Dissertations / Theses on the topic 'High throughput imaging'

This thesis presents three main strands of work concerned with developing digital imaging for high throughput beta autoradiography. These three strands comprise work with the image sensor technology, Monte Carlo simulation and the use of post-acquisition image analysis based on image registration. In this way, the complete autoradiography imaging chain is addressed. CCD and CMOS imaging technologies are presented as potential imaging alternatives to using conventional film in autoradiography. These digital technologies exhibit enhanced sensitivity, dynamic range and linearity compared to film using imaging methods developed at Surrey. These imaging methods address the different sources of noise typically present in CCD and CMOS technologies. Tissue imaging using 3H, 35S and 121I, the typical radioisotopes used by the Drug Addiction Group in the School of Biomedical and Biological Sciences, is presented. The first successful images of 3H-labelled tissue sections using CCD and CMOS technologies operating at room temperature are presented as one of the main achievements of this work. To better understand the image creation process some preliminary Monte Carlo simulations, using the GEANT4 toolkit, have been undertaken, demonstrating intrinsic and extrinsic key parameters of these digital sensors that can be used to optimise spatial resolution. These simulations demonstrate that each radioisotope requires a different optimum detector architecture. In this work these optimum architectures are analysed. To support the high sensitivity (i.e. fast) imaging produced by the sensor technology, automated post-acquisition analysis is also considered, using an atlas-based image registration approach, by previously aligning automatically segmented biological landmarks using a feature-based extraction approach, region growing. This has the potential to speed up the post-acquisition analysis aspects of the imaging chain. Thus a computer-based tool designed to semi-automatically elastically register a radiogram with an atlas has been developed.

Rapid testing of electrocatalysts and corrosion resistant alloys accelerates discovery of promising new materials. Imaging amperometry, based on the deployment of colloidal particles as probes of the local current density, allows simultaneous electrochemical characterization of the entire composition space represented in a thin-film alloy "library" electrode. Previous work has shown that nanometer scale variations in particle-electrode distance for single particles in electric fields can be measured optically and translated into local current density, independent of electrical measurements. Implementation of this method to enable simultaneous measurements across non-uniform samples involves using a sparse, uniform layer of particles, which requires modification of previously existing theory and methods. Imaging individual particles for this application is infeasible at the low magnification levels needed to image an entire macroscopic (~1 square cm) sample. Mapping of electrochemical activity across the surface can be achieved nevertheless by imaging the entire electrode surface and gridding the resulting images into a mosaic of square “patch” areas 100 μm to a side, each containing 15-30 particles. The work presented in this dissertation shows that the integrated light intensity in each patch is the sum of the light scattering from all of the particles present in that patch, and that this total measured intensity can be used to infer the current density in the patch during electrochemical experiments. In addition to scaling the imaging ammeter up to ensembles of particles, the theory for translating measured particle motion to current density has been substantially improved. These improvements involve proper modeling of the current distribution on the electrode below the particles, which has a profound impact on the forces acting on each particle. This work demonstrates that the use of realistic kinetic models for the imaging ammeter is both vital and a discovered opportunity to increase its sensitivity. Finite element analysis was used to explore the variable space of the parameters involved, to better understand the impact of factors such as the current density and solution conductivity on the motion of the particles. Going forward, this information will be leveraged to improve the accuracy of the macroscopic imaging ammeter. To complete the groundwork for the imaging ammeter laid out in this thesis, proof of concept experiments using a nickel/iron composition spread alloy film were performed. In a 1×5 mm2 area containing alloy compositions from 20% iron to 100% iron, expected trends in electrochemical activity were observed during experiments, i.e. the current density as a function of voltage increased with increasing nickel content on the electrode surface. Future work will probe Fe/Ni alloy compositions with less iron, subsequently moving on to other binary and eventually ternary alloy systems.

Visual knowledge plays an important role in many highly skilled applications, such as medical diagnosis, geospatial image analysis and pathology diagnosis. Medical practitioners are able to interpret and reason about diagnostic images based on not only primitive-level image features such as color, texture, and spatial distribution but also their experience and tacit knowledge which are seldom articulated explicitly. This reasoning process is dynamic and closely related to real-time human cognition. Due to a lack of visual knowledge management and sharing tools, it is difficult to capture and transfer such tacit and hard-won expertise to novices. Moreover, many mission-critical applications require the ability to process such tacit visual knowledge in real time. Precisely how to index this visual knowledge computationally and systematically still poses a challenge to the computing community.

My dissertation research results in novel computational approaches for highthroughput visual knowledge analysis and retrieval from large-scale databases using latest technologies in big data ecosystems. To provide a better understanding of visual reasoning, human gaze patterns are qualitatively measured spatially and temporally to model observers’ cognitive process. These gaze patterns are then indexed in a NoSQL distributed database as a visual knowledge repository, which is accessed using various unique retrieval methods developed through this dissertation work. To provide meaningful retrievals in real time, deep-learning methods for automatic annotation of visual activities and streaming similarity comparisons are developed under a gaze-streaming framework using Apache Spark.

This research has several potential applications that offer a broader impact among the scientific community and in the practical world. First, the proposed framework can be adapted for different domains, such as fine arts, life sciences, etc. with minimal effort to capture human reasoning processes. Second, with its real-time visual knowledge search function, this framework can be used for training novices in the interpretation of domain images, by helping them learn experts’ reasoning processes. Third, by helping researchers to understand human visual reasoning, it may shed light on human semantics modeling. Finally, integrating reasoning process with multimedia data, future retrieval of media could embed human perceptual reasoning for database search beyond traditional content-based media retrievals.

Focal plane array (FPA) FTIR imaging spectroscopy provides unprecedented levels of spatially resolvable chemical information for analysis of samples at the micrometer scale. This study evaluates the quantitative performance characteristics of the individual detector elements comprising the FPA camera, and applies them to making analytical measurements of a custom designed microfluidic multichannel transmission cell. Statistical descriptions are provided for the response distributions among the FPA's detector elements; RMS noise, peak response, and linear regression parameters. It was found that individual detector elements of the FPA allowed for accurate milli-absorbance measurements, however the variability was large when contrasting detector elements due to FPA detector non-uniformity issues. When applied to the microfluidic multichannel sampling system designed for the monitoring of four fluid streams, it was found that the detector elements covering the fluid stream could be averaged to generate a very repeatable response between streams – thus allowing for milli-absorbance measurements of 4 samples simultaneously with the current design.
L'imagerie par spectroscopie IRTF dans la matrice plane focale (MPF) offre des niveaux de résolution spatiale sans précédent des informations chimique dans le domaine spatial pour une analyse des échantillons à l'échelle du micromètre. L'étude actuelle examine l'ensemble des applications de la spectroscopie IRTF (MPF) avec l'utilisation d'un système micro-fluidique multicanaux de transmission de cellules conçut sur mesure comme une approche potentielle d'une analyse quantitative des échantillons liquides à haut débit. Des descriptions statistiques sont fournies selon la répartition des réponses parmi ces éléments individuels du détecteur. La réponse des éléments individuels du détecteur dans la MPF a été démontrée comme étant reproductible dans des unités de milli absorbance et ainsi, la plus importante variabilité de réponse à travers l'ensemble est due aux problèmes de non conformité associés à la MPF. La moyenne des réponses des éléments du détecteur sur lesquels les résultats de chaque canal est imagées dans de bonne reproductibilité inter-canal et ainsi compense de manière satisfaisante la non-uniformité des pixels. Les expériences qui prouvent ce concept impliquant des mesures analytiques sur quatre échantillons visualisés simultanément avec la conception actuel des cellules sont présentées.

