A Computational Framework for Ultrastructural Mapping of Neural Circuitry

Circuitry mapping of metazoan neural systems is difficult because canonical neural regions (regions containing one or more copies of all components) are large, regional borders are uncertain, neuronal diversity is high, and potential network topologies so numerous that only anatomical ground truth can resolve them. Complete mapping of a specific network requires synaptic resolution, canonical region coverage, and robust neuronal classification. Though transmission electron microscopy (TEM) remains the optimal tool for network mapping, the process of building large serial section TEM (ssTEM) image volumes is rendered difficult by the need to precisely mosaic distorted image tiles and register distorted mosaics. Moreover, most molecular neuronal class markers are poorly compatible with optimal TEM imaging. Our objective was to build a complete framework for ultrastructural circuitry mapping. This framework combines strong TEM-compliant small molecule profiling with automated image tile mosaicking, automated slice-to-slice image registration, and gigabyte-scale image browsing for volume annotation. Specifically we show how ultrathin molecular profiling datasets and their resultant classification maps can be embedded into ssTEM datasets and how scripted acquisition tools (SerialEM), mosaicking and registration (ir-tools), and large slice viewers (MosaicBuilder, Viking) can be used to manage terabyte-scale volumes. These methods enable large-scale connectivity analyses of new and legacy data. In well-posed tasks (e.g., complete network mapping in retina), terabyte-scale image volumes that previously would require decades of assembly can now be completed in months. Perhaps more importantly, the fusion of molecular profiling, image acquisition by SerialEM, ir-tools volume assembly, and data viewers/annotators also allow ssTEM to be used as a prospective tool for discovery in nonneural systems and a practical screening methodology for neurogenetics. Finally, this framework provides a mechanism for parallelization of ssTEM imaging, volume assembly, and data analysis across an international user base, enhancing the productivity of a large cohort of electron microscopists

Human-Centric Machine Vision

Recently, the algorithms for the processing of the visual information have greatly evolved, providing efficient and effective solutions to cope with the variability and the complexity of real-world environments. These achievements yield to the development of Machine Vision systems that overcome the typical industrial applications, where the environments are controlled and the tasks are very specific, towards the use of innovative solutions to face with everyday needs of people. The Human-Centric Machine Vision can help to solve the problems raised by the needs of our society, e.g. security and safety, health care, medical imaging, and human machine interface. In such applications it is necessary to handle changing, unpredictable and complex situations, and to take care of the presence of humans

Methods for Automated Neuron Image Analysis

Knowledge of neuronal cell morphology is essential for performing specialized analyses in the endeavor to understand neuron behavior and unravel the underlying principles of brain function. Neurons can be captured with a high level of detail using modern microscopes, but many neuroscientific studies require a more explicit and accessible representation than offered by the resulting images, underscoring the need for digital reconstruction of neuronal morphology from the images into a tree-like graph structure. This thesis proposes new computational methods for automated detection and reconstruction of neurons from fluorescence microscopy images. Specifically, the successive chapters describe and evaluate original solutions to problems such as the detection of landmarks (critical points) of the neuronal tree, complete tracing and reconstruction of the tree, and the detection of regions containing neurons in high-content screens

