Bioimage informatics in the context of drosophila research

Modern biological research relies heavily on microscopic imaging. The advanced genetic toolkit of drosophila makes it possible to label molecular and cellular components with unprecedented level of specificity necessitating the application of the most sophisticated imaging technologies. Imaging in drosophila spans all scales from single molecules to the entire populations of adult organisms, from electron microscopy to live imaging of developmental processes. As the imaging approaches become more complex and ambitious, there is an increasing need for quantitative, computer-mediated image processing and analysis to make sense of the imagery. Bioimage informatics is an emerging research field that covers all aspects of biological image analysis from data handling, through processing, to quantitative measurements, analysis and data presentation. Some of the most advanced, large scale projects, combining cutting edge imaging with complex bioimage informatics pipelines, are realized in the drosophila research community. In this review, we discuss the current research in biological image analysis specifically relevant to the type of systems level image datasets that are uniquely available for the drosophila model system. We focus on how state-of-the-art computer vision algorithms are impacting the ability of drosophila researchers to analyze biological systems in space and time. We pay particular attention to how these algorithmic advances from computer science are made usable to practicing biologists through open source platforms and how biologists can themselves participate in their further development

Machine Learning for Instance Segmentation

Volumetric Electron Microscopy images can be used for connectomics, the study of brain connectivity at the cellular level. A prerequisite for this inquiry is the automatic identification of neural cells, which requires machine learning algorithms and in particular efficient image segmentation algorithms. In this thesis, we develop new algorithms for this task. In the first part we provide, for the first time in this field, a method for training a neural network to predict optimal input data for a watershed algorithm. We demonstrate its superior performance compared to other segmentation methods of its category. In the second part, we develop an efficient watershed-based algorithm for weighted graph partitioning, the \emph{Mutex Watershed}, which uses negative edge-weights for the first time. We show that it is intimately related to the multicut and has a cutting edge performance on a connectomics challenge. Our algorithm is currently used by the leaders of two connectomics challenges. Finally, motivated by inpainting neural networks, we create a method to learn the graph weights without any supervision

Monitoring retinal changes with optical coherence tomography predicts neuronal loss in experimental autoimmune encephalomyelitis.

BACKGROUND:Retinal optical coherence tomography (OCT) is a clinical and research tool in multiple sclerosis, where it has shown significant retinal nerve fiber (RNFL) and ganglion cell (RGC) layer thinning, while postmortem studies have reported RGC loss. Although retinal pathology in experimental autoimmune encephalomyelitis (EAE) has been described, comparative OCT studies among EAE models are scarce. Furthermore, the best practices for the implementation of OCT in the EAE lab, especially with afoveate animals like rodents, remain undefined. We aimed to describe the dynamics of retinal injury in different mouse EAE models and outline the optimal experimental conditions, scan protocols, and analysis methods, comparing these to histology to confirm the pathological underpinnings. METHODS:Using spectral-domain OCT, we analyzed the test-retest and the inter-rater reliability of volume, peripapillary, and combined horizontal and vertical line scans. We then monitored the thickness of the retinal layers in different EAE models: in wild-type (WT) C57Bl/6J mice immunized with myelin oligodendrocyte glycoprotein peptide (MOG35-55) or with bovine myelin basic protein (MBP), in TCR2D2 mice immunized with MOG35-55, and in SJL/J mice immunized with myelin proteolipid lipoprotein (PLP139-151). Strain-matched control mice were sham-immunized. RGC density was counted on retinal flatmounts at the end of each experiment. RESULTS:Volume scans centered on the optic disc showed the best reliability. Retinal changes during EAE were localized in the inner retinal layers (IRLs, the combination of the RNFL and the ganglion cell plus the inner plexiform layers). In WT, MOG35-55 EAE, progressive thinning of IRL started rapidly after EAE onset, with 1/3 of total loss occurring during the initial 2 months. IRL thinning was associated with the degree of RGC loss and the severity of EAE. Sham-immunized SJL/J mice showed progressive IRL atrophy, which was accentuated in PLP-immunized mice. MOG35-55-immunized TCR2D2 mice showed severe EAE and retinal thinning. MBP immunization led to very mild disease without significant retinopathy. CONCLUSIONS:Retinal neuroaxonal damage develops quickly during EAE. Changes in retinal thickness mirror neuronal loss and clinical severity. Monitoring of the IRL thickness after immunization against MOG35-55 in C57Bl/6J mice seems the most convenient model to study retinal neurodegeneration in EAE

A curvature-enhanced random walker segmentation method for detailed capture of 3D cell surface Membranes

High-resolution 3D microscopy is a fast advancing field and requires new techniques in image analysis to handle these new datasets. In this work, we focus on detailed 3D segmentation of Dictyostelium cells undergoing macropinocytosis captured on an iSPIM microscope. We propose a novel random walker-based method with a curvature-based enhancement term, with the aim of capturing fine protrusions, such as filopodia and deep invaginations, such as macropinocytotic cups, on the cell surface. We tested our method on both real and synthetic 3D image volumes, demonstrating that the inclusion of the curvature enhancement term can improve the segmentation of the aforementioned features. We show that our method performs better than other state of the art segmentation methods in 3D images of Dictyostelium cells, and performs competitively against CNN-based methods in two Cell Tracking Challenge datasets, demonstrating the ability to obtain accurate segmentations without the requirement of large training datasets. We also present an automated seeding method for microscopy data, which, combined with the curvature-enhanced random walker method, enables the segmentation of large time series with minimal input from the experimenter

Doctor of Philosophy

dissertationNeuroscientists are developing new imaging techniques and generating large volumes of data in an effort to understand the complex structure of the nervous system. The complexity and size of this data makes human interpretation a labor intensive task. To aid in the analysis, new segmentation techniques for identifying neurons in these feature rich datasets are required. However, the extremely anisotropic resolution of the data makes segmentation and tracking across slices difficult. Furthermore, the thickness of the slices can make the membranes of the neurons hard to identify. Similarly, structures can change significantly from one section to the next due to slice thickness which makes tracking difficult. This thesis presents a complete method for segmenting many neurons at once in two-dimensional (2D) electron microscopy images and reconstructing and visualizing them in three-dimensions (3D). First, we present an advanced method for identifying neuron membranes in 2D, necessary for whole neuron segmentation, using a machine learning approach. The method described uses a series of artificial neural networks (ANNs) in a framework combined with a feature vector that is composed of image and context; intensities sampled over a stencil neighborhood. Several ANNs are applied in series allowing each ANN to use the classification context; provided by the previous network to improve detection accuracy. To improve the membrane detection, we use information from a nonlinear alignment of sequential learned membrane images in a final ANN that improves membrane detection in each section. The final output, the detected membranes, are used to obtain 2D segmentations of all the neurons in an image. We also present a method that constructs 3D neuron representations by formulating the problem of finding paths through sets of sections as an optimal path computation, which applies a cost function to the identification of a cell from one section to the next and solves this optimization problem using Dijkstras algorithm. This basic formulation accounts for variability or inconsistencies between sections and prioritizes cells based on the evidence of their connectivity. Finally, we present a tool that combines these techniques with a visual user interface that enables users to quickly segment whole neurons in large volumes