In the field of protein research in general and the pharmaceutical industry in particular, it is now necessary to perform measurements of the secondary structure of proteins on many samples simultaneously, for instance to screen for molecules that stabilize proteins or to evaluate the action of multiple environmental conditions. In this context, we have proposed a new approach to evaluate the secondary structure of proteins on a very large scale (approximately 2000 to 4000 samples / cm2), by combining infrared imaging and 2D printing of protein microarrays. In view of the large amount of data, in a first step, methods for automating the extraction of spectra of interest from microarray infrared images and for automating the processing of the spectra were developed. Since the estimation of the secondary structure from infrared spectra is based on the construction of prediction models by chemometric methods, a relevant set of proteins for calibration was mandatory. A protein bank consisting of 92 commercially available proteins, the structure of which was well characterized by X-ray crystallography, was established for this purpose. After the development of predictive models for secondary structure determination and the validation of the protein microarray approach, we tried to optimize the models to improve the secondary structure prediction by different approaches as secondary structure definition, partial deuteration or subtraction of side chain contribution to the spectra. On the other hand, dealing with non-native structures not present in the reference protein library was a challenge. We took the opportunity to analyze the structural modifications of a subset of our protein library subjected to moderate denaturation conditions. Multivariate curve resolution-alternating least squares (MCR-ALS) was used to model a new spectral component appearing in the protein set subjected to denaturing conditions, which could represent a potential spectroscopic marker of aggregation and could allow a semi-quantitative evaluation of the aggregation. While the assessment of secondary structure was well established in the first part of this work, tertiary structure and stability are also critical. Hydrogen / deuterium exchange (HDX) is a potential approach for studying the structure and dynamics of proteins. In the last part of this work, we built a device which allowed to follow the HDX exchange kinetics simultaneously on the entire microarray. In conclusion, protein microarray FTIR imaging opens the door to high throughput analysis of protein secondary structure without any labelling and would allow better understanding of three-dimensional structure and dynamics of proteins through recording HDX curves.
Dans le domaine de la recherche sur les protéines et de l'industrie pharmaceutique, il s’avère désormais nécessaire d'effectuer des mesures de la structure secondaire des protéines sur de nombreux échantillons simultanément, de cribler des molécules qui stabilisent les protéines, ou d'évaluer l'action de multiples conditions environnementales. Dans ce contexte, nous avons proposé une nouvelle approche pour évaluer la structure secondaire des protéines à très grande échelle (environ 2 000 à 4 000 échantillons / cm2), en associant l'imagerie infrarouge et l'impression 2D de damiers de protéines. Dans un premier temps, des méthodes d'automatisation de l'extraction des spectres d'intérêt à partir des images infrarouges des damiers et d'automatisation des spectres ont été développées. L'estimation de la structure secondaire à partir des spectres infrarouges étant basée sur la construction de modèles de prédiction à partir de méthodes chimiométriques, un ensemble pertinent de protéines pour l'étape de calibration était obligatoire. Une banque de protéines constituée de 92 protéines disponibles dans le commerce, dont la structure était bien caractérisée par cristallographie aux rayons X, a été constituée dans ce but. Après élaboration des modèles prédictifs de la structure secondaire et la validation de l'approche des damiers de protéines, nous avons tenté d'optimiser les modèles pour améliorer les prédictions de structure secondaire par différentes approches. D'autre part, traiter des protéines présentant une structure jamais rencontrée dans les structures natives de notre bibliothèque de protéines de référence constituait un défi. Nous avons saisi l'opportunité d'analyser les modifications structurales d'un sous-ensemble de notre bibliothèque de protéines, caractérisé par un contenu structurel secondaire très différent en le soumettant à des conditions de dénaturation modérées La méthode de résolution de courbes multivariées des moindres carrés alternés (MCR-ALS) a été utilisée pour modéliser une nouvelle composante spectrale apparaissant dans l'ensemble protéique soumis à des conditions dénaturantes, et a permis de révéler un marqueur spectroscopique potentiel d'agrégation protéique permettant une évaluation semi-quantitative de son contenu. Alors que l’évaluation de la structure secondaire a été bien établie dans la première partie de ce travail, la structure tertiaire et la stabilité sont également critiques. L'échange hydrogène / deutérium (HDX) est une approche potentielle pour l’étude de la structure et de la dynamique des protéines. Dans la dernière partie de ce travail, nous avons construit un dispositif qui a permis de suivre la cinétique d’échange HDX simultanément sur l'ensemble d’un damier. En conclusion, l'imagerie FTIR de damiers de protéines ouvre la porte à une analyse à haut débit de la structure secondaire des protéines et permettrait de mieux comprendre la structure et la dynamique tridimensionnelles grâce à l'enregistrement des courbes HDX.
Doctorat en Sciences
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Nearly three billion humans worldwide have helminth infections, which accounts for a global disease burden of 5.2 million disability-adjusted life years. Parasitic nematodes also have a great affect on agriculture, annually destroying 12.3% of global food crops and accounting for an annual loss of approximately $10 billion (USD) worldwide in the sheep and cattle industry. There is widespread resistance to all classes of anthelmintic drugs. The last drug to enter human clinical trials was 35 years ago. There is a great need for new anthelmintics and this concern has been strongly voiced by the World Health Organization and Gates Foundation. Anthelmintics are considered rare, which is why large chemical libraries are a necessity for anthelmintic discovery. I adapted my Caenorhabditis elegans high-throughput technique WormScan to overcome the bottleneck of whole organism screening for anthelmintic discovery. WormScan was used to screen a library of approximately 26,000 molecules. Hits were tested against diverse organisms to identify nematode specific compounds. I took into account preexisting drug resistance of the established anthelmintics. For every class of anthelmintics there is a corresponding C. elegans resistant strain. Screening the hits against these known resistant strains allowed me to determine if any of the compounds target a novel or established anthelmintic pathways. A forward genetic screen and C. elegans genetic tools were employed to identify the mechanism of action of the candidate D19. Molecular modelling was undertaken to elucidate the resistance mechanism and phylogenetic specificity of the D19. I undertook a structure activity relationship to identify structural moieties of D19 activity. The high throughput screen identified 14 new potential anthelmintics, 5 of which had nematode specificity, which indicates low toxic side effects in humans and limited environmental toxicity. Forward genetic screening and molecular modelling identified D19 to be a worm specific mitochondrial complex II inhibitor. The Structure Activity Relationship demonstrated the chemical features needed for D19 activity. C. elegans was used as a surrogate of parasitic nematodes for whole organism anthelmintic discovery. This thesis is a starting point to replenish the pipeline of potential nematicides. Future studies should focus on progressing these hits to a lead status.
Pharmaceutical Sciences, Faculty of
Graduate

