MOTIVATION: Advances in high-resolution microscopy have recently made possible the analysis of gene expression at the level of individual cells. The fixed lineage of cells in the adult worm Caenorhabditis elegans makes this organism an ideal model for studying complex biological processes like development and aging. However, annotating individual cells in images of adult C.elegans typically requires expertise and significant manual effort. Automation of this task is therefore critical to enabling high-resolution studies of a large number of genes. RESULTS: In this article, we describe an automated method for annotating a subset of 154 cells (including various muscle, intestinal and hypodermal cells) in high-resolution images of adult C.elegans. We formulate the task of labeling cells within an image as a combinatorial optimization problem, where the goal is to minimize a scoring function that compares cells in a test input image with cells from a training atlas of manually annotated worms according to various spatial and morphological characteristics. We propose an approach for solving this problem based on reduction to minimum-cost maximum-flow and apply a cross-entropy-based learning algorithm to tune the weights of our scoring function. We achieve 84% median accuracy across a set of 154 cell labels in this highly variable system. These results demonstrate the feasibility of the automatic annotation of microscopy-based images in adult C.elegans.

In a wide range of biological studies, it is highly desirable to visualize and analyze three-dimensional (3D) microscopic images. In this primer, we first introduce several major methods for visualizing typical 3D images and related multi-scale, multi-time-point, multi-color data sets. Then, we discuss three key categories of image analysis tasks, namely segmentation, registration, and annotation. We demonstrate how to pipeline these visualization and analysis modules using examples of profiling the single-cell gene-expression of C. elegans and constructing a map of stereotyped neurite tracts in a fruit fly brain.

MOTIVATION: Digital reconstruction, or tracing, of 3D neuron structures is critical toward reverse engineering the wiring and functions of a brain. However, despite a number of existing studies, this task is still challenging, especially when a 3D microscopic image has low signal-to-noise ratio (SNR) and fragmented neuron segments. Published work can handle these hard situations only by introducing global prior information, such as where a neurite segment starts and terminates. However, manual incorporation of such global information can be very time consuming. Thus, a completely automatic approach for these hard situations is highly desirable. RESULTS: We have developed an automatic graph algorithm, called the all-path pruning (APP), to trace the 3D structure of a neuron. To avoid potential mis-tracing of some parts of a neuron, an APP first produces an initial over-reconstruction, by tracing the optimal geodesic shortest path from the seed location to every possible destination voxel/pixel location in the image. Since the initial reconstruction contains all the possible paths and thus could contain redundant structural components (SC), we simplify the entire reconstruction without compromising its connectedness by pruning the redundant structural elements, using a new maximal-covering minimal-redundant (MCMR) subgraph algorithm. We show that MCMR has a linear computational complexity and will converge. We examined the performance of our method using challenging 3D neuronal image datasets of model organisms (e.g. fruit fly). AVAILABILITY: The software is available upon request. We plan to eventually release the software as a plugin of the V3D-Neuron package at http://penglab.janelia.org/proj/v3d. CONTACT: [email protected].

MOTIVATION: Automatic recognition of cell identities is critical for quantitative measurement, targeting and manipulation of cells of model animals at single-cell resolution. It has been shown to be a powerful tool for studying gene expression and regulation, cell lineages and cell fates. Existing methods first segment cells, before applying a recognition algorithm in the second step. As a result, the segmentation errors in the first step directly affect and complicate the subsequent cell recognition step. Moreover, in new experimental settings, some of the image features that have been previously relied upon to recognize cells may not be easy to reproduce, due to limitations on the number of color channels available for fluorescent imaging or to the cost of building transgenic animals. An approach that is more accurate and relies on only a single signal channel is clearly desirable. RESULTS: We have developed a new method, called simultaneous recognition and segmentation (SRS) of cells, and applied it to 3D image stacks of the model organism Caenorhabditis elegans. Given a 3D image stack of the animal and a 3D atlas of target cells, SRS is effectively an atlas-guided voxel classification process: cell recognition is realized by smoothly deforming the atlas to best fit the image, where the segmentation is obtained naturally via classification of all image voxels. The method achieved a 97.7% overall recognition accuracy in recognizing a key class of marker cells, the body wall muscle (BWM) cells, on a dataset of 175 C.elegans image stacks containing 14 118 manually curated BWM cells providing the 'ground-truth' for accuracy. This result was achieved without any additional fiducial image features. SRS also automatically identified 14 of the image stacks as involving ±90° rotations. With these stacks excluded from the dataset, the recognition accuracy rose to 99.1%. We also show SRS is generally applicable to other cell types, e.g. intestinal cells. AVAILABILITY: The supplementary movies can be downloaded from our web site http://penglab.janelia.org/proj/celegans_seganno. The method has been implemented as a plug-in program within the V3D system (http://penglab.janelia.org/proj/v3d), and will be released in the V3D plugin source code repository. CONTACT: [email protected].

