Adaptive Re-Segmentation Strategies for Accurate Bright Field Cell Tracking

Understanding complex interactions in cellular systems requires accurate tracking of individual cells observed in microscopic image sequence and acquired from multi-day in vitro experiments. To be effective, methods must follow each cell through the whole experimental sequence to recognize significant phenotypic transitions, such as mitosis, chemotaxis, apoptosis, and cell/cell interactions, and to detect the effect of cell treatments. However, high accuracy long-range cell tracking is difficult because the collection and detection of cells in images is error-prone, and single error in a one frame can cause a tracked cell to be lost. Detection of cells is especially difficult when using bright field microscopy images wherein the contrast difference between the cells and the background is very low. This work introduces a new method that automatically identifies and then corrects tracking errors using a combination of combinatorial registration, flow constraints, and image segmentation repair

Observing the Cell in Its Native State: Imaging Subcellular Dynamics in Multicellular Organisms

True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes that we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution, without inducing undue stress on either. We combined lattice light-sheet microscopy with adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments

Registration of serial sections: An evaluation method based on distortions of the ground truths

Registration of histological serial sections is a challenging task. Serial sections exhibit distortions and damage from sectioning. Missing information on how the tissue looked before cutting makes a realistic validation of 2D registrations extremely difficult. This work proposes methods for ground-truth-based evaluation of registrations. Firstly, we present a methodology to generate test data for registrations. We distort an innately registered image stack in the manner similar to the cutting distortion of serial sections. Test cases are generated from existing 3D data sets, thus the ground truth is known. Secondly, our test case generation premises evaluation of the registrations with known ground truths. Our methodology for such an evaluation technique distinguishes this work from other approaches. Both under- and over-registration become evident in our evaluations. We also survey existing validation efforts. We present a full-series evaluation across six different registration methods applied to our distorted 3D data sets of animal lungs. Our distorted and ground truth data sets are made publicly available.Comment: Supplemental data available under https://zenodo.org/record/428244

A Survey on Deep Learning in Medical Image Analysis

Deep learning algorithms, in particular convolutional networks, have rapidly become a methodology of choice for analyzing medical images. This paper reviews the major deep learning concepts pertinent to medical image analysis and summarizes over 300 contributions to the field, most of which appeared in the last year. We survey the use of deep learning for image classification, object detection, segmentation, registration, and other tasks and provide concise overviews of studies per application area. Open challenges and directions for future research are discussed.Comment: Revised survey includes expanded discussion section and reworked introductory section on common deep architectures. Added missed papers from before Feb 1st 201

Quantifying the Frequency and Orientation of Mitoses in Embryonic Epithelia

The miraculous birth of a new life starts by the formation of an embryo. The process by which an embryo is formed, embryogenesis, has been studied and shown to consist of three types of processes: mitosis, cell differentiation and morphogenetic movements. Scientists and medical doctors are still at a loss to explain the fundamental forces driving embryo development and the causes of birth defects remain largely unknown. Recent efforts by the Embryo Biomechanics Lab at the University of Waterloo have shown a relationship between morphogenetic movements that occur during embryo formation and the frequency and orientation of mitosis. To further study this relationship a means of automatically identifying the frequency and orientation of mitosis on time-lapse images of embryo epithelia is needed. Past efforts at identifying mitosis have been limited to the study of cell cultures and stained tissue segments. Two methods for identifying mitosis in contiguous sheets of cells are developed. The first method is based on local motion analysis and the second method is based on intensity analysis. These algorithms were tested on images of early and late stage embryos of the axolotl (Ambystoma mexicanum), a type of amphibian. The performance of the algorithms were measured using the F-Measure. The F-Measure determines the performance of the algorithm as the true mitosis detection rate penalized by the false mitosis detection rate. The motion based algorithm had performance rates of 68.2% on an early stage image set and 66.7% on a late stage image set, whereas the intensity based algorithm had a performance rates of 73.9% on early stage image set and 90.0% on late stage image set. The mitosis orientation errors for the motion based algorithm were 27.3 degrees average error with a standard deviation (std.) of 19.8 degrees for early stage set and 34.8 degrees average error with a std. of 23.5 degrees for the late stage set. For the intensity based algorithm the orientation errors were 39.8 degrees average with std. of 28.9 degrees for the early stage image set and 15.7 degrees average with std. of 18.9 degrees for the late stage image set. The intensity based algorithm had the best performance of the two algorithms presented, and the intensity based algorithm performs best on high-magnification images. Its performance is limited by mitoses in adjacent cells and by the presence of natural cell pigment variations. The algorithms presented here offer a powerful new set of tools for evaluating the role of mitoses in embryo morphogenesis