The exponential expansion in the field of biophotonics over the past half-century has been leading to ubiquitous basic science investigations, ranging from single cell to brain networking analysis. There is also one biophotonics technology used in clinic, which is optical coherence tomography, mostly for high-speed and high-resolution endoscopy. To keep up such momentum, new biophotonics technologies should be aiming at improving either the spatial resolution or temporal resolution of optical imaging. To this end, this thesis will address a new imaging technique which has an ultra-high temporal resolution. The applications and its cost-effective implementations will also be encompassed. In the first part, I will introduce an entirely new optical imaging modality coined as optical time-stretch microscopy. This technology allows ultra-fast real-time imaging capability with an unprecedented line-scan rate (~10 million frames per second). This ultrafast microscope is renowned as the world’s fastest camera. However, this imaging system is previously not specially designed for biophotonics applications. Through the endeavors of our group, we are able to demonstrate this optical time-stretch microscopy for biomedical applications with less biomolecules absorption and higher diffraction limited resolution (<2 μm). This ultrafast imaging technique is particularly useful for high-throughput and high-accuracy cells/drugs screening applications, such as imaging flow cytometry and emulsion encapsulated drugs imaging. In the second part, two cost-effective approaches for implementing optical time-stretch confocal microscopy are discussed in details. We experimentally demonstrate that even if we employ the two cost-effective approaches simultaneously, the images share comparable image quality to that of captured by costly specialty 1μm fiber and high-speed ( >16 GHz bandwidth) digitizer. In other words, the cost is drastically reduced while we can preserve similar image quality. At the end, I will be wrapping up my thesis by concluding all my work done and forecasting the future challenges concerning the development of optical time-stretch microscopy. In particular, three different research directions are discussed.
published_or_final_version
Electrical and Electronic Engineering
Master
Master of Philosophy

The objective of this research is to develop automated and integrated microsystems for high-resolution imaging and high-throughput phenotyping / laser ablation of C. elegans. These microsystems take advantage of microfluidic technology for precisely handling animals and computer-aid automation for high-throughput processing. We demonstrated automated and high-throughput imaging / sorting and laser ablation of C. elegans. This thesis work is divided into four parts: development of a microsystem for imaging and sorting, development of a microsystem for laser cell ablation, development of a novel temperature measurement method, and development of pressure measurement method in microchannels. First, a microsystem was developed for high-throughput microscopy at high resolution and sorting. The microfluidic chip integrates novel microfluidic components to trap, position, immobilize, and sort/release animals. To characterize device operation and aid design of the device numerical models were developed. The experimental results demonstrate that the device operates robustly in a completely automatable manner. Additionally, a sophisticated control algorithm developed by Matthew Crane (Dr. Hang Lu¡¯s lab) automates the entire process of image acquisition, analysis, and sorting, which allows the system to operate without human intervention. This microsystem sorted worms based on their fluorescent expression pattern with over 95% accuracy per round at a rate of several hundred worms per hour. Secondly, the technologies developed for the imaging/sorting system were adapted and further improved to develop a microsystem for high-throughput cell laser ablation of C. elegans. The multiplex ablation module combined with the embryo trap module enables robust manipulation of embryos/L1-stage C. elegans. In addition, software for image processing and automation was developed to allow high-throughput cell ablations. This system performed ablation of a large number of animals and demonstrated accurate ablation by showing behavioral defects of the ablated worms in a chemotaxis avoidance assay. Thirdly, to aid future development of the microdevices, a novel in situ method for three-dimensionally resolved temperature measurement in microchannels was developed. This method uses video-microscopy in combination with image analysis software (developed by Jaekyu Cho in Dr. Victor Breedveld¡¯s group) to measure Brownian diffusion of nanoparticles that is correlated to temperature. This method offers superior reproducibility and reduced systematic errors. In addition, we demonstrated that this method can be used to measure spatial temperature variations in three dimensions in situ. Lastly, a method for pressure measurement in microdevices was also developed through collaboration with Hyewon Lee (Dr. Hang Lu¡¯s lab) to aid further device optimization. These micro pressure-sensors are composed of two flow layers with a polydimethylsiloxane (PDMS) membrane in between. The membrane deforms as a function of pressure and its deformation is quantified by a simple image-based method. These sensors offer high-precision pressure measurement in broad sensing ranges. In addition, a pressure transduction scheme combined with imaging-based method enables multiplex pressure measurement for simultaneously detecting pressures in multiple locations in a microsystem. Overall, the technologies developed in this thesis will establish a solid basis for continuous improvement of the microsystems for multi-cellular model organisms. This high-throughput technology will facilitate a broad range of biological and medical research.

Gene expression is a fundamental process in the cell and is made up of two parts – the information flow from DNA to RNA, and from RNA to protein. Here, we examined specific sub-processes in Escherichia coli gene expression using newly available tools that permit genome-wide analysis. We begin our studies measuring mRNA and protein abundances in single cells by single-molecule fluorescence microscopy, and then focus our attention to studying RNA generation and degradation by high throughput sequencing. The details of the dynamics of gene expression can be observed from fluctuations in mRNA and protein copy numbers in a cell over time, or the variations in copy numbers in an isogenic cell population. We constructed a yellow fluorescent fusion protein library in E. coli and measured protein and mRNA abundances in single cells. At below ten proteins per cell, a simple model of gene expression is sufficient to explain the observed distributions. At higher expression levels, the distributions are dominated by extrinsic noise, which is the systematic heterogeneity between cells. Unlike proteins which can be stable over many hours, mRNA is made and degraded on the order of minutes in E. coli. To measure the dynamics of RNA generation and degradation, we developed a protocol using high throughput sequencing to measure steady-state RNA abundances, RNA polymerase elongation rates and RNA degradation rates simultaneously with high nucleotide-resolution genome-wide. Our data shows that RNA has similar lifetime at all positions throughout the length of the transcript. We also find that our polymerase elongation rates measured in vivo on a chromosome are generally slower than rates measured on plasmids by other groups. Studying nascent RNA will allow further understanding of RNA generation and degradation. To this end, we have developed a labeling protocol with a nucleoside analog that is compatible with high throughput sequencing.

Powerful tools for detailed cellular studies are emerging, increasing the knowledge ofthe ultimate target of all drugs: the living cell. Today, cells are commonly analyzed inensembles, i.e. thousands of cells per sample, yielding results on the average responseof the cells. However, cellular heterogeneity implies the importance of studying howindividual cells respond, one by one, in order to learn more about drug targeting andcellular behavior. In vitro assays offering low volume sampling and rapid analysis in ahigh-throughput manner are of great interest in a wide range of single-cellapplications. This work presents a microwell device in silicon and glass, developed using standardmicrofabrication techniques. The chip was designed to allow flow-cytometric cellsorting, a controlled way of analyzing and sorting individual cells for dynamic cultureand clone formation, previously shown in larger multiwell plates only. Dependent onthe application, minor modifications to the original device were made resulting in agroup of microwell devices suitable for various applications. Leukemic cancer cellswere analyzed with regard to their clonogenic properties and a method forinvestigation of drug response of critical importance to predict long-term clinicaloutcome, is presented. Stem cells from human and mouse were maintainedpluripotent in a screening assay, also shown useful in studies on neural differentiation.For integrated liquid handling, a fluidic system was integrated onto the chip fordirected and controlled addition of reagents in various cell-based assays. The chip wasproduced in a slide format and used as an imaging tool for low-volume sampling withthe ability to run many samples in parallel, demonstrated in a protein-binding assay fora novel bispecific affinity protein. Moving from cells and proteins into geneticanalysis, a method for screening genes from clones in a rapid manner was shown bygene amplification and mutation analysis in individual wells. In summary, a microwelldevice with associated methods were developed and applied in a range of biologicalinvestigations, particularly interesting from a cell-heterogeneity perspective.
QC 20100728

