Eliminating Unwanted Far-Field Excitation in Objective-Type TIRF. Part I. Identifying Sources of Nonevanescent Excitation Light

AbstractTotal internal reflection fluorescence microscopy (TIRFM) achieves subdiffraction axial sectioning by confining fluorophore excitation to a thin layer close to the cell/substrate boundary. However, it is often unknown how thin this light sheet actually is. Particularly in objective-type TIRFM, large deviations from the exponential intensity decay expected for pure evanescence have been reported. Nonevanescent excitation light diminishes the optical sectioning effect, reduces contrast, and renders TIRFM-image quantification uncertain. To identify the sources of this unwanted fluorescence excitation in deeper sample layers, we here combine azimuthal and polar beam scanning (spinning TIRF), atomic force microscopy, and wavefront analysis of beams passing through the objective periphery. Using a variety of intracellular fluorescent labels as well as negative staining experiments to measure cell-induced scattering, we find that azimuthal beam spinning produces TIRFM images that more accurately portray the real fluorophore distribution, but these images are still hampered by far-field excitation. Furthermore, although clearly measureable, cell-induced scattering is not the dominant source of far-field excitation light in objective-type TIRF, at least for most types of weakly scattering cells. It is the microscope illumination optical path that produces a large cell- and beam-angle invariant stray excitation that is insensitive to beam scanning. This instrument-induced glare is produced far from the sample plane, inside the microscope illumination optical path. We identify stray reflections and high-numerical aperture aberrations of the TIRF objective as one important source. This work is accompanied by a companion paper (Pt.2/2)

Doctor of Philosophy

dissertationTreatment and management of heart disease is challenging due to the heart's limited ability to self-repair. Although current approaches to manage heart disease, such as pharmacotherapy, medical devices, lifestyle changes, and heart transplantation, have improved and extended the quality of life for millions of individuals, they have inherent shortcomings. Future strategies to manage heart disease will likely be based upon a combination of biological and engineering approaches through cell therapy and tissue engineering strategies, both of which have the potential to regenerate the myocardium and improve cardiac function. However, a key hurdle in applying biological approaches is our limited ability to produce reliable tissue to study disease progression and tissue development, therapeutic intervention, drug discovery, or tissue replacement. Establishing hallmarks of the native myocardium in engineered cardiac tissue is a central goal and appears to be required for creating functional tissue that can serve as a surrogate for in vitro testing or the eventual replacement of diseased or injured myocardium. The objective of this research was to apply an engineering approach to develop tools and methods to produce engineered cardiac tissue and characterize both native and engineered cardiac tissue. Three phases of research included: 1) the development and utilization of a framework to characterize microstructure in living cardiac tissue using confocal microscopy and local dye delivery, 2) the development a next-generation bioreactor capable of continuously monitoring force-displacement in engineered tissue, and 3) the application of confocal imaging and image analysis to quantitatively describe features of the native myocardium, focusing on myocyte geometry and spatial distribution of a major gap junction protein connexin-43, in both engineered tissue and native tissue

Cellular 3D-reconstruction and analysis in the human cerebral cortex using automatic serial sections

Techniques involving three-dimensional (3D) tissue structure reconstruction and analysis provide a better understanding of changes in molecules and function. We have developed AutoCUTS-LM, an automated system that allows the latest advances in 3D tissue reconstruction and cellular analysis developments using light microscopy on various tissues, including archived tissue. The workflow in this paper involved advanced tissue sampling methods of the human cerebral cortex, an automated serial section collection system, digital tissue library, cell detection using convolution neural network, 3D cell reconstruction, and advanced analysis. Our results demonstrated the detailed structure of pyramidal cells (number, volume, diameter, sphericity and orientation) and their 3D spatial organization are arranged in a columnar structure. The pipeline of these combined techniques provides a detailed analysis of tissues and cells in biology and pathology

Using strain field mining to reveal the spatial distributions of tensile, fatigue, and fracture damage accumulation in paper

