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

Optimal-path approach for neural circuit reconstruction

Journal ArticleNeurobiologists are collecting large amounts of electron microscopy image data to gain a better understanding of neuron organization in the central nervous system. Image analysis plays an important role in extracting the connectivity present in these images; however, due to the large size of these datasets, manual analysis is essentially impractical. Automated analysis, however, is challenging because of the difficulty in reliably segmenting individual neurons in 3D. In this paper, we describe an automatic method for finding neurons in sequences of 2D sections. The proposed method formulates 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 Dijkstra's algorithm. This basic formulation allows us to account for variability or inconsistencies between sections and to prioritize cells based on the evidence of their connectivity

Energy Minimization of Discrete Protein Titration State Models Using Graph Theory

There are several applications in computational biophysics which require the optimization of discrete interacting states; e.g., amino acid titration states, ligand oxidation states, or discrete rotamer angles. Such optimization can be very time-consuming as it scales exponentially in the number of sites to be optimized. In this paper, we describe a new polynomial-time algorithm for optimization of discrete states in macromolecular systems. This algorithm was adapted from image processing and uses techniques from discrete mathematics and graph theory to restate the optimization problem in terms of "maximum flow-minimum cut" graph analysis. The interaction energy graph, a graph in which vertices (amino acids) and edges (interactions) are weighted with their respective energies, is transformed into a flow network in which the value of the minimum cut in the network equals the minimum free energy of the protein, and the cut itself encodes the state that achieves the minimum free energy. Because of its deterministic nature and polynomial-time performance, this algorithm has the potential to allow for the ionization state of larger proteins to be discovered

Detection of Neuron Membranes in Electron Microscopy Images Using Multi-scale Context and Radon-Like Features

Automated neural circuit reconstruction through electron microscopy (EM) images is a challenging problem. In this paper, we present a novel method that exploits multi-scale contextual information together with Radon-like features (RLF) to learn a series of discriminative models. The main idea is to build a framework which is capable of extracting information about cell membranes from a large contextual area of an EM image in a computationally efficient way. Toward this goal, we extract RLF that can be computed efficiently from the input image and generate a scale-space representation of the context images that are obtained at the output of each discriminative model in the series. Compared to a single-scale model, the use of a multi-scale representation of the context image gives the subsequent classifiers access to a larger contextual area in an effective way. Our strategy is general and independent of the classifier and has the potential to be used in any context based framework. We demonstrate that our method outperforms the state-of-the-art algorithms in detection of neuron membranes in EM images.Engineering and Applied Science

Improvements to the APBS biomolecular solvation software suite

The Adaptive Poisson-Boltzmann Solver (APBS) software was developed to solve the equations of continuum electrostatics for large biomolecular assemblages that has provided impact in the study of a broad range of chemical, biological, and biomedical applications. APBS addresses three key technology challenges for understanding solvation and electrostatics in biomedical applications: accurate and efficient models for biomolecular solvation and electrostatics, robust and scalable software for applying those theories to biomolecular systems, and mechanisms for sharing and analyzing biomolecular electrostatics data in the scientific community. To address new research applications and advancing computational capabilities, we have continually updated APBS and its suite of accompanying software since its release in 2001. In this manuscript, we discuss the models and capabilities that have recently been implemented within the APBS software package including: a Poisson-Boltzmann analytical and a semi-analytical solver, an optimized boundary element solver, a geometry-based geometric flow solvation model, a graph theory based algorithm for determining p$K_a$ values, and an improved web-based visualization tool for viewing electrostatics

A Computational Framework for Ultrastructural Mapping of Neural Circuitry

Circuitry mapping of metazoan neural systems is difficult because canonical neural regions (regions containing one or more copies of all components) are large, regional borders are uncertain, neuronal diversity is high, and potential network topologies so numerous that only anatomical ground truth can resolve them. Complete mapping of a specific network requires synaptic resolution, canonical region coverage, and robust neuronal classification. Though transmission electron microscopy (TEM) remains the optimal tool for network mapping, the process of building large serial section TEM (ssTEM) image volumes is rendered difficult by the need to precisely mosaic distorted image tiles and register distorted mosaics. Moreover, most molecular neuronal class markers are poorly compatible with optimal TEM imaging. Our objective was to build a complete framework for ultrastructural circuitry mapping. This framework combines strong TEM-compliant small molecule profiling with automated image tile mosaicking, automated slice-to-slice image registration, and gigabyte-scale image browsing for volume annotation. Specifically we show how ultrathin molecular profiling datasets and their resultant classification maps can be embedded into ssTEM datasets and how scripted acquisition tools (SerialEM), mosaicking and registration (ir-tools), and large slice viewers (MosaicBuilder, Viking) can be used to manage terabyte-scale volumes. These methods enable large-scale connectivity analyses of new and legacy data. In well-posed tasks (e.g., complete network mapping in retina), terabyte-scale image volumes that previously would require decades of assembly can now be completed in months. Perhaps more importantly, the fusion of molecular profiling, image acquisition by SerialEM, ir-tools volume assembly, and data viewers/annotators also allow ssTEM to be used as a prospective tool for discovery in nonneural systems and a practical screening methodology for neurogenetics. Finally, this framework provides a mechanism for parallelization of ssTEM imaging, volume assembly, and data analysis across an international user base, enhancing the productivity of a large cohort of electron microscopists