Ces dernières décennies, on a assisté à l’augmentation du nombre de technologies et de concepts permettant l’analyse des interactions intermoléculaires. Dans ce contexte, les puces à fluorescence restent les plus fréquemment utilisées. Cependant, cette technologie bien que très sensible et multiplexée, ne permet pas d’avoir accès aux paramètres cinétiques, indispensables au calcul des constantes d’affinité et la recherche de systèmes alternatifs s’impose. Dans cette optique, la résonance plasmonique de surface par imagerie (SPRi) est considérée comme une véritable option. Cette technologie se caractérise par l’absence de marquage et permet de suivre en temps réel d’infimes variations de masses consécutives à des interactions intermoléculaires sur la surface du prisme. L’obtention de constantes d’affinité est ainsi possible. En revanche, la SPRi présente un certain nombre de limites, principalement au niveau de la sensibilité et du multiplexage. Les objectifs de la thèse ont ainsi consisté à combler en partie ces différentes limites. La chimie de greffage basée sur l’utilisation d’oligonucléotides modifiés par un thiol a permis d’améliorer le multiplexage et de déposer plus de 1000 spots par cm² sur la surface d’or du prisme. Dans le même temps, la modification de la surface avec des colloïdes d’or et des dendrimères a permis pour des interactions ADN/ADN, d’atteindre une limite de détection de 2 nM (d’où un gain de 200%). En parallèle de ces travaux, diverses applications biologiques ont été effectuées. Une première étude a consisté à rechercher des ligands spécifiques des structures G-quadruplex des télomères. Une seconde étude s’est portée sur le complexe de partition bactérien. Par des études de criblage les bases impliquées dans l’interaction avec une protéine indispensable à la partition du plasmide F chez E.coli ont été identifiées. L’ensemble de ces travaux ont montré le fort potentiel de la SPRi et les applications potentielles qui en découlent sont nombreuses
During the last decades a large number of technologies have been developed to analyze intermolecular interactions. In this context, the fluorescence biochips remain the most frequently used. Although this technology is very sensitive and multiplexed, it does not allow access to the kinetic parameters, essential to the calculation of the constants of affinity. Therefore, the research for alternative systems is essential. In this way, the Surface Plasmon Resonance imaging (SPRi) is considered as an opportunity. It is an optical detection process that can occur when a polarized light hits a prism covered by a thin metal layer. Under certain conditions free electrons at the surface of the biochip absorb incident light photons and convert them into surface plasmon waves. Perturbations at the surface of the biochip, such as an interaction between probes immobilized on the chip and targets, induce a modification of resonance conditions which can be measured. It is a label free technology which allows intermolecular interactions in real time and gives access to the kinetics parameters. However, SPRi is limited in sensitivity and multiplexing. The objectives of my PhD were to circumvent these various limits. Thus, we validated the immobilization of DNA probes on gold surface using thiol-modified oligonucleotide probes. Deposition carried out on non-modified gold surface, does not require electrical stimulation and expensive specific robotic devices. The thiol modification of the probes was shown to be very stable at room temperature, contrary to pyrrole and diazonium probes that need to be prepared just prior to their spotting. We demonstrate that thiol-modified oligonucleotide probes spotted on a gold surface of the SPRi-prisms are very robust and reproducible. We also demonstrated that this simple chemistry is compatible with high density arrays fabrication bearing more than 1000 spots using a classical spotter. Furthermore, the modification of the prism surface with gold colloids and dendrimers allowed for DNA/DNA interactions, to reach a detection limit of 2 nM. In parallel of this work, various biological applications were carried out and validate our previous developments. A first study was to screen G-quadruplex specific ligands to inhibit telomerase activity. We demonstrated that SPRi technology is particularly well adapted to the screening of interaction of small molecules with DNA probes and is sensitive enough to permit distinction between interactions with different DNA structures. The second study was on the bacterial partition complex. We study the DNA binding requirement involved in SopB-sopC specific interactions and analysed at the nucleotide level the bases involved in the binding efficiency and essential for the partition All this PhD work improved the SPRi technology and demonstrated its great potential in biological applications

The goal of this thesis is the development of a tracking algorithm for populations of unmarked cancer cells that migrate in 3D in vitro gels. The tracking algorithm is intended to be a tool for analysing the motility of large population (i.e. hundreds) of cells in the context of the anti-migratory drug development and more specifically drug screening. In oncology, cancer cell migration plays pivotal roles in the spread of cancer cells from a primary tumor site to neighboring and secondary sites, i.e. the processes of tissue invasion and metastasis. Preventing such processes represents an important therapeutic approach to cancer treatment. Providing tools able to test potential anti-migratory drugs thus constitutes currently a real need in oncology therapy. The goal of drug screening in this context aims to rapidly and efficiently test the anti-migratory effects of many experimental conditions on cancer cell populations.

The focus in this thesis lies in two specific aspects that are important in anti-migratory drug screening: tracking cells inside an in vitro 3D environment and doing so using unmarked cells.
Doctorat en Sciences de l'ingénieur
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Les diverses classes d’acides gras (chaines carbonées saturées ou cis-/trans- insaturées) influencent la physiologie au niveau de la cellule et de l’organisme de façon différente. Curieusement, ces catégories distinctes ont un effet important (mais différent) sur l’autophagie, le mécanisme intracellulaire de dégradation qui maintient l’homéostasie énergétique et protège les cellules contre le stress. L’oléate, l’acide gras cis-insaturé endogène et alimentaire le plus abondant, possède la propriété atypique d’induire une redistribution de la protéine LC3 (signe particulier d’autophagie) de manière non-canonique et préférentiellement dans l’appareil de Golgi. Puisqu’il a été montré que, d’une part, les acides gras cis-insaturés présentent des effets bénéfiques pour la santé et que, d’autre part, les acides gras trans-insaturés et saturés induisent des effets toxiques, nous avons décidé d’explorer les mécanismes à la base de la redistribution de LC3 au niveau de l’appareil de Golgi induite par l’oléate. Cette analyse pourrait nous éclairer sur l’origine des différents effets des acides gras sur la santé. Pour cela, un criblage robotisé du génome entier par ARNs interférents a permis d’identifier plusieurs gènes impliqués dans le transport des protéines lié à l’appareil de Golgi, et également dans la réponse intégrée au stress.Des expériences supplémentaires ont montré que l’oléate impacte la morphologie subcellulaire de l’appareil de Golgi, en corrélation avec le blocage de la sécrétion protéique conventionnelle (dépendante du Golgi) lorsque que la cargaison est bloquée au niveau du réseau trans-golgien. L’inhibition de la sécrétion protéique a été observée dans plusieurs systèmes expérimentaux, tant in vitro qu’in vivo. De plus, un criblage visant à rechercher des agents chimiques capables d’induire les mêmes effets cellulaires que l’oléate, a permis d’identifier plusieurs composés appartenant à diverses classes pharmacologiques. De la même manière que l’oléate ces composés induisent un blocage de la sécrétion protéique conventionnelle, renforçant l’idée que cette voie de perturbation du Golgi joue un rôle pharmacologique important. En conclusion, ces résultats montrent que l’oléate représente une classe de molécules agissant sur l’appareil de Golgi pour y induire l’agrégation de LC3, tout en bloquant en même temps la sécrétion protéique
Distinct classes of fatty acids (FAs) (saturated or cis-/trans-unsaturated carbon chains) impact on cellular and organismal physiology in a different manner. Interestingly, these diverse categories have a profound (but different) effect on autophagy, the conserved intracellular degradation mechanism that maintains energy homeostasis and protects cells against stress. Oleate, the most abundant endogenous and dietary cis-unsaturated FA, has the atypical property to induce the redistribution of the LC3 protein (peculiar sign of autophagy) in a non-canonical fashion and preferentially to the Golgi apparatus. Intrigued by these observations, which might be related to the health-improving effects of cis-unsaturated FAs (and the notorious toxicity of trans-unsaturated and saturated FAs), we decided to explore the mechanisms causing the oleate-induced relocation of LC3 to the Golgi apparatus. To achieve this goal, a robotized RNA interference genome-wide screen led to the identification of multiple genes involved in the Golgi-related protein transport, as well as in the integrated stress response. Follow-up experiments revealed that oleate affected the subcellular morphology of the Golgi apparatus, correlating with a blockade of conventional (Golgi-dependent) protein secretion that caused secretory cargo to be stalled at the level of the trans-Golgi network. The inhibition of protein secretion was observed using several experimental systems, both in vitro and in vivo. Moreover, a systematic screen searching for other chemical entities that mimic the oleate-induced cellular effects led to the identification of several compounds belonging to rather different pharmacological classes. These “oleate mimetics” also shared with oleate the capacity to block conventional protein secretion, supporting the notion that this pathway of Golgi perturbation is indeed of pharmacological relevance. In conclusion, this research work shows that oleate represents a class of molecules that act on the Golgi apparatus to cause the recruitment of LC3 and to stall protein secretion