The most common nonwoven fiber composite material, paper, has a porous, heterogeneous fiber network structure and complicated mechanical properties. The mechanical properties of commercial, machine made papers are orthotropic and are sensitive to loading rate, moisture content, and temperature. Thus, defining the constitutive relationship of paper has remained as a challenge due to the stochastic nature of the structure and countless variables that affect the mechanics of paper. Moreover, the technology to non-destructively characterize the three-dimensional network topography at the fiber length scale is not readily available. This presents a critical barrier to establishing the structure-property relationships of paper. Here, I approached the problem with a fundamentally different strategy and used the structure of the strain fields as a proxy for the network topography. The strain fields of paper from tensile, fatigue, tearing experiments revealed new information about each damage mechanisms. During the tensile deformation, the interplay between the axial and the transverse motions in the fiber network resulted in specimen-orientation-dependent (MD and CD) parameters such as Poisson's ratio, hot spot length scales, and the degree of nonaffinity, D. These metrics were direct manifestation of the anisotropic fiber network in paper. Next, I used strain field mining to track the fatigue crack lengths and quantified crack growth rates during cyclic and constant loading conditions. The fracture profiles and the crack growth rates revealed that there was a unique fatigue damage mechanism in paper which induced the fiber fracture rather than the fiber pull-out. Moreover, I found that the pre-applied creep damage in paper can significantly reduce the fatigue crack growth rate and extend paper's high cycle fatigue life. Lastly, from the strain fields of tearing specimens, I was able to characterize paper's crack tip process zone and the zone of active plasticity (ZAP) whose shape depended on the orientation of the fiber network. Although paper has a completely different structure and failure mechanism from metals, I found that tearing of paper also followed a steady-state process, which was previously observed in thin sheet aluminum foils.Ph.D

AUTOMATIC ERROR DETECTION AND CORRECTION IN LASER METAL WIRE DEPOSITION - AN ADDITIVE MANUFACTURING TECHNOLOGY

Additive manufacturing (AM) technology involves building three-dimensional objects by adding material layer-upon-layer under computer control. Metal additive manufacturing offers new possibilities, not only in design, but also in the choice of materials. However, the additive process remains at a lower maturity level compared to the conventional subtractive processes such as milling, drilling and machining among others. Scientifically, there is a safety concern relating to the accuracy of the AM process, how printed products will perform over time and the consistency of their quality. Process accuracy and eventual part quality is compromised due to errors introduced by each of the building steps in the process.Laser metal deposition with wire (LMD-w) is an additive manufacturing technology that involves feeding metal wire through a nozzle and melting the wire with a high-power laser. The technology is being largely researched for use in the aerospace industry to fabricate large aircraft components. With efficient process control, i.e. sensing, processing, and feedback correction of errors, the LMD-w technology has the potential to change the course of manufacturing. However, a prominent limitation in LMD-w is the difficulty in controlling the process.This work proposes a method for detecting surface geometry errors in a deposited layer in the LMD-w process via laser height scanning and high-speed image processing. The controlled process is simplified into a linear system. The aim is to develop an effective sensing and correction module that automatically detects irregularities in each layer before proceeding to subsequent layers, which will reduce part porosity and improve inter-layer bond integrity

Quantitative Phase Imaging and Reconstruction For Biological Applications

Ph.DDOCTOR OF PHILOSOPH

Computational Depth-resolved Imaging and Metrology

In this thesis, the main research challenge boils down to extracting 3D spatial information of an object from 2D measurements using light. Our goal is to achieve depth-resolved tomographic imaging of transparent or semi-transparent 3D objects, and to perform topography characterization of rough surfaces. The essential tool we used is computational imaging, where depending on the experimental scheme, often indirect measurements are taken, and tailored algorithms are employed to perform image reconstructions. The computational imaging approach enables us to relax the hardware requirement of an imaging system, which is essential when using light in the EUV and x-ray regimes, where high-quality optics are not readily available. In this thesis, visible and infrared light sources are used, where computational imaging also offers several advantages. First of all, it often leads to a simple, flexible imaging system with low cost. In the case of a lensless configuration, where no lenses are involved in the final image-forming stage between the object and the detector, aberration-free image reconstructions can be obtained. More importantly, computational imaging provides quantitative reconstructions of scalar electric fields, enabling phase imaging, numerical refocus, as well as 3D imaging

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