Studies of Single-Molecule Dynamics in Microorganisms

Fluorescence microscopy is one of the most extensively used techniques in the life sciences. Considering the non-invasive sample preparation, enabling live-cell compliant imaging, and the specific fluorescence labeling, allowing for a specific visualization of virtually any cellular compound, it is possible to localize even a single molecule in living cells. This makes modern fluorescence microscopy a powerful toolbox. In the recent decades, the development of new, "super-resolution" fluorescence microscopy techniques, which surpass the diffraction limit, revolutionized the field. Single-Molecule Localization Microscopy (SMLM) is a class of super-resolution microscopy methods and it enables resolution of down to tens of nanometers. SMLM methods like Photoactivated Localization Microscopy (PALM), (direct) Stochastic Optical Reconstruction Microscopy ((d)STORM), Ground-State Depletion followed by Individual Molecule Return (GSDIM) and Point Accumulation for Imaging in Nanoscale Topography (PAINT) have allowed to investigate both, the intracellular spatial organization of proteins and to observe their real-time dynamics at the single-molecule level in live cells. The focus of this thesis was the development of novel tools and strategies for live-cell SingleParticle Tracking PALM (sptPALM) imaging and implementing them for biological research. In the first part of this thesis, I describe the development of new Photoconvertible Fluorescent Proteins (pcFPs) which are optimized for sptPALM lowering the phototoxic damage caused by the imaging procedure. Furthermore, we show that we can utilize them together with Photoactivatable Fluorescent Proteins (paFPs) to enable multi-target labeling and read-out in a single color channel, which significantly simplifies the sample preparation and imaging routines as well as data analysis of multi-color PALM imaging of live cells. In parallel to developing new fluorescent proteins, I developed a high throughput data analysis pipeline. I have implemented this pipeline in my second project, described in the second part of this thesis, where I have investigated the protein organization and dynamics of the CRISPR-Cas antiviral defense mechanism of bacteria in vivo at a high spatiotemporal level with the sptPALM approach. I was successful to show the differences in the target search dynamics of the CRISPR effector complexes as well as of single Cas proteins for different target complementarities. I have also first data describing longer-lasting bound-times between effector complex and their potential targets in vivo, for which only in vitro data has been available till today. In summary, this thesis is a significant contribution for both, the advances of current sptPALM imaging methods, as well as for the understanding of the native behavior of CRISPR-Cas systems in vivo

AI-Enabled Contextual Representations for Image-based Integration in Health and Safety

Recent advancements in the area of Artificial Intelligence (AI) have made it the field of choice for automatically processing and summarizing information in big-data domains such as high-resolution images. This approach, however, is not a one-size-fits-all solution, and must be tailored to each application. Furthermore, each application comes with its own unique set of challenges including technical variations, validation of AI solutions, and contextual information. These challenges are addressed in three human-health and safety related applications: (i) an early warning system of slope failures in open-pit mining operations; (ii) the modeling and characterization of 3D cell culture models imaged with confocal microscopy; and (iii) precision medicine of biomarker discovery from patients with glioblastoma multiforme through digital pathology. The methodologies and results in each of these domains show how tailor-made AI solutions can be used for automatically extracting and summarizing pertinent information from big-data applications for enhanced decision making

Automatic CNN channel selection and effective detection on face and rotated aerial objects