Light allows to non-invasively study the complex and dynamic biological phenomenon undergoing within cells and tissues in their native state. The development of super-resolution microscopes in recent years has helped to overcome the fundamental limitation imposed by Abbe’s diffraction limit, thereby revolutionizing the field of molecular and cellular biology. With the advancement of various super-resolution techniques (like STED, PALM, and 4Pi) it is now possible to visualize the nanometeric cellular structures and their dynamics in real time. The limitations of existing fluorescence microscopy techniques are: poor axial resolution when compared to their lateral counterpart, and their inability to produce high resolution images of dynamic samples. This thesis covers two broadly connected areas of fluorescence imaging techniques while addressing these limitations. First, the PSF engineering and spatial filtering technique for axial super-resolution microscopy and second, the integration of light sheet illumination PSF with microfluidic cytometry for imaging cells on-the-go. The first chapter gives an explicit description on the fundamentals of fluorescence imaging. This introductory chapter includes a variety of optical microscopes, PSF engineering, the resolution limit imposed by the wave nature of light, the photochemistry of the fluorescent dyes, and their proper selection for fluorescence experiments. In addition to the state-of-art imaging techniques, namely Laser Scanning Confocal Microscopy and Light Sheet Microscopy, this chapter also gives a brief explanation on the evolution of imaging cytometry techniques. Their high speed analytic capability (i.e sorting and counting) makes this technique an important tool in health care diagnosis and other various biomedical applications. The chapter ends with a discussion on the operating principle of the flow cytometers and their limitations. The second chapter in this thesis describes the spatial filtering technique for engineering the PSF to eliminate the side-lobes in the system PSF of the 4Pi Confocal Microscopes. Employing an amplitude mask with binary light transmission windows (also called binary filters), the incident light is structured to minimize the secondary lobes. These lobes are responsible for exciting the off-focal planes in the specimen, hence provide incorrect map of the fluorophore distribution in the object. The elimination of the side-lobes is essential for the artifact-free axial super-resolution microscopy. This second chapter describes the spatial filtering technique in details (its mathematical formulation, application in fluorescence microscopy for generation of desired PSF including Bessellike beam). Specifically, spatial filtering technique is employed in 4Pi type-C Confocal Microscope. The spatial mask used results in the reduction of the side-lobes in 1PE case while they are nearly eliminated in 2PE variant of the proposed technique. The side-lobes are reduced by 46% and 76% for 1PE and 2PE when compared to the existing 4Pi type-C Confocal Microscope system. Moreover, OTF of the proposed system confirms the presence of higher frequencies in the Fourier domain indicating high resolution imaging capability. Apart from the resolution in lateral and axial dimension, achieving high resolution while imaging dynamic samples is another challenge that is limiting the field of fluorescence microscopy to flourish. The third and fourth chapters are entirely dedicated towards the work that was carried out to develop imaging techniques on a microfluidic platform for imaging dynamic samples. The fusion of microscopy and flow cytometry has given rise to the celebrated field of imaging flow cytometry. In recent years, the focus has shifted towards miniaturized cytometry devices. Apart from the reduced cost of the sample reagents and the assays, portability and easy handling make the microfluidic devices more relevant to developing countries. The commercially available cytometers are bulky and quite costly. In addition to these practical concerns, they are complex in operation and limited in performance. Most of the existing cytometers use different inlets for sheath and sample flow to achieve the hydrodynamic focusing of the sample assays in a narrow and confined region. The laser beam in the illumination arm interrogates with the flowing samples at this region and the response is captured by the detection optics. The same principle is extensively used in most of the microfluidic based flow cytometers reported till date. Apart from the hydrodynamic force other effects like electro-osmotic, acoustic, and dielectrophoresis have also been exploited to achieve flow focusing in the microfluidic channel. Despite omitting the necessity of external syringe pump as required in pressure driven based cytometers, they all rely upon point-source based excitation scheme and thereby can not interrogate the cells flowing through the entire microfluidic channel. The third chapter describes the integration of light sheet illumination PSF with microfluidic flow cytometry for simultaneous counting and imaging cells on-the-go. The chapter starts with the description on photolithography procedure for preparing SU8 master and PDMS casting procedure adopted to prepare dedicated microfluidic chips for the developed imaging system. The research work reported here demonstrates the proof-ofprinciple of light sheet based imaging flow cytometer. A light sheet fills the entire microfluidic channel and thus omits the necessity of flow focusing and point-scanning based technology. Another advantage lies in the orthogonal detection geometry that totally cuts-off the incident light, thereby substantially reducing the background in the acquired images. Compared to the existing state-of-the-art techniques, the proposed technique shows marked improvement. Using fluorescently coated Saccharomyces cerevisiae cells, cell counting with throughput as high as 2090 cells/min was recorded. Overall the proposed system is cost-effective and simple in channel geometry. Apart from achieving efficient counting in operational regime of low flow rate, high contrast images of the dynamic samples are also acquired using the proposed cytometry technique. Further, visualization of intra-cellular organelles is achieved during flow in light sheet based high-throughput cytometry system. The fourth chapter demonstrates the proof of concept of light-sheet-based microfluidic cytometer in conjugation with 2π/3 detection system for high-throughput interrogation and high resolution imaging. This system interrogates the flow channel using a sheet of light rather than the existing point-scanning based techniques. This ensures single-shot scanning of specimens flowing through the microfluidic flow channel at variable flow rates. In addition to high throughput counting at low flow rate, visualization of the intra-cellular organelle (mitochondrial network in human cancerous cells) during flow is achieved with sub-cellular resolution. Using mitochondrial network tagged HeLa cells, a maximum count of 2400 cells/min at the optimized flow rate of 700 nl/min was recorded. The 2π/3 detection system ensures efficient photon collection and minimal background caused by scattered illumination light. The other advantage of this kind of detection system which includes 8f detection optics, is the capability to produce variable magnification using the same high NA objective. This thesis opens up in vivo imaging of sub-cellular structures and simultaneous cell counting in a miniaturized flow cytometry system. The developed imaging cytometry technique may find immediate applications in the diverse field of healthcare diagnostics, lab-on-chip technology, and fluorescence microscopy. The concluding chapter summarizes the results with a brief discussion on the future aspects of this field (e.g., live-cell imaging of infectious RBC in microfluidic device and 3D optical sectioning of flowing cells). The field of imaging flow cytometry has immense applications in the overlapping areas of physics and biology. The hydrodynamic forces which are used to achieve flow focusing of the sample assays can have an adverse effect in the cell morphology, thereby altering the cellular functions. Light sheet based cytometry system lifts off the requirement of flow focusing and ensures a single shot scanning of entire samples flowing through the microfluidic channel. The similar concept can be used to study the developmental biology of an entire organism, such as C. elegans. This enables the direct observation of developmental and physiological changes in the entire body. Such an organism can be kept alive for a longer duration in microfluidic chambers, and the neural development and mating behaviors can be extensively studied.