Interactive Learning for the Analysis of Biomedical and Industrial Imagery

In der vorliegenden Dissertation werden Methoden des überwachten Lernens untersucht und auf die Analyse und die Segmentierung digitaler Bilddaten angewendet, die aus diversen Forschungsgebieten stammen. Die Segmentierung und die Klassifikation spielen eine wichtige Rolle in der biomedizinischen und industriellen Bildverarbeitung, häufig basiert darauf weitere Erkennung und Quantifikation. Viele problemspezifische Ansätze existieren für die unterschiedlichsten Fragestellungen und nutzen meist spezifisches Vorwissen aus den jeweiligen Bilddaten aus. In dieser Arbeit wird ein überwachtes Lernverfahren vorgestellt, das mehrere Objekte und deren Klassen gleichzeitig segmentieren und unterscheiden kann. Die Methode ist generell genug um einen wichtigen Bereich von Anwendungen abzudecken, für deren Lösung lokale Merkmale eine Rolle spielen. Segmentierungsergebnisse dieses Ansatzes werden auf verschiedenen Datensätzen mit unterschiedlichen Problemstellungen gezeigt. Die Resultate unterstreichen die Anwendbarkeit der Lernmethode für viele biomedizinische und industrielle Anwendungen, ohne dass explizite Kenntnisse der Bildverarbeitung und Programmierung vorausgesetzt werden müssen. Der Ansatz basiert auf generellen Merkmalsklassen, die es erlauben lokal Strukturen wie Farbe, Textur und Kanten zu beschreiben. Zu diesem Zweck wurde eine interaktive Software implementiert, welche, für gewöhnliche Bildgrößen, in Echtzeit arbeitet und es somit einem Domänenexperten erlaubt Segmentierungs- und Klassifikationsaufgaben interaktiv zu bearbeiten. Dafür sind keine Kenntnisse in der Bildverarbeitung nötig, da sich die Benutzerinteraktion auf intuitives Markieren mit einem Pinselwerkzeug beschränkt. Das interaktiv trainierte System kann dann ohne weitere Benutzerinteraktion auf viele neue Bilder angewendet werden. Der Ansatz ist auf Segmentierungsprobleme beschränkt, für deren Lösung lokale diskriminative Merkmale ausreichen. Innerhalb dieser Einschränkung zeigt der Algorithmus jedoch erstaunlich gute Resultate, die in einer applikationsspezifischen Prozedur weiter verbessert werden können. Das Verfahren unterstützt bis zu vierdimensionale, multispektrale Bilddaten in vereinheitlichter Weise. Um die Anwendbar- und Übertragbarkeit der Methode weiter zu illustrieren wurden mehrere echte Anwendungsfälle, kommend aus verschiedenen bildgebenden Bereichen, untersucht. Darunter sind u. A. die Segmentierung von Tumorgewebe, aufgenommen mittelsWeitfeldmikroskopie, die Quantifikation von Zellwanderungen in konfokalmikroskopischen Aufnahmen für die Untersuchung der adulten Neurogenese, die Segmentierung von Blutgefäßen in der Retina des Auges, das Verfolgen von Kupferdrähten in einer Anwendung zur Produktauthentifikation und die Qualitätskontrolle von Mikroskopiebildern im Kontext von Hochdurchsatz-Experimenten. Desweiteren wurde eine neue Klassifikationsmethode basierend auf globalen Frequenzschätzungen für die Prozesskontrolle des Papieranlegers an Druckmaschinen entwickelt

A Modular and Open-Source Framework for Virtual Reality Visualisation and Interaction in Bioimaging

Life science today involves computational analysis of a large amount and variety of data, such as volumetric data acquired by state-of-the-art microscopes, or mesh data from analysis of such data or simulations. The advent of new imaging technologies, such as lightsheet microscopy, has resulted in the users being confronted with an ever-growing amount of data, with even terabytes of imaging data created within a day. With the possibility of gentler and more high-performance imaging, the spatiotemporal complexity of the model systems or processes of interest is increasing as well. Visualisation is often the first step in making sense of this data, and a crucial part of building and debugging analysis pipelines. It is therefore important that visualisations can be quickly prototyped, as well as developed or embedded into full applications. In order to better judge spatiotemporal relationships, immersive hardware, such as Virtual or Augmented Reality (VR/AR) headsets and associated controllers are becoming invaluable tools. In this work we present scenery, a modular and extensible visualisation framework for the Java VM that can handle mesh and large volumetric data, containing multiple views, timepoints, and color channels. scenery is free and open-source software, works on all major platforms, and uses the Vulkan or OpenGL rendering APIs. We introduce scenery's main features, and discuss its use with VR/AR hardware and in distributed rendering. In addition to the visualisation framework, we present a series of case studies, where scenery can provide tangible benefit in developmental and systems biology: With Bionic Tracking, we demonstrate a new technique for tracking cells in 4D volumetric datasets via tracking eye gaze in a virtual reality headset, with the potential to speed up manual tracking tasks by an order of magnitude. We further introduce ideas to move towards virtual reality-based laser ablation and perform a user study in order to gain insight into performance, acceptance and issues when performing ablation tasks with virtual reality hardware in fast developing specimen. To tame the amount of data originating from state-of-the-art volumetric microscopes, we present ideas how to render the highly-efficient Adaptive Particle Representation, and finally, we present sciview, an ImageJ2/Fiji plugin making the features of scenery available to a wider audience.:Abstract Foreword and Acknowledgements Overview and Contributions Part 1 - Introduction 1 Fluorescence Microscopy 2 Introduction to Visual Processing 3 A Short Introduction to Cross Reality 4 Eye Tracking and Gaze-based Interaction Part 2 - VR and AR for System Biology 5 scenery — VR/AR for Systems Biology 6 Rendering 7 Input Handling and Integration of External Hardware 8 Distributed Rendering 9 Miscellaneous Subsystems 10 Future Development Directions Part III - Case Studies C A S E S T U D I E S 11 Bionic Tracking: Using Eye Tracking for Cell Tracking 12 Towards Interactive Virtual Reality Laser Ablation 13 Rendering the Adaptive Particle Representation 14 sciview — Integrating scenery into ImageJ2 & Fiji Part IV - Conclusion 15 Conclusions and Outlook Backmatter & Appendices A Questionnaire for VR Ablation User Study B Full Correlations in VR Ablation Questionnaire C Questionnaire for Bionic Tracking User Study List of Tables List of Figures Bibliography Selbstständigkeitserklärun

