DeepSynth: Three-dimensional nuclear segmentation of biological images using neural networks trained with synthetic data

The scale of biological microscopy has increased dramatically over the past ten years, with the development of new modalities supporting collection of high-resolution fluorescence image volumes spanning hundreds of microns if not millimeters. The size and complexity of these volumes is such that quantitative analysis requires automated methods of image processing to identify and characterize individual cells. For many workflows, this process starts with segmentation of nuclei that, due to their ubiquity, ease-of-labeling and relatively simple structure, make them appealing targets for automated detection of individual cells. However, in the context of large, three-dimensional image volumes, nuclei present many challenges to automated segmentation, such that conventional approaches are seldom effective and/or robust. Techniques based upon deep-learning have shown great promise, but enthusiasm for applying these techniques is tempered by the need to generate training data, an arduous task, particularly in three dimensions. Here we present results of a new technique of nuclear segmentation using neural networks trained on synthetic data. Comparisons with results obtained using commonly-used image processing packages demonstrate that DeepSynth provides the superior results associated with deep-learning techniques without the need for manual annotation

Nucleus segmentation : towards automated solutions

Single nucleus segmentation is a frequent challenge of microscopy image processing, since it is the first step of many quantitative data analysis pipelines. The quality of tracking single cells, extracting features or classifying cellular phenotypes strongly depends on segmentation accuracy. Worldwide competitions have been held, aiming to improve segmentation, and recent years have definitely brought significant improvements: large annotated datasets are now freely available, several 2D segmentation strategies have been extended to 3D, and deep learning approaches have increased accuracy. However, even today, no generally accepted solution and benchmarking platform exist. We review the most recent single-cell segmentation tools, and provide an interactive method browser to select the most appropriate solution.Peer reviewe

Cancer Tissue Engineering: development of new 3D models and technologies to support cancer research

The activity of research of this thesis focuses on the relevance that appropriate models reproducing the in vivo tumor microenvironment are essential for improving cancer biology knowledge and for testing new anticancer compounds. Animal models are proven not to be entirely compatible with the human system, and the success rates between animal and human studies are still unsatisfactory. On the other hand, 2D cell cultures fail to reproduce some aspects of tumor system. These limitations have a significant weight especially during the screening of novel antitumor drugs, as it was demonstrated that cells are less sensitive to treatments when in contact with their microenvironment. To obtain the same tumor cell inhibition levels observed in vivo, the culture environment has to reflect the 3D natural environment. Natural or synthetic hydrogels reported successful outcomes in mimicking ECM environment. During this PhD, I developed different gel-based scaffolds to be use as substrates for the culture of breast cancer cells. In detail, I developed different gels for low and highly aggressive cancer cell lines (i.e. MCF-7 and MDA-MB-231), obtaining significant results as regards the reproduction of key features normally present into the in vivo environment. Considering the importance of the metastasis process in breast cancer evolution, I then focused on a new set-up for the observation of cancer cell motility and invasion. In particular, I combined a bioreactor-based bioengineering approach with single cell analysis of Circulating Tumor Cells (CTCs). This part of work was carried out at the Dipartiment of Biomedicine of the University of Basel (CH) that, among its equipment, has a cell celector machine for single cell analysis. At the end of this work, I provided a proof-of-concept that the approach can work, as well as evidence that the cells can be extracted from the device and used for molecular analysis

On Improving Generalization of CNN-Based Image Classification with Delineation Maps Using the CORF Push-Pull Inhibition Operator

Deployed image classification pipelines are typically dependent on the images captured in real-world environments. This means that images might be affected by different sources of perturbations (e.g. sensor noise in low-light environments). The main challenge arises by the fact that image quality directly impacts the reliability and consistency of classification tasks. This challenge has, hence, attracted wide interest within the computer vision communities. We propose a transformation step that attempts to enhance the generalization ability of CNN models in the presence of unseen noise in the test set. Concretely, the delineation maps of given images are determined using the CORF push-pull inhibition operator. Such an operation transforms an input image into a space that is more robust to noise before being processed by a CNN. We evaluated our approach on the Fashion MNIST data set with an AlexNet model. It turned out that the proposed CORF-augmented pipeline achieved comparable results on noise-free images to those of a conventional AlexNet classification model without CORF delineation maps, but it consistently achieved significantly superior performance on test images perturbed with different levels of Gaussian and uniform noise

Improving the Tractography Pipeline: on Evaluation, Segmentation, and Visualization

Recent advances in tractography allow for connectomes to be constructed in vivo. These have applications for example in brain tumor surgery and understanding of brain development and diseases. The large size of the data produced by these methods lead to a variety problems, including how to evaluate tractography outputs, development of faster processing algorithms for tractography and clustering, and the development of advanced visualization methods for verification and exploration. This thesis presents several advances in these fields. First, an evaluation is presented for the robustness to noise of multiple commonly used tractography algorithms. It employs a Monte–Carlo simulation of measurement noise on a constructed ground truth dataset. As a result of this evaluation, evidence for obustness of global tractography is found, and algorithmic sources of uncertainty are identified. The second contribution is a fast clustering algorithm for tractography data based on k–means and vector fields for representing the flow of each cluster. It is demonstrated that this algorithm can handle large tractography datasets due to its linear time and memory complexity, and that it can effectively integrate interrupted fibers that would be rejected as outliers by other algorithms. Furthermore, a visualization for the exploration of structural connectomes is presented. It uses illustrative rendering techniques for efficient presentation of connecting fiber bundles in context in anatomical space. Visual hints are employed to improve the perception of spatial relations. Finally, a visualization method with application to exploration and verification of probabilistic tractography is presented, which improves on the previously presented Fiber Stippling technique. It is demonstrated that the method is able to show multiple overlapping tracts in context, and correctly present crossing fiber configurations

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

Texture and Colour in Image Analysis

Research in colour and texture has experienced major changes in the last few years. This book presents some recent advances in the field, specifically in the theory and applications of colour texture analysis. This volume also features benchmarks, comparative evaluations and reviews

Microscopy Conference 2017 (MC 2017) - Proceedings

Das Dokument enthält die Kurzfassungen der Beiträge aller Teilnehmer an der Mikroskopiekonferenz "MC 2017", die vom 21. bis 25.08.2017, in Lausanne stattfand