Image Processing and Simulation Toolboxes of Microscopy Images of Bacterial Cells

Recent advances in microscopy imaging technology have allowed the characterization of the dynamics of cellular processes at the single-cell and single-molecule level. Particularly in bacterial cell studies, and using the E. coli as a case study, these techniques have been used to detect and track internal cell structures such as the Nucleoid and the Cell Wall and fluorescently tagged molecular aggregates such as FtsZ proteins, Min system proteins, inclusion bodies and all the different types of RNA molecules. These studies have been performed with using multi-modal, multi-process, time-lapse microscopy, producing both morphological and functional images. To facilitate the finding of relationships between cellular processes, from small-scale, such as gene expression, to large-scale, such as cell division, an image processing toolbox was implemented with several automatic and/or manual features such as, cell segmentation and tracking, intra-modal and intra-modal image registration, as well as the detection, counting and characterization of several cellular components. Two segmentation algorithms of cellular component were implemented, the first one based on the Gaussian Distribution and the second based on Thresholding and morphological structuring functions. These algorithms were used to perform the segmentation of Nucleoids and to identify the different stages of FtsZ Ring formation (allied with the use of machine learning algorithms), which allowed to understand how the temperature influences the physical properties of the Nucleoid and correlated those properties with the exclusion of protein aggregates from the center of the cell. Another study used the segmentation algorithms to study how the temperature affects the formation of the FtsZ Ring. The validation of the developed image processing methods and techniques has been based on benchmark databases manually produced and curated by experts. When dealing with thousands of cells and hundreds of images, these manually generated datasets can become the biggest cost in a research project. To expedite these studies in terms of time and lower the cost of the manual labour, an image simulation was implemented to generate realistic artificial images. The proposed image simulation toolbox can generate biologically inspired objects that mimic the spatial and temporal organization of bacterial cells and their processes, such as cell growth and division and cell motility, and cell morphology (shape, size and cluster organization). The image simulation toolbox was shown to be useful in the validation of three cell tracking algorithms: Simple Nearest-Neighbour, Nearest-Neighbour with Morphology and DBSCAN cluster identification algorithm. It was shown that the Simple Nearest-Neighbour still performed with great reliability when simulating objects with small velocities, while the other algorithms performed better for higher velocities and when there were larger clusters present

Recent Advances in Morphological Cell Image Analysis

This paper summarizes the recent advances in image processing methods for morphological cell analysis. The topic of morphological analysis has received much attention with the increasing demands in both bioinformatics and biomedical applications. Among many factors that affect the diagnosis of a disease, morphological cell analysis and statistics have made great contributions to results and effects for a doctor. Morphological cell analysis finds the cellar shape, cellar regularity, classification, statistics, diagnosis, and so forth. In the last 20 years, about 1000 publications have reported the use of morphological cell analysis in biomedical research. Relevant solutions encompass a rather wide application area, such as cell clumps segmentation, morphological characteristics extraction, 3D reconstruction, abnormal cells identification, and statistical analysis. These reports are summarized in this paper to enable easy referral to suitable methods for practical solutions. Representative contributions and future research trends are also addressed

Quantitative microscopy workflows for the study of cellular receptor trafficking

The trafficking and signalling of cellular receptors are complex, intertwined processes with many feedback mechanisms. Confocal microscopy is a powerful tool to study the trafficking of receptors. The aim of this thesis was to report and develop workflows to quantify the spatio-temporal dynamics of receptor trafficking and colocalization using confocal microscopy. Importantly, the workflows should be reproducible and unbiased, as well as automated and accurate. A 4D level set approach is developed to enable accurate cellular segmentation. Temporal constraints are introduced to further improve segmentation accuracy. This novel approach is thoroughly validated, and statistically significant performance increase over equivalent 2D and 3D approaches is demonstrated. We present a confocal microscopy based RNAi depletion screen. Specifically, quantitative workflows to identify genes which perturb the trafficking of receptor are described. Finally, a critical review of current approaches to the quantification of colocalization between receptors and endosomes is presented. Improvements to existing techniques and complete workflows are provided for 4D data (3D time-lapse). Together the described protocols provide a complete microscopy based platform to identify and investigate regulators of receptor signalling and trafficking

