Micro Phase Shifting

We consider the problem of shape recovery for real world scenes, where a variety of global illumination (inter-reflections, subsurface scattering, etc.) and illumination defocus effects are present. These effects introduce systematic and often significant errors in the recovered shape. We introduce a structured light technique called Micro Phase Shifting, which overcomes these problems. The key idea is to project sinusoidal patterns with frequencies limited to a narrow, high-frequency band. These patterns produce a set of images over which global illumination and defocus effects remain constant for each point in the scene. This enables high quality reconstructions of scenes which have traditionally been considered hard, using only a small number of images. We also derive theoretical lower bounds on the number of input images needed for phase shifting and show that Micro PS achieves the bound

Super-resolution and super-localization microscopy: a novel tool for imaging chemical and biological processes

Optical microscopy imaging of single molecules and single particles is an essential method for studying fundamental biological and chemical processes at the molecular and nanometer scale. The best spatial resolution (~ λ/2) achievable in traditional optical microscopy is governed by the diffraction of light. However, single molecule-based super-localization and super-resolution microscopy imaging techniques have emerged in the past decade. Individual molecules can be localized with nanometer scale accuracy and precision for studying of biological and chemical processes. The obtained spatial resolution for plant cell imaging is not yet as good as that achieved in mammalian cell imaging. Numerous technical challenges, including the generally high fluorescence background due to significant autofluorescence of endogenous components, and the presence of the cell wall (\u3e 250 nm thickness) limit the potential of super-resolution imaging in studying the cellular processes in plants. Here variable-angle epi-fluorescence microscopy (VAEM) was combined with localization based super-resolution imaging, direct stochastic optical reconstruction microscopy (dSTORM), to demonstrate imaging of cortical microtubule (CMT) network in the Arabidopsis thaliana root cells with 20 – 40 nm spatial resolution for the first time. With such high spatial resolution, the subcellular organizations of CMTs within single cells, and different cells in many regions along the root, were analyzed quantitatively. Nearly all of these technical advances in super-localization and super-resolution microscopy imaging were originally developed for biological studies. More recently, however, efforts in super-resolution chemical imaging started to gain momentum. New discoveries that were previously unattainable with conventional diffraction-limited techniques have been made, such as a) super-resolution mapping of catalytic reactions on single nanocatalysts and b) mechanistic insight into protein ion-exchange adsorptive separations. Furthermore, single molecules and single particles were localized with nanometer precision for resolving the dynamic behavior of single molecules in porous materials. This work uncovered the heterogeneous properties of the pore structures. In this dissertation, the coupling of molecular transport and catalytic reaction at the single molecule and single particle level in multilayer mesoporous nanocatalysts was elucidated. Most previous studies dealt with these two important phenomena separately. A fluorogenic oxidation reaction of non-fluorescent amplex red to highly fluorescent resorufin was tested. The diffusion behavior of single resorufin molecules in aligned nanopores was studied using total internal reflection fluorescence microscopy (TIRFM). To fully understand the working mechanisms of biological processes such as stepping of motor proteins requires resolving both the translational movement and the rotational motions of biological molecules or molecular complexes. Nanoparticle optical probes have been widely used to study biological processes such as membrane diffusion, endocytosis, and so on. The greatly enhanced absorption and scattering cross sections at the surface plasmon resonance (SPR) wavelength make nanoparticles an ideal probe for high precision tracking. Furthermore, gold nanorods (AuNRs) were used for resolving orientation changes in all three dimensions. The translational and rotational motions of AuNRs in glycerol solutions were tracked with fast imaging rate up to 500 frames per second (fps) in reflected light sheet microscopy (RLSM). The effect of imaging rates on resolving details of single AuNR motions was studied

Evaluation of yarn characteristics using computer vision and image processing

Irregularity, hairiness and twist are among the most important characteristics that define yarn quality. This thesis describes computer vision and image processing techniques developed to evaluate these characteristics. The optical and electronic aspects such as the illumination, lens parameters and aberrations play crucial role on the quality of yam images and on the overall performance of image processing. The depth of field limitation being the most important restraint in yam imaging as well as image distortion in line scan cameras arising from digitisation and yam movement are modelled mathematically and verified through experiments both for front-lit and back-lit illuminations. Various light sources and arrangements are tested and relative advantages and disadvantages are discussed based on the image quality. Known problems in defining the hair-core boundaries and determining the total hairiness from yam images are addressed and image enhancement and processing algorithms developed to overcome these problems are explained. A method to simulate various yam scanning resolution conditions is described. Using this method, the minimum scanning resolution limits to measure the hairiness and irregularity are investigated. [Continues.

Depth from Defocus Technique: A Simple Calibration-Free Approach for Dispersion Size Measurement

Dispersed particle size measurement is crucial in a variety of applications, be it in the sizing of spray droplets, tracking of particulate matter in multiphase flows, or the detection of target markers in machine vision systems. Further to sizing, such systems are characterised by extracting quantitative information like spatial position and associated velocity of the dispersed phase particles. In the present study we propose an imaging based volumetric measurement approach for estimating the size and position of spherically dispersed particles. The approach builds on the 'Depth from Defocus' (DFD) technique using a single camera approach. The simple optical configuration, consisting of a shadowgraph setup and a straightforward calibration procedure, makes this method readily deployable and accessible for broader applications

Quantitative Phase Imaging and Reconstruction For Biological Applications

Ph.DDOCTOR OF PHILOSOPH

New quantitative phase imaging modalities on standard microscope platforms

Three new reconstruction methods for quantitative phase imaging, including two interrelated two-dimensional methods, called multifilter phase imaging with partially coherent light and phase optical transfer function recovery, which lead to a third three-dimensional method, called tomographic deconvolution phase microscopy, were developed in response to a growing need among biomedical end users for solutions which can be integrated on standard microscope platforms. The performance of these new methods were evaluated using modelling and simulation as well as experimentation with known test cases. In addition to the development of new methods, existing methods for quantitative phase imaging were applied to characterize the effects of manufacturing, cleaving, and fusion splicing in large-mode-area erbium- and ytterbium-doped optical fibers.Ph.D

Large Field of View Electron Ptychography

Electron ptychography can overcome the limits of the conventional electron microscopy in terms of both resolution and phase quantitative measurements. There are two ways to implement ptychography with electrons. One employs a focused probe and the other uses a large probe. The advantage of focused probe electron ptychography is allowing to analyse spectrum while collecting the data. The biggest advantage of large probe electron ptychography is much larger field of view with the same scanning positions. In this thesis, we investigate the applications of the large probe ptychography in three modes, which are a transmission electron microscope in the selected area diffraction mode (SAD ptychography), a scanning electron microscope in the transmission mode (SEM ptychography), and a scanning transmission electron microscope (STEM ptychography). The thesis includes the detailed experimental procedures to collect ptychographic data in the three modes, as well as the investigation and evaluation of the experimental parameters. It presents extensive experimental data and results, which includes the decomposition of a partially coherent electron source via the modal decomposition ptychography with the SAD ptychography, the improvement of the delocalization issue with the SEM ptychography, and the atomic resolution reconstruction with the STEM ptychography. The challenges of the implementation and the reconstruction of ptychography in the three modes are also discussed. The main achievement of the thesis is the modal decomposition of matter wave, which has never been done before