This thesis describes the design, fabrication, and characterization of the Implantable Fluorescence Imager (IFI): a camera chip with a needle-like form factor designed for imaging neuronal activity in the deep brain. It is fabricated with a complementary metal oxide semiconductor (CMOS) process, allowing for hundreds or thousands of single- photon-sensitive photodetectors to be densely packed onto a device width comparable to a single-channel fiber optic cannula (~100 μm). The IFI uses a combination of spectral and temporal filters as a fluorescence emission filter, and per-pixel Talbot gratings for 3D light-field imaging. The IFI has the potential to overcome the imaging depth limit of multi-photon microscopes imposed by the scattering and absorption of photons in brain tissue, and the resolution limit of noninvasive imaging techniques, such as functional magnetic resonance imaging and photoacoustic imaging. It competes with graded index lens-based miniaturized microscopes in imaging depth, but offers several comparative advantages. First, its cross sectional area is at least an order of magnitude smaller for an equal field of view. Second, the distribution of pixels along its entire length allows the study of multi- layer or multi-region dynamics. Finally, the scalability advantage of silicon integrated circuit technology in system miniaturization and data bandwidth may allow thousands of such imaging shanks to be simultaneously deployed for large-scale volumetric recording.

As the major component of the plant cell wall, cellulose is produced by all plant species at an annual rate of over a hundred billion tonnes, making it the most abundant biopolymer on Earth. The hierarchical assembly of cellulose glucan chains into crystalline fibrils, bundles and higher-order networks endows cellulose with its high mechanical strength, but makes it challenging to breakdown and produce cellulose-based nanomaterials and renewable biofuels. In order to fully leverage the potential of cellulose as a sustainable resource, it is important to study the supramolecular structure and hydrolysis of this biomaterial from the nano- to the microscale. In this thesis, we develop new chemical strategies for fluorescently labeling cellulose and employ advanced imaging techniques to study its supramolecular structure at the singlefibril level. The developed labeling method provides a simple and efficient route for fluorescently tagging cellulose nanomaterials with commercially available dyes, yielding high degrees of labeling without altering the native properties of the nanocelluloses. This allowed the preparation of samples that were optimal for super-resolution fluorescence microscopy (SRFM), which was used to provide for the first time, a direct visualization of periodic disorder along the crystalline structure of individual cellulose fibrils. The alternating disordered and crystalline structure observed in SFRM was corroborated with time-lapsed acid hydrolysis experiments to propose a mechanism for the acid hydrolysis of cellulose fibrils. To gain insight on the ultrastructural origin of these regions, we applied a correlative super-resolution light and electron microscopy (SR-CLEM) workflow and observed that the disordered regions were associated nanostructural defects present along cellulose fibrils. Overall, the findings presented in this work provide significant advancements in our understanding of the hierarchical structure and depolymerization of cellulose, which will be useful for the development of new and efficient ways of breaking down this polymer for the production of renewable nanomaterials and bio-based products like biofuels and bioplastics. / Thesis / Doctor of Philosophy (PhD) / In this dissertation, we have studied in unprecedented detail the structure of cellulose – a polymer that is found in every plant. As the main structural component of the plant cell wall, cellulose endows trees with their strength and resilience while storing sunlight energy in its chemical bonds. Since plant biomass represents eighty percent of all living matter on Earth, cellulose is an abundant resource that can be used to produce sustainable and environmentally benign nanomaterials and bioproducts, like biofuels and bioplastics. Our ability to use cellulose as a renewable source of structural materials and energy is intimately linked to our capacity to break apart its tight structural packing. Deconstructing cellulose into various forms demands that we understand the multi-level organization of its structure and the susceptible regions within it. To gain this information, in this thesis we develop new labeling methods and apply state-of-the-art microscopy tools to directly visualize the arrangement of cellulose fibrils at the nanoscale (comparable to 1/10,000 the width of a human hair) and study their breakdown by acid treatment. The findings presented in this work furthers our fundamental understanding of the natural structure of cellulose, which has important implications on the development of industrial strategies to break down this abundant and renewable biomaterial.

This thesis is intended as an introductory lab manual on the experimental methods using fluorescence microscopy in modern radiobiology research. It is written for those who are unfamiliar with biology research. It first covers the proper use of laboratory equipment and growth of cell cultures in the lab. Subsequent chapters provide overviews of relevant modern experimental techniques for the quantification of radiation induced DNA damage in cells, and detailed protocols for performing these procedures. Techniques covered include immunostaining with fluorescent antibodies, the comet assay, and plasmid DNA transfections. Results of some straightforward experiments using these techniques are presented.