Doctor of Philosophy

dissertationElectron microscopy can visualize synapses at nanometer resolution, and can thereby capture the fine structure of these contacts. However, this imaging method lacks three key elements: temporal information, protein visualization, and large volume reconstruction. For my dissertation, I developed three methods in electron microscopy that overcame these limitations. First, I developed a method to freeze neurons at any desired time point after a stimulus to study synaptic vesicle cycle. Second, I developed a method to couple super-resolution fluorescence microscopy and electron microscopy to pinpoint the location of proteins in electron micrographs at nanometer resolution. Third, I collaborated with computer scientists to develop methods for semi-automated reconstruction of nervous system. I applied these techniques to answer two fundamental questions in synaptic biology. Which vesicles fuse in response to a stimulus? How are synaptic vesicles recovered at synapses after fusion? Only vesicles that are in direct contact with plasma membrane fuse upon stimulation. The active zone in C. elegans is broad, but primed vesicles are concentrated around the dense projection. Following exocytosis of synaptic vesicles, synaptic vesicle membrane was recovered rapidly at two distinct locations at a synapse: the dense projection and adherens junctions. These studies suggest that there may be a novel form of ultrafast endocytosis

Doctor of Philosophy

dissertationIt is imperative to obtain a complete network graph of at least one representative retina if we are to fully understand vertebrate vision. Synaptic connectomics endeavors to construct such graphs. Though previously prevented by hardware and software limitations, the creation of customized viewing and analysis software, affordable data storage, and advances in electron imaging platform control now permit connectome assembly and analysis. The optimal strategy for building complete connectomes utilizes automated transmission electron imaging with 2 nm or better resolution, molecular tags for cell identification, open access data volumes for navigation, and annotation with open source tools to build three-dimensional cell libraries, complete network diagrams, and connectivity databases. In a few years, the first retinal connectome analyses reveal that many well-studied cells participate in much richer networks than expected. Collectively, these results impel a refactoring of the inner plexiform layer, while providing proof of concept for connectomics as a game-changing approach for a new era of scientific discovery

Segmentation and Characterization of Small Retinal Vessels in Fundus Images Using the Tensor Voting Approach