Biological research and Clinical Diagnostics heavily rely on Optical Microscopy for analyzing properties of cells. The experimental protocol for con-ducting a microscopy based diagnostic test consists of several manual steps, like sample extraction, slide preparation and inspection. Recent advances in optical microscopy have predominantly focused on resolution enhancement. Whereas, the aspect of automating the manual steps and enhancing imaging throughput were relatively less explored. Cost-e ective automation of clinical microscopy would potentially enable the creation of diagnostic devices with a wide range of medical and biological applications. Further, automation plays an important role in enabling diagnostic testing in resource-limited settings. This thesis presents a novel optofluidics based approach for automation of clinical diagnostic microscopy. A system-level integrated optofluidic architecture, which enables the automation of overall diagnostic work- ow has been proposed. Based on the proposed architecture, three different prototypes, which can enable point-of-care (POC) imaging cytometry have been developed. The characterization of these prototypes has been performed. Following which, the applicability of the platform for usage in diagnostic testing has been validated. The prototypes were used to demonstrate applications like Cell Viability Assay, Red Blood Cell Counting, Diagnosis of Malaria and Spherocytosis. An important performance metric of the device is the throughput (number of cells imaged per second). A novel microfluidic channel design, capable of enabling imaging throughputs of about 2000 cells per second has been incorporated into the instrument. Further, material properties of the sample handling component (microfluidic device) determine several functional aspects of the instrument. Ultrafast-laser inscription (ULI) based glass microfluidic devices have been identi ed and tested as viable alternatives to Polydimethylsiloxane (PDMS) based microfluidic chips. Cellular imaging with POC platforms has thus far been limited to acquisition of 2D morphology. To potentially enable 3D cellular imaging with POC platforms, a novel slanted channel microfluidic chip design has been proposed. The proposed design has been experimentally validated by performing 3D imaging of fluorescent microspheres and cells. It is envisaged that the proposed innovation would aid to the current e orts towards implementing good quality health-care in rural scenarios. The thesis is organized in the following manner : The overall thesis can be divided into two parts. The first part (chapters 2, 3) of the thesis deals with the optical aspects of the proposed Optofluidic instrument (development, characterization and validations demonstrating its use in poc diagnostic applications). The second part (chapters 4,5,6) of the thesis details the microfluidic sample handling aspects implemented with the help of custom fabricated microfludic devices, the integration of the prototype, func-tional framework of the device. Chapter 2 introduces the proposed optofluidic architecture for implementing the POC tool. Further, it details the first implementation of the proposed platform, based on the philosophy of adapting ubiquitously available electronic imaging devices to perform cellular diagnostic testing. The characterization of the developed prototypes is also detailed. Chapter 3 details the development of a stand-alone prototype based on the proposed architecture using inexpensive o -the-shelf, low frame-rate image sensors. The characterization of the developed prototype and its performance evaluation for application in malaria diagnostic testing are also presented. The chapter concludes with a comparative evaluation of the developed prototypes, so far. Chapter 4 presents a novel microfludic channel design, which enables the enhancement of imaging throughput, even while employing an inexpensive low frame-rate imaging modules. The design takes advantage of radial arrangement of microfludic channels for enhancing the achievable imaging throughput. The fabrication of the device and characterization of achievable throughputs is presented. The stand-alone optofluidic imaging system was then integrated into a single functional unit, with the proposed microfluidic channel design, a viscoelastic effect based micro uidic mixer and a suction-based microfluidic pumping mechanism. Chapter 5 brings into picture the aspect of the material used to fabricate the sample handling unit, the robustness of which determines certain functional aspects of the device. An investigative study on the applicability of glass microfluidic devices, fabricated using ultra-fast laser inscription in the context of the microfluidics based imaging flow cytometry is presented. As detailed in the introduction, imaging in poc platforms, has thus far been limited to acquisition of 2D images. The design and implementation of a novel slanted channel microfluidic chip, which can potentially enable 3D imaging with simplistic optical imaging systems (such as the one reported in the earlier chapters of this thesis) is detailed. A example application of the proposed microfludic chip architecture for imaging 3D fluorescence imaging of cells in flow is presented. Chapter 6 introduces a diagnostic assessment framework for the use of the developed of m in an actual clinical diagnostic scenario. The chapter presents the use of computational signatures (extracted from cell images) to be employed for cell recognition, as part of the proposed framework. The experimental results obtained while employing the framework to identify cells from three different leukemia cell lines have been presented in this chapter. Chapter 7 summarizes the contributions reported in this thesis. Potential future scope of the work is also detailed.

碩士
國立交通大學
電子研究所
107
Radar technology and its developments have been important issues for decades. In recent years, related applications are becoming more and more popular such as automobile safety system and speed measurement. With the growth of semiconductor processing technology, the development of circuit design related to THz technology has gradually been noted. Imaging radar system with THz technology has many advantages so that it can be applied on security scan, because of the characteristics of THz such as penetrability and reflection on conducting material. However, there are ultra-long series in the application of wideband radar system with high sampling rate. While realizing the ultra-long FFT, it would introduce some design challenges such as high hardware complexity and large area cost. On the other hand, it needs to achieve a high throughput rate to meet the requirement of real-time processing. In this thesis, we implement a 4-parallel 64K-point FFT hardware architecture based on the 2-epoch FFT algorithm for the application of THz imaging radar system. With the proposed middle twiddle factor generator, we can reduce a large number of storages for twiddle factor coefficients, so the area of ROM is reduced. In addition, the maximum operating frequency will not be limited by the long access time of a large size ROM. We implement the 64K-point FFT architecture in TSMC 90 nm CMOS technology with high-Vt standard cell library. The maximum operating frequency of the system is 390 MHz, and the throughput rate reaches 1.57 GS/s. Its high throughput rate meets the requirement of real-time processing in our THz imaging radar system with a mechanical scan. When operating at the maximum clock rate, it consumes 0.2811 W (@0.9 V), and the total gate counts of this work are around 3974.1k.