Designing Deep Learning Frameworks for Plant Biology

In recent years the parallel progress in high-throughput microscopy and deep learning drastically widened the landscape of possible research avenues in life sciences. In particular, combining high-resolution microscopic images and automated imaging pipelines powered by deep learning dramatically reduced the manual annotation work required for quantitative analysis. In this work, we will present two deep learning frameworks tailored to the needs of life scientists in the context of plant biology. First, we will introduce PlantSeg, a software for 2D and 3D instance segmentation. The PlantSeg pipeline contains several pre-trained models for different microscopy modalities and multiple popular graph-based instance segmentation algorithms. In the second part, we will present CellTypeGraph, a benchmark for quantitatively evaluating graph neural networks. The benchmark is designed to test the ability of machine learning methods to classify the types of cells in an \textit{Arabidopsis thaliana} ovules. CellTypeGraph's prime aim is to give a valuable tool to the geometric learning community, but at the same time it also offers a framework for plant biologists to perform fast and accurate cell type inference on new data

Learning Instance Segmentation from Sparse Supervision

Instance segmentation is an important task in many domains of automatic image processing, such as self-driving cars, robotics and microscopy data analysis. Recently, deep learning-based algorithms have brought image segmentation close to human performance. However, most existing models rely on dense groundtruth labels for training, which are expensive, time consuming and often require experienced annotators to perform the labeling. Besides the annotation burden, training complex high-capacity neural networks depends upon non-trivial expertise in the choice and tuning of hyperparameters, making the adoption of these models challenging for researchers in other fields. The aim of this work is twofold. The first is to make the deep learning segmentation methods accessible to non-specialist. The second is to address the dense annotation problem by developing instance segmentation methods trainable with limited groundtruth data. In the first part of this thesis, I bring state-of-the-art instance segmentation methods closer to non-experts by developing PlantSeg: a pipeline for volumetric segmentation of light microscopy images of biological tissues into cells. PlantSeg comes with a large repository of pre-trained models and delivers highly accurate results on a variety of samples and image modalities. We exemplify its usefulness to answer biological questions in several collaborative research projects. In the second part, I tackle the dense annotation bottleneck by introducing SPOCO, an instance segmentation method, which can be trained from just a few annotated objects. It demonstrates strong segmentation performance on challenging natural and biological benchmark datasets at a very reduced manual annotation cost and delivers state-of-the-art results on the CVPPP benchmark. In summary, my contributions enable training of instance segmentation models with limited amounts of labeled data and make these methods more accessible for non-experts, speeding up the process of quantitative data analysis

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

Doctor of Philosophy

dissertationThe nervous system is comprised of an estimated 100 billion individual neurons, which are connected to one another to form a network that senses environmental stimuli and coordinates the organism's behavior. Because of the complexity of the nervous system, deciphering the developmental processes and adult wiring diagram has proved challenging. A number of axon guidance molecules have been identified; however, the means by which they guide billions of axons to their target cells in vivo remains poorly understood. Several axon guidance molecules have been found to be bifunctional, meaning they can elicit different growth cone responses depending on the presence or absence of other molecules, such as growth cone receptors, intracellular signal transduction molecules, or extracellular modulators. Axon sorting within axon tracts is perhaps a means by which axons are presorted to make a precise connection on their target cells. The zebrafish, Danio rerio, is an ideal model organism to study vertebrate axon guidance and axon sorting due to its external fertilization, optical transparency, amenability to forward genetics, and ease of making transgenic lines. In order to study axon guidance within the zebrafish retinotectal system, I developed a new method of misexpressing genes. Local misexpression can be induced by using a modified soldering iron in transgenic zebrafish in which a gene of interest is driven by a heat shock promoter. This method allowed me to examine the mechanisms by which Slit1a and Slit2 guide axons from the retina to the optic tectum. I determined the expression pattern of Slits in the zebrafish and used antisense morpholino technology to knock down Slit1a. The iv resultant axon guidance errors indicated that Slit1a acts to guide retinal axons through the optic tract. I then misexpressed Slit1a and Slit2 near the optic tract to observe their effect on axons. I found that both proteins appeared to attract retinal axons. Additionally, I saw that Slit2 seems to attract retinal axons earlier in the retinotectal pathway, at the optic chiasm. I also report on a new method, to whose development I contributed, for automated tracking of axons through electron microscopy datasets. Taken together, my results add new methods to the endeavor of mapping neural connectivity and development, and suggest a new role for Slits in axon guidance