RÉSUMÉ La rétine permet de visualiser facilement une partie du réseau vasculaire humain. Elle offre ainsi un aperçu direct sur le développement et le résultat de certaines maladies liées au réseau vasculaire dans son entier. Chaque complication visible sur la rétine peut avoir un impact sur la capacité visuelle du patient. Les plus petits vaisseaux sanguins sont parmi les premières structures anatomiques affectées par la progression d’une maladie, être capable de les analyser est donc crucial. Les changements dans l’état, l’aspect, la morphologie, la fonctionnalité, ou même la croissance des petits vaisseaux indiquent la gravité des maladies. Le diabète est une maladie métabolique qui affecte des millions de personnes autour du monde. Cette maladie affecte le taux de glucose dans le sang et cause des changements pathologiques dans différents organes du corps humain. La rétinopathie diabétique décrit l’en- semble des conditions et conséquences du diabète au niveau de la rétine. Les petits vaisseaux jouent un rôle dans le déclenchement, le développement et les conséquences de la rétinopa- thie. Dans les dernières étapes de cette maladie, la croissance des nouveaux petits vaisseaux, appelée néovascularisation, présente un risque important de provoquer la cécité. Il est donc crucial de détecter tous les changements qui ont lieu dans les petits vaisseaux de la rétine dans le but de caractériser les vaisseaux sains et les vaisseaux anormaux. La caractérisation en elle-même peut faciliter la détection locale d’une rétinopathie spécifique. La segmentation automatique des structures anatomiques comme le réseau vasculaire est une étape cruciale. Ces informations peuvent être fournies à un médecin pour qu’elles soient considérées lors de son diagnostic. Dans les systèmes automatiques d’aide au diagnostic, le rôle des petits vaisseaux est significatif. Ne pas réussir à les détecter automatiquement peut conduire à une sur-segmentation du taux de faux positifs des lésions rouges dans les étapes ultérieures. Les efforts de recherche se sont concentrés jusqu’à présent sur la localisation précise des vaisseaux de taille moyenne. Les modèles existants ont beaucoup plus de difficultés à extraire les petits vaisseaux sanguins. Les modèles existants ne sont pas robustes à la grande variance d’apparence des vaisseaux ainsi qu’à l’interférence avec l’arrière-plan. Les modèles de la littérature existante supposent une forme générale qui n’est pas suffisante pour s’adapter à la largeur étroite et la courbure qui caractérisent les petits vaisseaux sanguins. De plus, le contraste avec l’arrière-plan dans les régions des petits vaisseaux est très faible. Les méthodes de segmentation ou de suivi produisent des résultats fragmentés ou discontinus. Par ailleurs, la segmentation des petits vaisseaux est généralement faite aux dépends de l’amplification du bruit. Les modèles déformables sont inadéquats pour segmenter les petits vaisseaux. Les forces utilisées ne sont pas assez flexibles pour compenser le faible contraste, la largeur, et vii la variance des vaisseaux. Enfin, les approches de type apprentissage machine nécessitent un entraînement avec une base de données étiquetée. Il est très difficile d’obtenir ces bases de données dans le cas des petits vaisseaux. Cette thèse étend les travaux de recherche antérieurs en fournissant une nouvelle mé- thode de segmentation des petits vaisseaux rétiniens. La détection de ligne à échelles multiples (MSLD) est une méthode récente qui démontre une bonne performance de segmentation dans les images de la rétine, tandis que le vote tensoriel est une méthode proposée pour reconnecter les pixels. Une approche combinant un algorithme de détection de ligne et de vote tensoriel est proposée. L’application des détecteurs de lignes a prouvé son efficacité à segmenter les vais- seaux de tailles moyennes. De plus, les approches d’organisation perceptuelle comme le vote tensoriel ont démontré une meilleure robustesse en combinant les informations voisines d’une manière hiérarchique. La méthode de vote tensoriel est plus proche de la perception humain que d’autres modèles standards. Comme démontré dans ce manuscrit, c’est un outil pour segmenter les petits vaisseaux plus puissant que les méthodes existantes. Cette combinaison spécifique nous permet de surmonter les défis de fragmentation éprouvés par les méthodes de type modèle déformable au niveau des petits vaisseaux. Nous proposons également d’utiliser un seuil adaptatif sur la réponse de l’algorithme de détection de ligne pour être plus robuste aux images non-uniformes. Nous illustrons également comment une combinaison des deux méthodes individuelles, à plusieurs échelles, est capable de reconnecter les vaisseaux sur des distances variables. Un algorithme de reconstruction des vaisseaux est également proposé. Cette dernière étape est nécessaire car l’information géométrique complète est requise pour pouvoir utiliser la segmentation dans un système d’aide au diagnostic. La segmentation a été validée sur une base de données d’images de fond d’oeil à haute résolution. Cette base contient des images manifestant une rétinopathie diabétique. La seg- mentation emploie des mesures de désaccord standards et