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

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

Variational image fusion

The main goal of this work is the fusion of multiple images to a single composite that offers more information than the individual input images. We approach those fusion tasks within a variational framework. First, we present iterative schemes that are well-suited for such variational problems and related tasks. They lead to efficient algorithms that are simple to implement and well-parallelisable. Next, we design a general fusion technique that aims for an image with optimal local contrast. This is the key for a versatile method that performs well in many application areas such as multispectral imaging, decolourisation, and exposure fusion. To handle motion within an exposure set, we present the following two-step approach: First, we introduce the complete rank transform to design an optic flow approach that is robust against severe illumination changes. Second, we eliminate remaining misalignments by means of brightness transfer functions that relate the brightness values between frames. Additional knowledge about the exposure set enables us to propose the first fully coupled method that jointly computes an aligned high dynamic range image and dense displacement fields. Finally, we present a technique that infers depth information from differently focused images. In this context, we additionally introduce a novel second order regulariser that adapts to the image structure in an anisotropic way.Das Hauptziel dieser Arbeit ist die Fusion mehrerer Bilder zu einem Einzelbild, das mehr Informationen bietet als die einzelnen Eingangsbilder. Wir verwirklichen diese Fusionsaufgaben in einem variationellen Rahmen. Zunächst präsentieren wir iterative Schemata, die sich gut für solche variationellen Probleme und verwandte Aufgaben eignen. Danach entwerfen wir eine Fusionstechnik, die ein Bild mit optimalem lokalen Kontrast anstrebt. Dies ist der Schlüssel für eine vielseitige Methode, die gute Ergebnisse für zahlreiche Anwendungsbereiche wie Multispektralaufnahmen, Bildentfärbung oder Belichtungsreihenfusion liefert. Um Bewegungen in einer Belichtungsreihe zu handhaben, präsentieren wir folgenden Zweischrittansatz: Zuerst stellen wir die komplette Rangtransformation vor, um eine optische Flussmethode zu entwerfen, die robust gegenüber starken Beleuchtungsänderungen ist. Dann eliminieren wir verbleibende Registrierungsfehler mit der Helligkeitstransferfunktion, welche die Helligkeitswerte zwischen Bildern in Beziehung setzt. Zusätzliches Wissen über die Belichtungsreihe ermöglicht uns, die erste vollständig gekoppelte Methode vorzustellen, die gemeinsam ein registriertes Hochkontrastbild sowie dichte Bewegungsfelder berechnet. Final präsentieren wir eine Technik, die von unterschiedlich fokussierten Bildern Tiefeninformation ableitet. In diesem Kontext stellen wir zusätzlich einen neuen Regularisierer zweiter Ordnung vor, der sich der Bildstruktur anisotrop anpasst