In the field of cell biology, there is an increasing use of time-lapse data to understand cellular function. Using automated microscopes, large numbers of images can be acquired, delivering videos of cell samples over time. Analysing the images manually is extremely time consuming as there are typically thousands of individual images in any given sequence. Additionally, decisions made by those analysing the images, e.g. labelling a mitotic phase (one of a set of distinct sequential stages of cell division) can be subjective, especially around transition boundaries between phases, leading to inconsistencies in the annotation. There is therefore a need for tools which facilitate automated high-throughput analysis. In this thesis we develop systems to automatically detect, track and analyse sub-cellular structures in image sequences to address biological research needs in three areas: (i) Mitotic phase labelling, (ii) Mitotic defect detection, and (iii) Cell volume estimation. We begin by presenting a system for automated segmentation and mitotic phase labelling using temporal models. This work takes the novel approach of using temporal features evaluated over the whole of the mitotic phases rather than over single frames, thereby capturing the distinctive behaviour over the phases. We compare and contrast three different temporal models: Dynamic Time Warping, Hidden Markov Models, and Semi Markov Models. A new loss function is proposed for the Semi Markov model to make it more robust to inconsistencies in data annotation near transition boundaries. We then present an approach for detecting subtle chromosome segregation errors in mitosis in embryonic stem cells, targeting two cases: misaligned chromosomes in a metaphase cell, and lagging chromosomes between anaphase cells. We additionally explore an unsupervised approach to detect unusual mitotic occurrences and test its applicability to detecting misaligned metaphase chromosomes. Finally, we describe a fully automated method, suited to high-throughput analysis, for estimating the volume of spherical mitotic cells based on a learned membrane classifier and a circular Hough transform. We also describe how it is being used further in biological research.

Fluorescence microscopy is an important tool for biological research. However, the resolution of a standard fluorescence microscope is limited by diffraction, which makes it difficult to observe small details of a specimen’s structure. We have developed two fluorescence microscopy methods that achieve resolution beyond the classical diffraction limit. The first method represents an extension of localisation microscopy. We used nonnegative matrix factorisation (NMF) to model a noisy dataset of highly overlapping fluorophores with intermittent intensities. We can recover images of individual sources from the optimised model, despite their high mutual overlap in the original dataset. This allows us to consider blinking quantum dots as bright and stable fluorophores for localisation microscopy. Moreover, NMF allows recovery of sources each having a unique shape. Such a situation can arise, for example, when the sources are located in different focal planes, and NMF can potentially be used for three dimensional superresolution imaging. We discuss the practical aspects of applying NMF to real datasets, and show super-resolution images of biological samples labelled with quantum dots. It should be noted that this technique can be performed on any wide-field epifluorescence microscope equipped with a camera, which makes this super-resolution method very accessible to a wide scientific community. The second optical microscopy method we discuss in this thesis is a member of the growing family of structured illumination techniques. Our main goal is to apply structured illumination to thick fluorescent samples generating a large out-of-focus background. The out-of-focus fluorescence background degrades the illumination pattern, and the reconstructed images suffer from the influence of noise. We present a combination of structured illumination microscopy and line scanning. This technique reduces the out-of-focus fluorescence background, which improves the quality of the illumination pattern and therefore facilitates reconstruction. We present super-resolution, optically sectioned images of a thick fluorescent sample, revealing details of the specimen’s inner structure. In addition, in this thesis we also discuss a theoretical resolution limit for noisy and pixelated data. We correct a previously published expression for the so-called fundamental resolution measure (FREM) and derive FREM for two fluorophores with intermittent intensity. We show that the intensity intermittency of the sources (observed for quantum dots, for example) can increase the “resolution” defined in terms of FREM.

"When cells are exposed to environmental stress, they respond by compartmentalizing mRNA and translation proteins in stress granulates to protect mRNA. However, the mechanism through which external stress is communicated into the cell to form stress granules is unknown. Phospholipase Cβ (PLCβ) is activated by Gq on the plasma membrane in response to sensory stimuli to initiate calcium signals resulting in a variety of cellular responses. Here, we show that PLCβ binds to major proteins that organize stress granules as well as the main component of the RNA-induced silencing machinery, Argonaute-2 (Ago2). Under stress, PLCβ moves from the plasma membrane to the cytosol to escort Ago2 into stress granules and potentially inhibit mRNA degradation by regulating microRNAs (miRs) expression. Using a model muscle cell line functionally adapted to handle stress, we find that upon osmotic stress, the movement of PLCβ into the cytosol to move Ago2 into stress granules changes the population and distribution of miRs, and in particular, members of the let family. The impact of changes in let is to acutely affect glucose metabolism allowing cells to adapt to stress conditions. Our studies present a model in which PLCβ relays information about external stress to promote stress granule formation and protect mRNAs."

