The Discriminative Generalized Hough Transform for Localization of Highly Variable Objects and its Application for Surveillance Recordings

This work is about the localization of arbitrary objects in 2D images in general and the localization of persons in video surveillance recordings in particular. More precisely, it is about localizing specific landmarks. Thereby the possibilities and limitations of localization approaches based on the Generalized Hough Transform (GHT), especially of the Discriminative Generalized Hough Transform (DGHT) will be evaluated. GHT-based approaches determine the number of matching model and feature points and the most likely target point position is given by the highest number of matching model and feature points. Additionally, the DGHT comprises a statistical learning approach to generate optimal DGHT-models achieving good results on medical images. This work will show that the DGHT is not restricted to medical tasks but has issues with large target object variabilities, which are frequent in video surveillance tasks. As all GHT-based approaches also the DGHT only considers the number of matching model-feature-point-combinations, which means that all model points are treated independently. This work will show that model points are not independent of each other and considering them independently will result in high error rates. This drawback is analyzed and a universal solution, which is not only applicable for the DGHT but all GHT-based approaches, is presented. This solution is based on an additional classifier that takes the whole set of matching model-feature-point-combinations into account to estimate a confidence score. On all tested databases, this approach could reduce the error rates drastically by up to 94.9%. Furthermore, this work presents a general approach for combining multiple GHT-models into a deeper model. This can be used to combine the localization results of different object landmarks such as mouth, nose, and eyes. Similar to Convolutional Neural Networks (CNNs) this will split the target object variability into multiple and smaller variabilities. A comparison of GHT-based approaches with CNNs and a description of the advantages, disadvantages, and potential application of both approaches will conclude this work.Diese Arbeit beschäftigt sich im Allgemeinen mit der Lokalisierung von Objekten in 2D Bilddaten und im Speziellen mit der Lokalisierung von Personen in Videoüberwachungsaufnahmen. Genauer gesagt handelt es sich hierbei um die Lokalisierung spezieller Landmarken. Dabei werden die Möglichkeiten und Limiterungen von Lokalisierungsverfahren basierend auf der Generalisierten Hough Transformation (GHT) untersucht, insbesondere die der Diskriminativen Generalisierten Hough Transformation (DGHT). Bei GHT-basierten Ansätze wird die Anzahl an übereinstimmenden Modelpunkten und Merkmalspunkten ermittelt und die wahrscheinlicheste Objekt-Position ergibt sich aus der höchsten Anzahl an übereinstimmenden Model- und Merkmalspunkte. Die DGHT umfasst darüber hinaus noch ein statistisches Lernverfahren, um optimale DGHT-Modele zu erzeugen und erzielte damit auf medizinischen Bilder und Anwendungen sehr gute Erfolge. Wie sich in dieser Arbeit zeigen wird, ist die DGHT nicht auf medizinische Anwendungen beschränkt, hat allerdings Schwierigkeiten große Variabilität der Ziel-Objekte abzudecken, wie sie in Überwachungsszenarien zu erwarten sind. Genau wie alle GHT-basierten Ansätze leidet auch die DGHT unter dem Problem, dass lediglich die Anzahl an übereinstimmenden Model- und Merkmalspunkten ermittelt wird, was bedeutet, dass alle Modelpunkte unabhängig voneinander betrachtet werden. Dass Modelpunkte nicht unabhängig voneinander sind, wird im Laufe dieser Arbeit gezeigt werden, und die unabhängige Betrachtung führt gerade bei sehr variablen Zielobjekten zu einer hohen Fehlerrate. Dieses Problem wird in dieser Arbeit grundlegend untersucht und ein allgemeiner Lösungsansatz vorgestellt, welcher nicht nur für die DGHT sondern grundsätzlich für alle GHT-basierten Verfahren Anwendung finden kann. Die Lösung basiert auf der Integration eines zusätzlichen Klassifikators, welcher die gesamte Menge an übereinstimmenden Model- und Merkmalspunkten betrachtet und anhand dessen ein zusätzliches Konfidenzmaß vergibt. Dadurch konnte auf allen getesteten Datenbanken eine deutliche Reduktion der Fehlerrate erzielt werden von bis zu 94.9%. Darüber hinaus umfasst die Arbeit einen generellen Ansatz zur Kombination mehrere GHT-Model in einem tieferen Model. Dies kann dazu verwendet werden, um die Lokalisierungsergebnisse verschiedener Objekt-Landmarken zu kombinieren, z. B. die von Mund, Nase und Augen. Ähnlich wie auch bei Convolutional Neural Networks (CNNs) ist es damit möglich über mehrere Ebenen unterschiedliche Bereiche zu lokalisieren und somit die Variabilität des Zielobjektes in mehrere, leichter zu handhabenden Variabilitäten aufzuspalten. Abgeschlossen wird die Arbeit durch einen Vergleich von GHT-basierten Ansätzen mit CNNs und einer Beschreibung der Vor- und Nachteile und mögliche Einsatzfelder beider Verfahren

From nanometers to centimeters: Imaging across spatial scales with smart computer-aided microscopy