Variational image fusion

The main goal of this work is the fusion of multiple images to a single composite that offers more information than the individual input images. We approach those fusion tasks within a variational framework. First, we present iterative schemes that are well-suited for such variational problems and related tasks. They lead to efficient algorithms that are simple to implement and well-parallelisable. Next, we design a general fusion technique that aims for an image with optimal local contrast. This is the key for a versatile method that performs well in many application areas such as multispectral imaging, decolourisation, and exposure fusion. To handle motion within an exposure set, we present the following two-step approach: First, we introduce the complete rank transform to design an optic flow approach that is robust against severe illumination changes. Second, we eliminate remaining misalignments by means of brightness transfer functions that relate the brightness values between frames. Additional knowledge about the exposure set enables us to propose the first fully coupled method that jointly computes an aligned high dynamic range image and dense displacement fields. Finally, we present a technique that infers depth information from differently focused images. In this context, we additionally introduce a novel second order regulariser that adapts to the image structure in an anisotropic way.Das Hauptziel dieser Arbeit ist die Fusion mehrerer Bilder zu einem Einzelbild, das mehr Informationen bietet als die einzelnen Eingangsbilder. Wir verwirklichen diese Fusionsaufgaben in einem variationellen Rahmen. Zunächst präsentieren wir iterative Schemata, die sich gut für solche variationellen Probleme und verwandte Aufgaben eignen. Danach entwerfen wir eine Fusionstechnik, die ein Bild mit optimalem lokalen Kontrast anstrebt. Dies ist der Schlüssel für eine vielseitige Methode, die gute Ergebnisse für zahlreiche Anwendungsbereiche wie Multispektralaufnahmen, Bildentfärbung oder Belichtungsreihenfusion liefert. Um Bewegungen in einer Belichtungsreihe zu handhaben, präsentieren wir folgenden Zweischrittansatz: Zuerst stellen wir die komplette Rangtransformation vor, um eine optische Flussmethode zu entwerfen, die robust gegenüber starken Beleuchtungsänderungen ist. Dann eliminieren wir verbleibende Registrierungsfehler mit der Helligkeitstransferfunktion, welche die Helligkeitswerte zwischen Bildern in Beziehung setzt. Zusätzliches Wissen über die Belichtungsreihe ermöglicht uns, die erste vollständig gekoppelte Methode vorzustellen, die gemeinsam ein registriertes Hochkontrastbild sowie dichte Bewegungsfelder berechnet. Final präsentieren wir eine Technik, die von unterschiedlich fokussierten Bildern Tiefeninformation ableitet. In diesem Kontext stellen wir zusätzlich einen neuen Regularisierer zweiter Ordnung vor, der sich der Bildstruktur anisotrop anpasst

Fehlerkaschierte Bildbasierte Darstellungsverfahren

Creating photo-realistic images has been one of the major goals in computer graphics since its early days. Instead of modeling the complexity of nature with standard modeling tools, image-based approaches aim at exploiting real-world footage directly,as they are photo-realistic by definition. A drawback of these approaches has always been that the composition or combination of different sources is a non-trivial task, often resulting in annoying visible artifacts. In this thesis we focus on different techniques to diminish visible artifacts when combining multiple images in a common image domain. The results are either novel images, when dealing with the composition task of multiple images, or novel video sequences rendered in real-time, when dealing with video footage from multiple cameras.Fotorealismus ist seit jeher eines der großen Ziele in der Computergrafik. Anstatt die Komplexität der Natur mit standardisierten Modellierungswerkzeugen nachzubauen, gehen bildbasierte Ansätze den umgekehrten Weg und verwenden reale Bildaufnahmen zur Modellierung, da diese bereits per Definition fotorealistisch sind. Ein Nachteil dieser Variante ist jedoch, dass die Komposition oder Kombination mehrerer Quellbilder eine nichttriviale Aufgabe darstellt und häufig unangenehm auffallende Artefakte im erzeugten Bild nach sich zieht. In dieser Dissertation werden verschiedene Ansätze verfolgt, um Artefakte zu verhindern oder abzuschwächen, welche durch die Komposition oder Kombination mehrerer Bilder in einer gemeinsamen Bilddomäne entstehen. Im Ergebnis liefern die vorgestellten Verfahren neue Bilder oder neue Ansichten einer Bildsammlung oder Videosequenz, je nachdem, ob die jeweilige Aufgabe die Komposition mehrerer Bilder ist oder die Kombination mehrerer Videos verschiedener Kameras darstellt

Advances in Image Processing, Analysis and Recognition Technology

For many decades, researchers have been trying to make computers’ analysis of images as effective as the system of human vision is. For this purpose, many algorithms and systems have previously been created. The whole process covers various stages, including image processing, representation and recognition. The results of this work can be applied to many computer-assisted areas of everyday life. They improve particular activities and provide handy tools, which are sometimes only for entertainment, but quite often, they significantly increase our safety. In fact, the practical implementation of image processing algorithms is particularly wide. Moreover, the rapid growth of computational complexity and computer efficiency has allowed for the development of more sophisticated and effective algorithms and tools. Although significant progress has been made so far, many issues still remain, resulting in the need for the development of novel approaches