Biological model representation and analysis

In this thesis, we discuss solutions of phenotype description based on the microscopy image analysis to deal with biological problems both in 2D and 3D space. Our description of patterns goes beyond conventional features and helps to visualize the unseen in feature dataset. These solutions share several common processes which are based on similar principles. Furthermore, we notice that advanced features and classier strategies can help us improve the performance of the solutions. The biological problems that we have studied include the endocytosis routing using high-throughput screening in 2D and time and 3D geometrical representation from biological structures.China Scholarship CouncilComputer Systems, Imagery and Medi

Automated retinal layer segmentation and pre-apoptotic monitoring for three-dimensional optical coherence tomography

The aim of this PhD thesis was to develop segmentation algorithm adapted and optimized to retinal OCT data that will provide objective 3D layer thickness which might be used to improve diagnosis and monitoring of retinal pathologies. Additionally, a 3D stack registration method was produced by modifying an existing algorithm. A related project was to develop a pre-apoptotic retinal monitoring based on the changes in texture parameters of the OCT scans in order to enable treatment before the changes become irreversible; apoptosis refers to the programmed cell death that can occur in retinal tissue and lead to blindness. These issues can be critical for the examination of tissues within the central nervous system. A novel statistical model for segmentation has been created and successfully applied to a large data set. A broad range of future research possibilities into advanced pathologies has been created by the results obtained. A separate model has been created for choroid segmentation located deep in retina, as the appearance of choroid is very different from the top retinal layers. Choroid thickness and structure is an important index of various pathologies (diabetes etc.). As part of the pre-apoptotic monitoring project it was shown that an increase in proportion of apoptotic cells in vitro can be accurately quantified. Moreover, the data obtained indicates a similar increase in neuronal scatter in retinal explants following axotomy (removal of retinas from the eye), suggesting that UHR-OCT can be a novel non-invasive technique for the in vivo assessment of neuronal health. Additionally, an independent project within the computer science department in collaboration with the school of psychology has been successfully carried out, improving analysis of facial dynamics and behaviour transfer between individuals. Also, important improvements to a general signal processing algorithm, dynamic time warping (DTW), have been made, allowing potential application in a broad signal processing field.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Mathematical Morphology for Quantification in Biological & Medical Image Analysis

Mathematical morphology is an established field of image processing first introduced as an application of set and lattice theories. Originally used to characterise particle distributions, mathematical morphology has gone on to be a core tool required for such important analysis methods as skeletonisation and the watershed transform. In this thesis, I introduce a selection of new image analysis techniques based on mathematical morphology. Utilising assumptions of shape, I propose a new approach for the enhancement of vessel-like objects in images: the bowler-hat transform. Built upon morphological operations, this approach is successful at challenges such as junctions and robust against noise. The bowler-hat transform is shown to give better results than competitor methods on challenging data such as retinal/fundus imagery. Building further on morphological operations, I introduce two novel methods for particle and blob detection. The first of which is developed in the context of colocalisation, a standard biological assay, and the second, which is based on Hilbert-Edge Detection And Ranging (HEDAR), with regard to nuclei detection and counting in fluorescent microscopy. These methods are shown to produce accurate and informative results for sub-pixel and supra-pixel object counting in complex and noisy biological scenarios. I propose a new approach for the automated extraction and measurement of object thickness for intricate and complicated vessels, such as brain vascular in medical images. This pipeline depends on two key technologies: semi-automated segmentation by advanced level-set methods and automatic thickness calculation based on morphological operations. This approach is validated and results demonstrating the broad range of challenges posed by these images and the possible limitations of this pipeline are shown. This thesis represents a significant contribution to the field of image processing using mathematical morphology and the methods within are transferable to a range of complex challenges present across biomedical image analysis