Balancing accuracy and computational cost is a challenging task in computer vision. This is especially true for convolutional neural networks (CNNs), which required far larger scale of processing power than traditional learning algorithms. This thesis is aimed at the development of new CNN structures and loss functions to tackle the unbalanced accuracy-effciency issue in image classification and object detection, which are two fundamental yet challenging tasks of computer vision. For a CNN based object detector, the main computational cost is caused by the feature extractor (backbone), which has been originally applied to image classification.;Optimising the structure of CNN applied to image classification will bring benefits when it is applied to object detection. Although the outputs of detectors may vary across detection tasks, the challenges and the design principles among detectors are similar. Therefore, this thesis will start with face detection (i.e. a single object detection task), which is a significant branch of objection detection and has been widely used in real life. After that, object detection on aerial image will be investigated, which is a more challenging detection task.;Specifically, the objectives of this thesis are: 1. Optimising the CNN structures for image classification; 2. Developing a face detector which enables a trade-off between computational cost and accuracy; and 3. Proposing an object detector for aerial images, which suppresses the background noise without damaging the inference efficiency.;For the first target, this thesis aims to automatically optimise the topology of CNNs to generate the structure of fixed-length models, in which unnecessary convolutional kernels are removed. Experimental results have demonstrated that the optimised model can achieve comparable accuracy to the state-of-the-art models, across a broad range of datasets, whilst significantly reducing the number of parameters.;To tackle the unbalanced accuracy-effciency challenge in face detection, a novel context enhanced approach is proposed which improves the performance of the face detector in terms of both loss function and structure. For loss function optimisation, a hierarchical loss, referred to as 'triple loss' in this thesis, is introduced to optimise the feature pyramid network (FPN) based face detector. For structural optimisation, this thesis proposes a context-sensitive structure to increase the capacity of the network prediction. Experimental results indicate that the proposed method achieves a good balance between the accuracy and computational cost of face detection.;To suppress the background noise in aerial image object detection, this thesis presents a two-stage detector, named as 'SAFDet'. To be more specific, a rotation anchor-free-branch (RAFB) is proposed to regress the precise rectangle boundary. Asthe RAFB is anchor free, the computational cost is negligible during training. Meanwhile,a centre prediction module (CPM) is introduced to enhance the capabilities oftarget localisation and noise suppression from the background. As the CPM is only deployed during training, it does not increase the computational cost of inference. Experimental results indicate that the proposed method achieves a good balance between the accuracy and computational cost, and it effectively suppresses the background noise at the same time.Balancing accuracy and computational cost is a challenging task in computer vision. This is especially true for convolutional neural networks (CNNs), which required far larger scale of processing power than traditional learning algorithms. This thesis is aimed at the development of new CNN structures and loss functions to tackle the unbalanced accuracy-effciency issue in image classification and object detection, which are two fundamental yet challenging tasks of computer vision. For a CNN based object detector, the main computational cost is caused by the feature extractor (backbone), which has been originally applied to image classification.;Optimising the structure of CNN applied to image classification will bring benefits when it is applied to object detection. Although the outputs of detectors may vary across detection tasks, the challenges and the design principles among detectors are similar. Therefore, this thesis will start with face detection (i.e. a single object detection task), which is a significant branch of objection detection and has been widely used in real life. After that, object detection on aerial image will be investigated, which is a more challenging detection task.;Specifically, the objectives of this thesis are: 1. Optimising the CNN structures for image classification; 2. Developing a face detector which enables a trade-off between computational cost and accuracy; and 3. Proposing an object detector for aerial images, which suppresses the background noise without damaging the inference efficiency.;For the first target, this thesis aims to automatically optimise the topology of CNNs to generate the structure of fixed-length models, in which unnecessary convolutional kernels are removed. Experimental results have demonstrated that the optimised model can achieve comparable accuracy to the state-of-the-art models, across a broad range of datasets, whilst significantly reducing the number of parameters.;To tackle the unbalanced accuracy-effciency challenge in face detection, a novel context enhanced approach is proposed which improves the performance of the face detector in terms of both loss function and structure. For loss function optimisation, a hierarchical loss, referred to as 'triple loss' in this thesis, is introduced to optimise the feature pyramid network (FPN) based face detector. For structural optimisation, this thesis proposes a context-sensitive structure to increase the capacity of the network prediction. Experimental results indicate that the proposed method achieves a good balance between the accuracy and computational cost of face detection.;To suppress the background noise in aerial image object detection, this thesis presents a two-stage detector, named as 'SAFDet'. To be more specific, a rotation anchor-free-branch (RAFB) is proposed to regress the precise rectangle boundary. Asthe RAFB is anchor free, the computational cost is negligible during training. Meanwhile,a centre prediction module (CPM) is introduced to enhance the capabilities oftarget localisation and noise suppression from the background. As the CPM is only deployed during training, it does not increase the computational cost of inference. Experimental results indicate that the proposed method achieves a good balance between the accuracy and computational cost, and it effectively suppresses the background noise at the same time

Morphological Changes of Neuronal Growth Cones by Intracellular Signaling and Cell Culture Conditions