aussi des mesures basées sur la perception. En considérant juste les petits vaisseaux dans les images de la base de données, l’amélioration dans le taux de sensibilité que notre méthode apporte par rapport à la méthode standard de détection multi-niveaux de lignes est de 6.47%. En utilisant les mesures basées sur la perception, l’amélioration est de 7.8%. Dans une seconde partie du manuscrit, nous proposons également une méthode pour caractériser les rétines saines ou anormales. Certaines images contiennent de la néovascula- risation. La caractérisation des vaisseaux en bonne santé ou anormale constitue une étape essentielle pour le développement d’un système d’aide au diagnostic. En plus des défis que posent les petits vaisseaux sains, les néovaisseaux démontrent eux un degré de complexité encore plus élevé. Ceux-ci forment en effet des réseaux de vaisseaux à la morphologie com- plexe et inhabituelle, souvent minces et à fortes courbures. Les travaux existants se limitent viii à l’utilisation de caractéristiques de premier ordre extraites des petits vaisseaux segmentés. Notre contribution est d’utiliser le vote tensoriel pour isoler les jonctions vasculaires et d’uti- liser ces jonctions comme points d’intérêts. Nous utilisons ensuite une statistique spatiale de second ordre calculée sur les jonctions pour caractériser les vaisseaux comme étant sains ou pathologiques. Notre méthode améliore la sensibilité de la caractérisation de 9.09% par rapport à une méthode de l’état de l’art. La méthode développée s’est révélée efficace pour la segmentation des vaisseaux réti- niens. Des tenseurs d’ordre supérieur ainsi que la mise en œuvre d’un vote par tenseur via un filtrage orientable pourraient être étudiés pour réduire davantage le temps d’exécution et résoudre les défis encore présents au niveau des jonctions vasculaires. De plus, la caractéri- sation pourrait être améliorée pour la détection de la rétinopathie proliférative en utilisant un apprentissage supervisé incluant des cas de rétinopathie diabétique non proliférative ou d’autres pathologies. Finalement, l’incorporation des méthodes proposées dans des systèmes d’aide au diagnostic pourrait favoriser le dépistage régulier pour une détection précoce des rétinopathies et d’autres pathologies oculaires dans le but de réduire la cessité au sein de la population.----------ABSTRACT As an easily accessible site for the direct observation of the circulation system, human retina can offer a unique insight into diseases development or outcome. Retinal vessels are repre- sentative of the general condition of the whole systematic circulation, and thus can act as a "window" to the status of the vascular network in the whole body. Each complication on the retina can have an adverse impact on the patient’s sight. In this direction, small vessels’ relevance is very high as they are among the first anatomical structures that get affected as diseases progress. Moreover, changes in the small vessels’ state, appearance, morphology, functionality, or even growth indicate the severity of the diseases. This thesis will focus on the retinal lesions due to diabetes, a serious metabolic disease affecting millions of people around the world. This disorder disturbs the natural blood glucose levels causing various pathophysiological changes in different systems across the human body. Diabetic retinopathy is the medical term that describes the condition when the fundus and the retinal vessels are affected by diabetes. As in other diseases, small vessels play a crucial role in the onset, the development, and the outcome of the retinopathy. More importantly, at the latest stage, new small vessels, or neovascularizations, growth constitutes a factor of significant risk for blindness. Therefore, there is a need to detect all the changes that occur in the small retinal vessels with the aim of characterizing the vessels to healthy or abnormal. The characterization, in turn, can facilitate the detection of a specific retinopathy locally, like the sight-threatening proliferative diabetic retinopathy. Segmentation techniques can automatically isolate important anatomical structures like the vessels, and provide this information to the physician to assist him in the final decision. In comprehensive systems for the automatization of DR detection, small vessels role is significant as missing them early in a CAD pipeline might lead to an increase in the false positive rate of red lesions in subsequent steps. So far, the efforts have been concentrated mostly on the accurate localization of the medium range vessels. In contrast, the existing models are weak in case of the small vessels. The required generalization to adapt an existing model does not allow the approaches to be flexible, yet robust to compensate for the increased variability in the appearance as well as the interference with the background. So far, the current template models (matched filtering, line detection, and morphological processing) assume a general shape for the vessels that is not enough to approximate the narrow, curved, characteristics of the small vessels. Additionally, due to the weak contrast in the small vessel regions, the current segmentation and the tracking methods