Microscopes have been an invaluable tool throughout the history of the life sciences, as they allow researchers to observe the miniscule details of living systems in space and time. However, modern biology studies complex and non-obvious phenotypes and their distributions in populations and thus requires that microscopes evolve from visual aids for anecdotal observation into instruments for objective and quantitative measurements. To this end, many cutting-edge developments in microscopy are fuelled by innovations in the computational processing of the generated images. Computational tools can be applied in the early stages of an experiment, where they allow for reconstruction of images with higher resolution and contrast or more colors compared to raw data. In the final analysis stage, state-of-the-art image analysis pipelines seek to extract interpretable and humanly tractable information from the high-dimensional space of images. In the work presented in this thesis, I performed super-resolution microscopy and wrote image analysis pipelines to derive quantitative information about multiple biological processes. I contributed to studies on the regulation of DNMT1 by implementing machine learning-based segmentation of replication sites in images and performed quantitative statistical analysis of the recruitment of multiple DNMT1 mutants. To study the spatiotemporal distribution of DNA damage response I performed STED microscopy and could provide a lower bound on the size of the elementary spatial units of DNA repair. In this project, I also wrote image analysis pipelines and performed statistical analysis to show a decoupling of DNA density and heterochromatin marks during repair. More on the experimental side, I helped in the establishment of a protocol for many-fold color multiplexing by iterative labelling of diverse structures via DNA hybridization. Turning from small scale details to the distribution of phenotypes in a population, I wrote a reusable pipeline for fitting models of cell cycle stage distribution and inhibition curves to high-throughput measurements to quickly quantify the effects of innovative antiproliferative antibody-drug-conjugates. The main focus of the thesis is BigStitcher, a tool for the management and alignment of terabyte-sized image datasets. Such enormous datasets are nowadays generated routinely with light-sheet microscopy and sample preparation techniques such as clearing or expansion. Their sheer size, high dimensionality and unique optical properties poses a serious bottleneck for researchers and requires specialized processing tools, as the images often do not fit into the main memory of most computers. BigStitcher primarily allows for fast registration of such many-dimensional datasets on conventional hardware using optimized multi-resolution alignment algorithms. The software can also correct a variety of aberrations such as fixed-pattern noise, chromatic shifts and even complex sample-induced distortions. A defining feature of BigStitcher, as well as the various image analysis scripts developed in this work is their interactivity. A central goal was to leverage the user's expertise at key moments and bring innovations from the big data world to the lab with its smaller and much more diverse datasets without replacing scientists with automated black-box pipelines. To this end, BigStitcher was implemented as a user-friendly plug-in for the open source image processing platform Fiji and provides the users with a nearly instantaneous preview of the aligned images and opportunities for manual control of all processing steps. With its powerful features and ease-of-use, BigStitcher paves the way to the routine application of light-sheet microscopy and other methods producing equally large datasets

[¹⁸F]fluorination of biorelevant arylboronic acid pinacol ester scaffolds synthesized by convergence techniques

Aim: The development of small molecules through convergent multicomponent reactions (MCR) has been boosted during the last decade due to the ability to synthesize, virtually without any side-products, numerous small drug-like molecules with several degrees of structural diversity.(1) The association of positron emission tomography (PET) labeling techniques in line with the “one-pot” development of biologically active compounds has the potential to become relevant not only for the evaluation and characterization of those MCR products through molecular imaging, but also to increase the library of radiotracers available. Therefore, since the [18F]fluorination of arylboronic acid pinacol ester derivatives tolerates electron-poor and electro-rich arenes and various functional groups,(2) the main goal of this research work was to achieve the 18F-radiolabeling of several different molecules synthesized through MCR. Materials and Methods: [18F]Fluorination of boronic acid pinacol esters was first extensively optimized using a benzaldehyde derivative in relation to the ideal amount of Cu(II) catalyst and precursor to be used, as well as the reaction solvent. Radiochemical conversion (RCC) yields were assessed by TLC-SG. The optimized radiolabeling conditions were subsequently applied to several structurally different MCR scaffolds comprising biologically relevant pharmacophores (e.g. β-lactam, morpholine, tetrazole, oxazole) that were synthesized to specifically contain a boronic acid pinacol ester group. Results: Radiolabeling with fluorine-18 was achieved with volumes (800 μl) and activities (≤ 2 GBq) compatible with most radiochemistry techniques and modules. In summary, an increase in the quantities of precursor or Cu(II) catalyst lead to higher conversion yields. An optimal amount of precursor (0.06 mmol) and Cu(OTf)2(py)4 (0.04 mmol) was defined for further reactions, with DMA being a preferential solvent over DMF. RCC yields from 15% to 76%, depending on the scaffold, were reproducibly achieved. Interestingly, it was noticed that the structure of the scaffolds, beyond the arylboronic acid, exerts some influence in the final RCC, with electron-withdrawing groups in the para position apparently enhancing the radiolabeling yield. Conclusion: The developed method with high RCC and reproducibility has the potential to be applied in line with MCR and also has a possibility to be incorporated in a later stage of this convergent “one-pot” synthesis strategy. Further studies are currently ongoing to apply this radiolabeling concept to fluorine-containing approved drugs whose boronic acid pinacol ester precursors can be synthesized through MCR (e.g. atorvastatin)