Proper wiring of neurons is key to the functionality of nervous system. This is achieved by a highly motile structure referred to as the neuronal growth cone located at the tip of axons and dendrites during both development and regeneration. The morphology of growth cone is a product of both intracellular signaling pathways and extracellular factors, and is closely linked with neuronal outgrowth. Studies of how growth cone morphology is changed by key regulatory proteins and by cell culture conditions are therefore important in elucidating the mechanisms of neurite regeneration. In the first part of this thesis, we identified a single tyrosine residue (Y499) in Aplysia cortactin that is important for regulating filopodia formation in growth cone. Overexpression of the 499F phospho-deficient cortactin mutant decreased filopodia length and density, whereas overexpression of the 499E phospho-mimetic mutant increased filopodia length, regardless of the phosphorylation state of Y505 or Y509. Using a custom-made antibody against cortactin phosphorylated at Y499, we showed that phosphorylated cortactin is enriched in the peripheral domain, specifically along the leading edge. We found that treatment with the Src inhibitor PP2 decreased cortactin phosphorylation, while overexpression of Src2 increased cortactin phosphorylation. We demonstrated that the leading edge localization of phosphorylated cortactin is F-actin independent, and important in promoting filopodia formation. Finally, by interfering both with cortactin phosphorylation and Arp2/3 activation, we found that Arp2/3 complex acts downstream of cortactin to regulate filopodia density but not length. In conclusion, we have characterized a tyrosine phosphorylation site in Aplysia cortactin that plays a major role in the Src/cortactin/Arp2/3 signaling pathway controlling filopodia formation. In the second part of this thesis, we offer a comprehensive cellular analysis of the motile behavior of Aplysia growth cones on two-dimensional (2D) culture beyond the pausing state. We found that average growth cone size decreased with cell culture time whereas average growth rate increased. This inverse correlation of growth rate and growth cone size was due to the occurrence of large growth cones with a peripheral domain larger than 100 μm2. The large pausing growth cones had central domains that were less consistently aligned with the direction of growth and could be converted into smaller, faster-growing growth cones by addition of a three-dimensional (3D) collagen gel. We conclude that the significant lateral expansion of lamellipodia and filopodia as observed during these culture conditions has a negative effect on neurite growth. Further, using the novel collagen gel and an easy-to-make microwell device, we developed a simple protocol for 3D culture of Aplysia bag cell neuron. We found that the morphology and growth pattern of bag cell growth cones in 3D culture closely resemble the ones of growth cones observed in vivo, and demonstrated the capability of our device for high-resolution imaging of cytoskeletal and signaling proteins as well as organelles. We expect that our microwell device will facilitate a wider adoption of 3D neuronal cultures to study the mechanisms of neurite regeneration

Experimental analysis of computer system dependability

This paper reviews an area which has evolved over the past 15 years: experimental analysis of computer system dependability. Methodologies and advances are discussed for three basic approaches used in the area: simulated fault injection, physical fault injection, and measurement-based analysis. The three approaches are suited, respectively, to dependability evaluation in the three phases of a system's life: design phase, prototype phase, and operational phase. Before the discussion of these phases, several statistical techniques used in the area are introduced. For each phase, a classification of research methods or study topics is outlined, followed by discussion of these methods or topics as well as representative studies. The statistical techniques introduced include the estimation of parameters and confidence intervals, probability distribution characterization, and several multivariate analysis methods. Importance sampling, a statistical technique used to accelerate Monte Carlo simulation, is also introduced. The discussion of simulated fault injection covers electrical-level, logic-level, and function-level fault injection methods as well as representative simulation environments such as FOCUS and DEPEND. The discussion of physical fault injection covers hardware, software, and radiation fault injection methods as well as several software and hybrid tools including FIAT, FERARI, HYBRID, and FINE. The discussion of measurement-based analysis covers measurement and data processing techniques, basic error characterization, dependency analysis, Markov reward modeling, software-dependability, and fault diagnosis. The discussion involves several important issues studies in the area, including fault models, fast simulation techniques, workload/failure dependency, correlated failures, and software fault tolerance

Object Recognition

Vision-based object recognition tasks are very familiar in our everyday activities, such as driving our car in the correct lane. We do these tasks effortlessly in real-time. In the last decades, with the advancement of computer technology, researchers and application developers are trying to mimic the human's capability of visually recognising. Such capability will allow machine to free human from boring or dangerous jobs

How much BiGAN and CycleGAN-learned hidden features are effective for COVID-19 detection from CT images? A comparative study

Bidirectional generative adversarial networks (BiGANs) and cycle generative adversarial networks (CycleGANs) are two emerging machine learning models that, up to now, have been used as generative models, i.e., to generate output data sampled from a target probability distribution. However, these models are also equipped with encoding modules, which, after weakly supervised training, could be, in principle, exploited for the extraction of hidden features from the input data. At the present time, how these extracted features could be effectively exploited for classification tasks is still an unexplored field. Hence, motivated by this consideration, in this paper, we develop and numerically test the performance of a novel inference engine that relies on the exploitation of BiGAN and CycleGAN-learned hidden features for the detection of COVID-19 disease from other lung diseases in computer tomography (CT) scans. In this respect, the main contributions of the paper are twofold. First, we develop a kernel density estimation (KDE)-based inference method, which, in the training phase, leverages the hidden features extracted by BiGANs and CycleGANs for estimating the (a priori unknown) probability density function (PDF) of the CT scans of COVID-19 patients and, then, in the inference phase, uses it as a target COVID-PDF for the detection of COVID diseases. As a second major contribution, we numerically evaluate and compare the classification accuracies of the implemented BiGAN and CycleGAN models against the ones of some state-of-the-art methods, which rely on the unsupervised training of convolutional autoencoders (CAEs) for attaining feature extraction. The performance comparisons are carried out by considering a spectrum of different training loss functions and distance metrics. The obtained classification accuracies of the proposed CycleGAN-based (resp., BiGAN-based) models outperform the corresponding ones of the considered benchmark CAE-based models of about 16% (resp., 14%)