produce fragmented or discontinued results. Alternatively, the small vessel segmentation can be accomplished at the expense of x background noise magnification, in the case of using thresholding or the image derivatives methods. Furthermore, the proposed deformable models are not able to propagate a contour to the full extent of the vasculature in order to enclose all the small vessels. The deformable model external forces are ineffective to compensate for the low contrast, the low width, the high variability in the small vessel appearance, as well as the discontinuities. Internal forces, also, are not able to impose a global shape constraint to the contour that could be able to approximate the variability in the appearance of the vasculature in different categories of vessels. Finally, machine learning approaches require the training of a classifier on a labelled set. Those sets are difficult to be obtained, especially in the case of the smallest vessels. In the case of the unsupervised methods, the user has to predefine the number of clusters and perform an effective initialization of the cluster centers in order to converge to the global minimum. This dissertation expanded the previous research work and provides a new segmentation method for the smallest retinal vessels. Multi-scale line detection (MSLD) is a recent method that demonstrates good segmentation performance in the retinal images, while tensor voting is a method first proposed for reconnecting pixels. For the first time, we combined the line detection with the tensor voting framework. The application of the line detectors has been proved an effective way to segment medium-sized vessels. Additionally, perceptual organization approaches like tensor voting, demonstrate increased robustness by combining information coming from the neighborhood in a hierarchical way. Tensor voting is closer than standard models to the way human perception functions. As we show, it is a more powerful tool to segment small vessels than the existing methods. This specific combination allows us to overcome the apparent fragmentation challenge of the template methods at the smallest vessels. Moreover, we thresholded the line detection response adaptively to compensate for non-uniform images. We also combined the two individual methods in a multi-scale scheme in order to reconnect vessels at variable distances. Finally, we reconstructed the vessels from their extracted centerlines based on pixel painting as complete geometric information is required to be able to utilize the segmentation in a CAD system. The segmentation was validated on a high-resolution fundus image database that in- cludes diabetic retinopathy images of varying stages, using standard discrepancy as well as perceptual-based measures. When only the smallest vessels are considered, the improve- ments in the sensitivity rate for the database against the standard multi-scale line detection method is 6.47%. For the perceptual-based measure, the improvement is 7.8% against the basic method. The second objective of the thesis was to implement a method for the characterization of isolated retinal areas into healthy or abnormal cases. Some of the original images, from which xi these patches are extracted, contain neovascularizations. Investigation of image features for the vessels characterization to healthy or abnormal constitutes an essential step in the direction of developing CAD system for the automatization of DR screening. Given that the amount of data will significantly increase under CAD systems, the focus on this category of vessels can facilitate the referral of sight-threatening cases to early treatment. In addition to the challenges that small healthy vessels pose, neovessels demonstrate an even higher degree of complexity as they form networks of convolved, twisted, looped thin vessels. The existing work is limited to the use of first-order characteristics extracted from the small segmented vessels that limits the study of patterns. Our contribution is in using the tensor voting framework to isolate the retinal vascular junctions and in turn using those junctions as points of interests. Second, we exploited second-order statistics computed on the junction spatial distribution to characterize the vessels as healthy or neovascularizations. In fact, the second-order spatial statistics extracted from the junction distribution are combined with widely used features to improve the characterization sensitivity by 9.09% over the state of art. The developed method proved effective for the segmentation of the retinal vessels. Higher order tensors along with the implementation of tensor voting via steerable filtering could be employed to further reduce the execution time, and resolve the challenges at vascular junctions. Moreover, the characterization could be advanced to the detection of prolifera- tive retinopathy by extending the supervised learning to include non-proliferative diabetic retinopathy cases or other pathologies. Ultimately, the incorporation of the methods into CAD systems could facilitate screening for the effective reduction of the vision-threatening diabetic retinopathy rates, or the early detection of other than ocular pathologies