MRI is a versatile tool for systematically assessing anatomical and functional changes in small animal models of human disease. Its noninvasive nature makes MRI an ideal candidate for longitudinal evaluation of disease progression in mice; however achieving the desired level of statistical power can be expensive in terms of imaging time. This is particularly true for cancer studies, where dynamic contrast-enhanced (DCE-) MRI, which involves the repeated acquisition of anatomical images before, during, and after the injection of a paramagnetic contrast agent, is used to monitor changes in tumor vasculature. A means of reducing the overall time required to scan multiple cohorts of animals in distinct experimental groups is therefore highly desirable. Multiple-mouse MRI, in which several animals are simultaneously scanned in a common MRI system, has been successfully used to improve study throughput. However, to best utilize the next generation of small-animal MRI systems that will be equipped with an increased number of receive channels, a paradigm shift from simultaneously scanning as many animals as possible to scanning a more manageable number, at a faster rate, must be considered. Given a small-animal MRI system with 16 available receive channels, the simulations described in this work explore the tradeoffs between the number of animals scanned at once and the number of array elements dedicated to each animal for maximizing throughput. An array system consisting of 15 receive and 5 transmit coils allows throughput-optimized acceleration of a DCE-MRI protocol by a combination of multi-animal and parallel imaging techniques. The array system was designed and fabricated for use on a 7.0-T / 30-cm MRI system, and tested for high-throughput imaging performance in phantoms. Results indicate that up to a nine-fold throughput improvement is possible without sacrificing image quality compared to standard single-animal imaging hardware. A DCE-MRI study throughput improvement of just over six times that achieved with conventional single-mouse imaging was realized. This system will lower the barriers for DCE-MRI in preclinical research and enable more thorough sampling of disease pathologies that progress rapidly over time.
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Vibrational imaging approaches including Raman microscopy and IR-absorption micro-spectroscopy can provide rich chemical information about biological samples. This dissertation contributes to improve the capabilities of vibrational microscopy in three aspects each with corresponding biological applications. First, along the line of combining electronic resonant effect with stimulated Raman scattering (SRS), we studied the spectroscopic characteristics for on-resonant SRS case and demonstrated the utility of electronic pre-resonant SRS on super-multiplexed imaging for live cells and tissue sections. Second, we provided a new light-matter interaction as a hybrid technique of Raman and fluorescence, called stimulated Raman excited fluorescence (SREF), bringing the long-sought-after goal of detecting single-molecule Raman scattering without plasmonic enhancement into view. Coupling SREF with vibrational sensing, local electric field and hydrogen-bonding environment can thus be visualized in situ. Third, we brought small vibrational probes into mid-infrared imaging for the goal of rich-information-content, high-throughput metabolic imaging. Chapter 1 introduces some basis of Raman scattering, and provides an overview of state-of-art SRS microscopy. Chapter 2 explores on the rigorous electronic resonant region with SRS (er-SRS) through suppression of electronic background and subsequent retrieval of vibrational peaks. In agreement with theoretical prediction, changing of vibrational band shapes from normal Lorentzian, through dispersive shapes, to inverted Lorentzian is observed when approaching electronic resonance. As large as 10-23 cm2 of resonance Raman cross section is estimated in er-SRS. In Chapter 3, a new light-matter interaction called stimulated Raman excited fluorescence (SREF) is studied. Through stimulated Raman pumping to an intermediate vibrational eigenstate followed by an upconversion to an electronic fluorescent state, SREF encodes vibrational resonance into the excitation spectrum of fluorescence emission. By leveraging superb sensitivity of SREF, we achieved all-far-field single-molecule Raman spectroscopy and imaging without plasmonic enhancement. Chapter 4 details the development of SREF into a novel water-sensing tool, by coupling with vibrational solvatochromism of environment-sensitive Raman mode. This new technique allows direct visualization on spatially-resolved distribution of water states inside single mammalian cells. Interesting intracellular heterogeneity of water states between nucleus and cytoplasm has been revealed. Chapter 5 demonstrates the utility of epr-SRS in super-multiplexed imaging with either commercial fluorophores in lives cells or our MARS probes on tissue sections. Multiplex protein-based tissue imaging is completed with newly-designed functional MARS dye with up to 12 channels simultaneously. Chapter 6 focus on metabolic imaging by mid-infrared (MIR) microscopy with vibrational probes. Raman scattering microscopy has made a major advance in metabolic imaging utilizing vibrational probes, yet is limited to relatively low throughput. As an alternative solution, MIR microscopy provides significantly higher cross section and exhibits as a rich-information-content, high-throughput technique with recent rapid technical advances. We introduced three types of small vibrational probes as azide, 13C and carbon-deuterium for studying dynamic metabolic activities of protein, lipids and carbohydrates in cells, small organisms and mice for the first time. Two MIR microscopy platforms as Fourier transform infrared (FTIR) absorption microscopy and discrete frequency mid-infrared (DFIR) microscopy were utilized to validate the generality of our vibrational probes and applicability for single-cell metabolic profiling and metabolism study on large-scale tissues.

Development of a single cell into an adult organism is accomplished through an elaborate and complex cascade of spatiotemporal gene expression. While methods exist for capturing spatiotemporal expression patterns---in situ hybridization, reporter constructs, fluorescent tags---these methods have been highly laborious, and results are frequently assessed by subjective qualitative comparisons. To address these issues, methods must be developed for automating the capture of images, as well as for the normalization and quantification of the resulting data. In this thesis, I design computational approaches for high throughput image analysis which can be grouped into three main areas. First, I develop methods for the capture of high resolution images from high throughput platforms. In addition to the informatics aspect of this problem, I also devise a novel multiscale probabilistic model that allows us to identify and segment objects in an automated fashion. Second, high resolution images must be registered and normalized to a common frame of reference for cross image comparisons. To address these issues, I implement approaches for image registration using statistical shape models and non-rigid registration. Lastly, I validate the spatial expression data obtained from microscopy images to other known spatial expression methods, and develop methods for comparing and calculating the significance between spatial expression patterns. I demonstrate these methods on two model developmental organisms: Arabidopsis and Drosophila.

Dissertation

Wide field-of-view (FOV) microscopy is of high importance to biological research and clinical diagnosis where a high-throughput screening of samples is needed. This thesis presents the development of several novel wide FOV imaging technologies and demonstrates their capabilities in longitudinal imaging of living organisms, on the scale of viral plaques to live cells and tissues.

The ePetri Dish is a wide FOV on-chip bright-field microscope. Here we applied an ePetri platform for plaque analysis of murine norovirus 1 (MNV-1). The ePetri offers the ability to dynamically track plaques at the individual cell death event level over a wide FOV of 6 mm × 4 mm at 30 min intervals. A density-based clustering algorithm is used to analyze the spatial-temporal distribution of cell death events to identify plaques at their earliest stages. We also demonstrate the capabilities of the ePetri in viral titer count and dynamically monitoring plaque formation, growth, and the influence of antiviral drugs.

We developed another wide FOV imaging technique, the Talbot microscope, for the fluorescence imaging of live cells. The Talbot microscope takes advantage of the Talbot effect and can generate a focal spot array to scan the fluorescence samples directly on-chip. It has a resolution of 1.2 μm and a FOV of ~13 mm². We further upgraded the Talbot microscope for the long-term time-lapse fluorescence imaging of live cell cultures, and analyzed the cells’ dynamic response to an anticancer drug.