Towards Accurate and Efficient Cell Tracking During Fly Wing Development

Understanding the development, organization, and function of tissues is a central goal in developmental biology. With modern time-lapse microscopy, it is now possible to image entire tissues during development and thereby localize subcellular proteins. A particularly productive area of research is the study of single layer epithelial tissues, which can be simply described as a 2D manifold. For example, the apical band of cell adhesions in epithelial cell layers actually forms a 2D manifold within the tissue and provides a 2D outline of each cell. The Drosophila melanogaster wing has become an important model system, because its 2D cell organization has the potential to reveal mechanisms that create the final fly wing shape. Other examples include structures that naturally localize at the surface of the tissue, such as the ciliary components of planarians. Data from these time-lapse movies typically consists of mosaics of overlapping 3D stacks. This is necessary because the surface of interest exceeds the field of view of todays microscopes. To quantify cellular tissue dynamics, these mosaics need to be processed in three main steps: (a) Extracting, correcting, and stitching individ- ual stacks into a single, seamless 2D projection per time point, (b) obtaining cell characteristics that occur at individual time points, and (c) determine cell dynamics over time. It is therefore necessary that the applied methods are capable of handling large amounts of data efficiently, while still producing accurate results. This task is made especially difficult by the low signal to noise ratios that are typical in live-cell imaging. In this PhD thesis, I develop algorithms that cover all three processing tasks men- tioned above and apply them in the analysis of polarity and tissue dynamics in large epithelial cell layers, namely the Drosophila wing and the planarian epithelium. First, I introduce an efficient pipeline that preprocesses raw image mosaics. This pipeline accurately extracts the stained surface of interest from each raw image stack and projects it onto a single 2D plane. It then corrects uneven illumination, aligns all mosaic planes, and adjusts brightness and contrast before finally stitching the processed images together. This preprocessing does not only significantly reduce the data quantity, but also simplifies downstream data analyses. Here, I apply this pipeline to datasets of the developing fly wing as well as a planarian epithelium. I additionally address the problem of determining cell polarities in chemically fixed samples of planarians. Here, I introduce a method that automatically estimates cell polarities by computing the orientation of rootlets in motile cilia. With this technique one can for the first time routinely measure and visualize how tissue polarities are established and maintained in entire planarian epithelia. Finally, I analyze cell migration patterns in the entire developing wing tissue in Drosophila. At each time point, cells are segmented using a progressive merging ap- proach with merging criteria that take typical cell shape characteristics into account. The method enforces biologically relevant constraints to improve the quality of the resulting segmentations. For cases where a full cell tracking is desired, I introduce a pipeline using a tracking-by-assignment approach. This allows me to link cells over time while considering critical events such as cell divisions or cell death. This work presents a very accurate large-scale cell tracking pipeline and opens up many avenues for further study including several in-vivo perturbation experiments as well as biophysical modeling. The methods introduced in this thesis are examples for computational pipelines that catalyze biological insights by enabling the quantification of tissue scale phenomena and dynamics. I provide not only detailed descriptions of the methods, but also show how they perform on concrete biological research projects

A dynamic-shape-prior guided snake model with application in visually tracking dense cell populations

This paper proposes a dynamic-shape-prior guided snake (DSP G-snake) model that is designed to improve the overall stability of the point-based snake model. The dynamic shape prior is first proposed for snakes, that efficiently unifies different types of high-level priors into a new force term. To be specific, a global-topology regularity is first introduced that settles the inherent self-intersection problem with snakes. The problem that a snake’s snaxels tend to unevenly distribute along the contour is also handled, leading to good parameterization. Unlike existing methods that employ learning templates or commonly enforce hard priors, the dynamic-template scheme strongly respects the deformation flexibility of the model, while retaining a decent global topology for the snake. It is verified by experiments that the proposed algorithm can effectively prevent snakes from selfcrossing, or automatically untie an already self-intersected contour. In addition, the proposed model is combined with existing forces and applied to the very challenging task of tracking dense biological cell populations. The DSP G-snake model has enabled an improvement of up to 30% in tracking accuracy with respect to regular model-based approaches. Through experiments on real cellular datasets, with highly dense populations and relatively large displacements, it is confirmed that the proposed approach has enabled superior performance, in comparison to modern active-contour competitors as well as the state-of-the-art cell tracking frameworks