζουν μια σειρά ιδιοτήτων που τον έχουν καταστήσει ιδανική λύση ως κατασκευαστικό και μονωτικό υλικό εδώ και δεκαετίες. Αμιαντούχα υλικά σήμερα βρίσκονται σχεδόν παντού. Εξαιτίας της σύνδεσής του με βλάβες στην ανθρώπινη υγεία (αμιάντωση, μεσοθηλίωμα, καρκίνος πνεύμονα) έχει απαγορευθεί η εμπορία και η χρήση του στην Ελλάδα από 1-1-2005. Οι κίνδυνοι περιορίζονται πλέον κατά την εργασία αφαίρεσης αμιάντου και στις συντηρήσεις και ανακαινίσεις κτιρίων, πλοίων και τρένων. Η νομοθεσία που σχετίζεται με τον αμίαντο και την προστασία εργαζομένων, καταναλωτικού κοινού και περιβάλλοντος εξελίσσεται, επακόλουθο των εξελίξεων στην ιατρική και στην τεχνολογία. Η μέθοδος μικροσκοπίας αντίθεσης φάσης χρησιμοποιήθηκε για τον υπολογισμό της συγκέντρωσης ινών στο χώρο εργασίας λατομείου. Τα αποτελέσματα των αναλύσεων μπορούν μελλοντικά να αξιοποιηθούν κατάλληλα, όπως γενικά και η χρήση της μεθόδου σε ανάλογες περιπτώσεις. / -