We present two wide FOV endoscopes for tissue imaging, named the AnCam and the PanCam. The AnCam is based on the contact image sensor (CIS) technology, and can scan the whole anal canal within 10 seconds with a resolution of 89 μm, a maximum FOV of 100 mm × 120 mm, and a depth-of-field (DOF) of 0.65 mm. We also demonstrate the performance of the AnCam in whole anal canal imaging in both animal models and real patients. In addition to this, the PanCam is based on a smartphone platform integrated with a panoramic annular lens (PAL), and can capture a FOV of 18 mm × 120 mm in a single shot with a resolution of 100─140 μm. In this work we demonstrate the PanCam’s performance in imaging a stained tissue sample.

Stored red blood cell (sRBC) morphology is currently scored manually by technicians in a slow labor intensive process prone to error. This project proposes a way to simplify, automate, and expedite the morphology scoring process by using a novel microfluidic device that I designed to facilitate the flow of a single layer of red blood cells (RBCs). The appearance of this flow allows for the capture of a series of high clarity images captured via digital camera coupled to a microscope that are ideally suited for image analysis algorithm-based morphological scoring. During storage, RBCs heterogeneously shift from the form of discocyte to the reversibly altered form of discoechinocyte as storage lesion progresses. Beyond this level of degradation, the cell assumes the form of a spheroechinocyte or spherocyte and becomes irreparably damaged. The microfluidic device and image analysis algorithm developed in this research classified the individual morphology of 5000 RBCs taken from storage into the physiologically relevant category of either “discocyte,” “reversibly changed,” or “irreversibly changed.” This process took only 15 minutes. The accuracy in classification was verified as 92.6% in a separate trial when compared against classification of the same sample images via manual inspection. The morphological distribution of the RBC population remained consistent in both cases. The findings of this project suggest that microfluidic device assisted automated image analysis can provide a quick and effective way to quantitatively estimate the viability of a sRBC population and the extent of storage lesion endured. This technology could provide augmented RBC storage and transfusion research capabilities and have clinical applications, such as the ability to conveniently differentiate between the transfusion qualities of two sRBC units of the same age.
acase@tulane.edu

Research Doctorate - Doctor of Philosophy (PhD)
This thesis is quite diverse in nature. The first section is about three dimensional imaging, the second about high throughput phenotyping and the third genetic sequencing. It is all driven towards trying to understand how sugars are stored in stems. Each section contained within has an explanation of how and why this diversity was conducted. It has coalesced into novel techniques, inventions and genetic targets that when combined will undoubtedly help drive understanding of the post phloem pathway.

Systems biology studies the complex interactions between components of biological systems. One major goal of systems biology is to reconstruct the network of interactions between genes in response to normal and perturbed conditions. In order to accomplish this goal, large-scale data are needed. Accordingly, diverse powerful and high-throughput methods must be developed for this purpose. We have developed novel high-throughput technologies focusing on cellular phenotype profiling and now provide additional genome-scale analysis of gene and protein function. Few high-throughput methods can perform large-scale and high-throughput cellular phenotype profiling. However, analyzing gene expression patterns and protein behaviors in their cellular context will provide insights into important aspects of gene function. To complement current genomic approaches, we developed two technologies, the spotted cell microarray (cell chip) and the yeast spheroplast microarray, which allow high-throughput and highly-parallel cellular phenotype profiling including cell morphology and protein localization. These methods are based on printing collections of cells, combined with automated high-throughput microscopy, allowing systematic cellular phenotypic characterization. We used spotted cell microarrays to identify 15 new genes involved in the response of yeast to mating pheromone, 80 proteins associated with shmoo-tip 'localizome' upon pheromone stimulation and 5 genes involved in regulating the localization pattern of a group II intron encoded reverse transcriptase, LtrA, in Escherichia coli. Furthermore, in addition to morphology assays, yeast spheroplast microarrays were built for high-throughput immunofluorescence microscopy, allowing large-scale protein and RNA localization studies. In order to identify additional cell cycle genes, especially those difficult to identify in loss-of-function studies, we performed a genome-scale screen to identify yeast genes with overexpression-induced defects in cell cycle progression. After measuring the fraction of cells in G1 and G2/M phases of the cell cycle via high-throughput flow cytometry for each of ~5,800 ORFs and performing the validation and secondary assays, we observed that overexpression of 108 genes leads to reproducible and significant delay in the G1 or G2/M phase. Of 108 genes, 82 are newly implicated in the cell cycle and are likely to affect cell cycle progression via a gain-of-function mechanism. The G2/M category consists of 87 genes that showed dramatic enrichment in the regulation of mitotic cell cycle and related biological processes. YPR015C and SHE1 in the G2/M category were further characterized for their roles in cell cycle progression. We found that the G2/M delay caused by the overexpression of YPR015C and SHE1 likely results from the malfunction of spindle and chromosome segregation, which was supported by the observations of highly elevated population of large-budded cells in the pre-M phase, super-sensitivity to nocodazole, and high chromosome loss rates in these two overexpression strains. While the genes in the G2/M category were strongly enriched for cell cycle associated functions, no pathway was significantly enriched in the G1 category that is composed of 21 genes. However, the strongest enrichment for the G1 category consists of the genes involved in negative regulation of transcription. For instance, the overexpression of SKO1, a transcription repressor, resulted in strong cell cycle delay at G1 phase. Moreover, we found that the overexpression of SKO1 results in cell morphology changes that resembles mating yeast cells (shmoos) and activates the mating pheromone response pathway, thus explaining the G1 cell cycle arrest phenotype of SKO1 ORF strains.

Hyperspectral imaging has become one of the most popular technologies in plant phenotyping because it can efficiently and accurately predict numerous plant physiological features such as plant biomass, leaf moisture content, and chlorophyll content. Various hyperspectral imaging systems have been deployed in both greenhouse and field phenotyping activities. However, the hyperspectral imaging quality is severely affected by the continuously changing environmental conditions such as cloud cover, temperature and wind speed that induce noise in plant spectral data. Eliminating these environmental effects to improve imaging quality is critically important. In this thesis, two approaches were taken to address the imaging noise issue in greenhouse and field separately. First, a computational simulation model was built to simulate the greenhouse microclimate changes (such as the temperature and radiation distributions) through a 24-hour cycle in a research greenhouse. The simulated results were used to optimize the movement of an automated conveyor in the greenhouse: the plants were shuffled with the conveyor system with optimized frequency and distance to provide uniform growing conditions such as temperature and lighting intensity for each individual plant. The results showed the variance of the plants’ phenotyping feature measurements decreased significantly (i.e., by up to 83% in plant canopy size) in this conveyor greenhouse. Secondly, the environmental effects (i.e., sun radiation) on aerial hyperspectral images in field plant phenotyping were investigated and modeled. An artificial neural network (ANN) method was proposed to model the relationship between the image variation and environmental changes. Before the 2019 field test, a gantry system was designed and constructed to repeatedly collect time-series hyperspectral images with 2.5 minutes intervals of the corn plants under varying environmental conditions, which included sun radiation, solar zenith angle, diurnal time, humidity, temperature and wind speed. Over 8,000 hyperspectral images of corn (Zea mays L.) were collected with synchronized environmental data throughout the 2019 growing season. The models trained with the proposed ANN method were able to accurately predict the variations in imaging results (i.e., 82.3% for NDVI) caused by the changing environments. Thus, the ANN method can be used by remote sensing professionals to adjust or correct raw imaging data for changing environments to improve plant characterization.