UNLABELLED: Volume-object annotation system (VANO) is a cross-platform image annotation system that enables one to conveniently visualize and annotate 3D volume objects including nuclei and cells. An application of VANO typically starts with an initial collection of objects produced by a segmentation computation. The objects can then be labeled, categorized, deleted, added, split, merged and redefined. VANO has been used to build high-resolution digital atlases of the nuclei of Caenorhabditis elegans at the L1 stage and the nuclei of Drosophila melanogaster's ventral nerve cord at the late embryonic stage. AVAILABILITY: Platform independent executables of VANO, a sample dataset, and a detailed description of both its design and usage are available at research.janelia.org/peng/proj/vano. VANO is open-source for co-development.

MOTIVATION: Caenorhabditis elegans, a roundworm found in soil, is a widely studied model organism with about 1000 cells in the adult. Producing high-resolution fluorescence images of C.elegans to reveal biological insights is becoming routine, motivating the development of advanced computational tools for analyzing the resulting image stacks. For example, worm bodies usually curve significantly in images. Thus one must 'straighten' the worms if they are to be compared under a canonical coordinate system. RESULTS: We develop a worm straightening algorithm (WSA) that restacks cutting planes orthogonal to a 'backbone' that models the anterior-posterior axis of the worm. We formulate the backbone as a parametric cubic spline defined by a series of control points. We develop two methods for automatically determining the locations of the control points. Our experimental methods show that our approaches effectively straighten both 2D and 3D worm images.

Feature selection is an important problem for pattern classification systems. We study how to select good features according to the maximal statistical dependency criterion based on mutual information. Because of the difficulty in directly implementing the maximal dependency condition, we first derive an equivalent form, called minimal-redundancy-maximal-relevance criterion (mRMR), for first-order incremental feature selection. Then, we present a two-stage feature selection algorithm by combining mRMR and other more sophisticated feature selectors (e.g., wrappers). This allows us to select a compact set of superior features at very low cost. We perform extensive experimental comparison of our algorithm and other methods using three different classifiers (naive Bayes, support vector machine, and linear discriminate analysis) and four different data sets (handwritten digits, arrhythmia, NCI cancer cell lines, and lymphoma tissues). The results confirm that mRMR leads to promising improvement on feature selection and classification accuracy.

Document Image Recognition (DIR), a very useful technique in office automation and digital library applications, is to find the most similar template for any input document image in a prestored template document image data set. Existing methods use both local features and global layout information. In this paper, we propose a novel algorithm based on the global matching of Component Block Projections (CBP), which are the concatenated directional projection vectors of the component blocks of a document image. Compared to those existing methods, CBP-based template-matching methods possess two major advantages: (1) The spatial relationship among the component blocks of a document image is better represented, hence a very high matching accuracy can be obtained even for a large template set and seriously distorted input images; and (2) the effective matching distance of each template and the triangle inequality are proposed to significantly reduce the computational cost. Our experimental results confirm these advantages and show that the CBP-based template-matching methods are very suitable for DIR applications.

A novel hierarchical distributed genetic algorithm is proposed for image segmentation. Firstly, a technique of histogram dichotomy is proposed to explore the statistical property of input image and produce a hierarchical quantization image. Then a hierarchical distributed genetic algorithm (HDGA) is imposed on the quantized image to explore the spatial connectivity and produce final segmentation result. HDGA is a major improvement of the original distributed genetic algorithm (DGA) and multiscale distributed genetic algorithm (MDGA) in four aspects: (1) HDGA does not require the a priori number of image regions, however it can effectively and adaptively control the segmentation quality; (2) the chromosome structure is revised from the original label (multilabel)-condition-fitness format to a more compact (storage-efficient) label-fitness format; (3) the fitness function is revised to utilize the spatial connectivity, but not the original "reconstruction" error; (4) three revised genetic operations are presented to make the algorithm computation-efficient. Our experiments give proofs for the advantages of HDGA.

Document image matching is the key technique for document registration and retrieval. In this paper, a new matching algorithm based on document component block list and component block tree is proposed. Our method can effectively make use of the local information of each page block and the global information of page layout, while it is also robust to image distortion, filled-in text, and noises. This algorithm is then refined and applied to automatic data extraction of column forms. A demonstrating software package has been developed.