All animals harbor microorganisms that interact with each other and with their hosts. These microorganisms play important roles in health, disease, and defense against pathogens. The microbial communities in the intestine are particularly important in preventing colonization by pathogens; however, this defense mechanism and the means by which pathogens overcome it remain largely unknown. Moreover, while the composition of animal-associated microbial communities has been studied in great depth, the spatial and temporal dynamics of these communities has only recently begun to be explored. Here, we use a transparent model organism, larval zebrafish, to study how a human pathogen, Vibrio cholerae, invades intestinal communities. We pay particular attention to a bacterial competition mechanism, the type VI secrection system (T6SS), in this process. In vivo 3D fluorescence imaging and differential contrast imaging of transparent host tissue allow us to establish that V. cholerae can use the T6SS to modulate the intestinal mechanics of its host to displace established bacterial communities, and we demonstrate that one part of the T6SS apparatus, the actin crosslinking domain, is responsible for this function. Next, we develop an automated high-throughput light sheet fluorescence microscope to allow rapid imaging of bacterial communities and host cells in live larval zebrafish. Light sheet fluorescence microscopy (LSFM) has been limited in the past by low throughput and tedious sample preparation, and our new microscope features an integrated fluidic circuit and automated positioning and imaging to address these issues and allow faster collection of larger datasets, which will considerably expand the use of LSFM in the life sciences. This microscope could also be used for future experiments related to bacterial communities and the immune system. The overarching theme of the work in this dissertation is the use and development of advanced imaging techniques to make new biological discoveries, and the conclusions of this work point the way toward understanding pathogenic invasion, maximizing the use of LSFM in the life sciences, and gaining a better grasp of host-associated bacterial community dynamics. This dissertation includes previously published and unpublished co-authored material.

As optical microscopy techniques continue to improve, most notably the development of super-resolution optical microscopy which garnered the Nobel Prize in Chemistry in 2014, renewed emphasis has been placed on the development and use of fluorescence microscopy techniques. Of particular note is a renewed interest in multiphoton excitation due to a number of inherent properties of the technique including simplified optical filtering, increased sample penetration, and inherently confocal operation. With this renewed interest in multiphoton fluorescence microscopy, comes an increased demand for robust non-linear fluorescent markers, and characterization of the associated tool set. These factors have led to an experimental setup to allow a systematized approach for identifying and characterizing properties of fluorescent probes in the hopes that the tool set will provide researchers with additional information to guide their efforts in developing novel fluorophores suitable for use in advanced optical microscopy techniques as well as identifying trends for their synthesis. Hardware was setup around a software control system previously developed [1]. Three experimental tool sets were set up, characterized, and applied over the course of this work. These tools include scanning multiphoton fluorescence microscope with single molecule sensitivity, an interferometric autocorrelator for precise determination of the bandwidth and pulse width of the ultrafast Titanium Sapphire excitation source, and a simplified fluorescence microscope for the measurement of two-photon absorption cross sections. Resulting values for two-photon absorption cross sections and two-photon absorption action cross sections for two standardized fluorophores, four commercially available fluorophores, and ten novel fluorophores are presented as well as absorption and emission spectra.

Cancer morbidity and mortality is greatly reduced when the disease is diagnosed and treated early in its development. Tissue biopsies are the gold standard for cancer diagnosis, and an accurate diagnosis requires a biopsy from the malignant portion of an organ. Light, guided through a fiber optic probe, could be used to inspect regions of interest and provide real-time feedback to determine the optimal tissue site for biopsy. This approach could increase the diagnostic accuracy of current biopsy procedures. The studies in this thesis have characterized changes in tissue optical signals with carcinogenesis, increasing our understanding of the sensitivity of optical techniques for cancer detection. All in vivo studies were conducted on the dimethylbenz[alpha]anthracene treated hamster cheek pouch model of epithelial carcinogenesis. Multiphoton microscopy studies in the near infrared wavelength region quantified changes in tissue morphology and fluorescence with carcinogenesis in vivo. Statistically significant morphological changes with precancer included increased epithelial thickness, loss of stratification in the epithelium, and increased nuclear diameter. Fluorescence changes included a statistically significant decrease in the epithelial fluorescence intensity per voxel at 780 nm excitation, a decrease in the fluorescence lifetime of protein-bound nicotinamide adenine dinucleotide (NADH, an electron donor in oxidative phosphorylation), and an increase in the fluorescence lifetime of protein-bound flavin adenine dinucleotide (FAD, an electron acceptor in oxidative phosphorylation) with precancer. The redox ratio (fluorescence intensity of FAD/NADH, a measure of the cellular oxidation-reduction state) did not significantly change with precancer. Cell culture experiments (MCF10A cells) indicated that the decrease in protein-bound NADH with precancer could be due to increased levels of glycolysis. Point measurements of diffuse reflectance and fluorescence spectra in the ultraviolet to visible wavelength range indicated that the most diagnostic optical signals originate from sub-surface tissue layers. Optical properties extracted from these spectroscopy measurements showed a significant decrease in the hemoglobin saturation, absorption coefficient, reduced scattering coefficient and fluorescence intensity (at 400 nm excitation) in neoplastic compared to normal tissues. The results from these studies indicate that multiphoton microscopy and optical spectroscopy can non-invasively provide information on tissue structure and function in vivo that is related to tissue pathology. / Dissertation

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2010. / Committee Chair: Griffin, Paul; Committee Member: Butera, Robert; Committee Member: Halpern, Michael; Committee Member: Nichols, Richard; Committee Member: Vidakovic, Brani. Part of the SMARTech Electronic Thesis and Dissertation Collection.