Thesis (Ph.D.)--Victoria University (Melbourne, Vic.), 2009.

Many biological specimens are transparent and in weak intensity contrast, making it invisible using conventional bright field microscopes. Therefore, the phase-based optical microscopy techniques play important roles in the development of the modern biomedical science. Furthermore, the ability to achieve quantitative phase measurement of the tiny structures of biomedical specimens is of great importance for many biomedical applications. Thus, quantitative phase imaging becomes an important technique to measure the phase variations due to the difference of refractive index and geometric thickness of various structures and materials within the biomedical specimens. In this thesis, a spectral modulation interferometry (SMI) is developed to achieve quantitative phase imaging. In SMI, the phase and amplitude information will simultaneously be modulated onto the interference spectrum of the broadband light. Full-field phase images can be obtained by scanning along the orthogonal direction only. SMI incorporates the advantages of low coherence from broadband light source, high sensitivity from spectral domain interferometry and the high speed from the spectral modulation technique to achieve quantitative phase measurement with free of speckle, high temporal sensitivity (~0.1nm) and fast imaging rate. The principles of SMI system and programming as well as some important image processing methods will be discussed in detail. Besides, the quantitative phase measurement of the reflective object (USAF resolution target) and the transmitted biological objects (Peranema, human cheek cells) will be shown. / Master of Science

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.

This thesis presents the recent progress of diffusion measurements in nanoporous host systems by micro-imaging. Interference microscopy is applied as a powerful tool to record transient, intracrystalline concentration profiles of different sorbate species in the porous framework of two different zeolites, viz. ZSM-5 (MFI) and ZSM-58 (DDR). These profiles, yielding high temporal and spatial resolutions of about 10 s and 0.45 μm, follow the change of the refractive index of the host-guest system during uptake and release of certain guest molecules. With the thus accessible changes of concentration and particle fluxes, mass transport parameters, such as intracrystalline diffusivity and surface permeability, can be obtained by the use of the very fundamental equations on diffusion. Additionally, in two examples of never before performed types of experiments, further insights into challenging fields of host-guest interactions are provided: The well known phase transition in MFI type zeolites covering high benzene loadings is investigated in a single crystal study, allowing to follow the change of the sorbate phase in great detail. Furthermore, in DDR zeolites, a new way of data analysis facilitates to study the uptake and release of binary mixtures. Here, from the two-dimension profiles obtained by interference microscopy, the local concentrations of the sorbate species could be retrieved by using the so-called ideal adsorbed solution theory.

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.

Biological nanoparticles have enormous utility as well as potential adverse impacts in biotechnology, human health, and medicine. The physical and chemical properties of these nanoparticles have strong implications on their distribution, circulation, and clearance in vivo. Accurate morphological visualization and chemical characterization of nanoparticles by label-free (direct) optical microscopy would provide valuable insights into their natural and intrinsic properties. However, three major challenges related to label-free nanoparticle imaging must be overcome: (i) weak contrast due to exceptionally small size and low-refractive-index difference with the surrounding medium, (ii) inadequate spatial resolution to discern nanoscale features, and (iii) lack of chemical specificity. Advances in common-path interferometric microscopy have successfully overcome the weak contrast limitation and enabled direct detection of low-index biological nanoparticles down to single proteins. However, interferometric light microscopy does not overcome the diffraction limit, and studying the nanoparticle morphology at sub-wavelength spatial resolution remains a significant challenge. Moreover, chemical signature and composition are inaccessible in these interferometric optical measurements. This dissertation explores innovations in common-path interferometric microscopy to provide enhanced spatial resolution and chemical specificity in high-throughput imaging of individual nanoparticles. The dissertation research effort focuses on a particular modality of interferometric imaging, termed “single-particle interferometric reflectance (SPIR) microscopy”, that uses an oxide-coated silicon substrate for enhanced coherent detection of the weakly scattered light. We seek to advance three specific aspects of SPIR microscopy: sensitivity, spatial resolution, and chemical specificity. The first one is to enhance particle visibility via novel optical and computational methods that push optical detection sensitivity. The second one is to improve the lateral resolution beyond the system's classical limit by a new computational imaging method with an engineered illumination function that accesses high-resolution spatial information at the nanoscale. The last one is to extract a distinctive chemical signature by probing the mid-infrared absorption-induced photothermal effect. To realize these goals, we introduce new theoretical models and experimental concepts. This dissertation makes the following four major contributions in the wide-field common-path interferometric microscopy field: (1) formulating vectorial-optics based linear forward model that describes interferometric light scattering near planar interfaces in the quasi-static limit, (2) developing computationally efficient image reconstruction methods from defocus images to detect a single 25 nm dielectric nanoparticle, (3) developing asymmetric illumination based computational microscopy methods to achieve direct morphological visualization of nanoparticles at 150 nm, and (4) developing bond-selective interferometric microscopy to enable multispectral chemical imaging of sub-wavelength nanoparticles in the vibrational fingerprint region. Collectively, through these research projects, we demonstrate significant advancement in the wide-field common-path interferometric microscopy field to achieve high-resolution and accurate visualization and chemical characterization of a broad size range of individual biological nanoparticles with high sensitivity.

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.

This thesis presents the recent progress of diffusion measurements in nanoporous host systems by micro-imaging. Interference microscopy is applied as a powerful tool to record transient, intracrystalline concentration profiles of different sorbate species in the porous framework of two different zeolites, viz. ZSM-5 (MFI) and ZSM-58 (DDR). These profiles, yielding high temporal and spatial resolutions of about 10 s and 0.45 μm, follow the change of the refractive index of the host-guest system during uptake and release of certain guest molecules. With the thus accessible changes of concentration and particle fluxes, mass transport parameters, such as intracrystalline diffusivity and surface permeability, can be obtained by the use of the very fundamental equations on diffusion. Additionally, in two examples of never before performed types of experiments, further insights into challenging fields of host-guest interactions are provided: The well known phase transition in MFI type zeolites covering high benzene loadings is investigated in a single crystal study, allowing to follow the change of the sorbate phase in great detail. Furthermore, in DDR zeolites, a new way of data analysis facilitates to study the uptake and release of binary mixtures. Here, from the two-dimension profiles obtained by interference microscopy, the local concentrations of the sorbate species could be retrieved by using the so-called ideal adsorbed solution theory.

The master´s thesis deals with the study of optical properties of thin transparent layers on the organic materials (PPV, P3HT, TiO2, DPP) and especially with the determination of dispersion dependences of refractive index of prepared thin layers. In the theoretical part there are described principles of deposition thin layers of the analyzed materials and their properties. In addition, there are also described methods of optical properties measurements (optical and interference microscopy and ellipsometry). The combination of interference microscope with digital camera was used for determination of refractive index. The image analysis was used for the determination of parameters (with help of the software HarFA). The images of thin layers surfaces were analyzed from the side of the metal contact as well as from the side of glass. In conclusion, there are presented results of the refractive index of the thin layers obtained from the measured values.