No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
A cell-centered database for electron tomographic data
Abstract
Electron tomography is providing a wealth of 3D structural data on biological components ranging from molecules to cells. We are developing a web-accessible database tailored to high-resolution cellular level structural and protein localization data derived from electron tomography. The Cell Centered Database or CCDB is built on an object-relational framework using Oracle 8i and is housed on a server at the San Diego Supercomputer Center at the University of California, San Diego. Data can be deposited and accessed via a web interface. Each volume reconstruction is stored with a full set of descriptors along with tilt images and any derived products such as segmented objects and animations. Tomographic data are supplemented by high-resolution light microscopic data in order to provide correlated data on higher-order cellular and tissue structure. Every object segmented from a reconstruction is included as a distinct entity in the database along with measurements such as volume, surface area, diameter, and length and amount of protein labeling, allowing the querying of image-specific attributes. Data sets obtained in response to a CCDB query are retrieved via the Storage Resource Broker, a data management system for transparent access to local and distributed data collections. The CCDB is designed to provide a resource for structural biologists and to make tomographic data sets available to the scientific community at large.
... The images show hippocampal CA1 molecular layer protoplasmic astrocytes loaded with a fluorescent dye (Lucifer yellow). The images are from the Cell Centered Database 38,138,139 at the National Center for Microscopy and Imaging Research (http://ccdb.ucsd.edu/ index.shtm) ...
... The region of overlap is on the order of ~5%. These images are from the Cell Centered Database 38,138,139 at the National Center for Microscopy and Imaging Research (http://ccdb.ucsd.edu/index.shtm) and have Accession #28. Khakh ...
Astrocytes tile the entire CNS. They are vital for neural circuit function, but have traditionally been viewed as simple, homogenous cells that serve the same essential supportive roles everywhere. Here, we summarize breakthroughs that instead indicate that astrocytes represent a population of complex and functionally diverse cells. Physiological diversity of astrocytes is apparent between different brain circuits and microcircuits, and individual astrocytes display diverse signaling in subcellular compartments. With respect to injury and disease, astrocytes undergo diverse phenotypic changes that may be protective or causative with regard to pathology in a context-dependent manner. These new insights herald the concept that astrocytes represent a diverse population of genetically tractable cells that mediate neural circuit-specific roles in health and disease.
... The clinical data for a VPH model should include neuropsychological test scores, molecular data, imaging, electrophysiological data, biomarkers detected from blood, patient history, and demographic data. While many tools have already been designed and developed to organize and analyze biological data [44,45], specialized Information and Communication Technologies (ICT) components should be used for the implementation of a VPH model, such as the Web Ontology Language (WOL), the Field Modeling Language (FieldML), and the OSIRIX Open-Source software (OsiriX MD 12.x (PIXMEO SARL, Geneva, Switzerland)) for the imaging data manipulation and storage [46][47][48][49][50][51][52][53][54][55]. ...
While frailty corresponds to a multisystem failure, geriatric assessment can recognize multiple pathophysiological lesions and age changes. Up to now, a few frailty indexes have been introduced, presenting definitions of psychological problems, dysregulations in nutritional intake, behavioral abnormalities, and daily functions, genetic, environmental, and cardiovascular comorbidities. The geriatric evaluation includes a vast range of health professionals; therefore, we describe a broad range of applications and frailty scales-biomarkers to investigate and formulate the relationship between frailty lesions, diagnosis, monitoring, and treatment. Additionally, artificial intelligence applications and computational tools are presented, targeting a more efficacy individualized geriatric management of healthy aging.
... Another class of biological data can be obtained in the form of images, which try to represent in a pictorial way the cells and molecules in various biological phenomena of interest. Currently there are some biological imaging databases such as Allen Brain Atlas (Lein et al, 2007), Cell Centered Database (Martone et al, 2002) and FlyEx Database (Poustelnikova, Pisarev, Blagov, Samsonova, & Reinitz, 2004). ...
The inference of Gene Regulatory Networks (GRNs) is a very challenging problem which has attracted increasing attention since the development of high-throughput sequencing and gene expression measurement technologies. Many models and algorithms have been developed to identify GRNs using mainly gene expression profile as data source. As the gene expression data usually has limited number of samples and inherent noise, the integration of gene expression with several other sources of information can be vital for accurately inferring GRNs. For instance, some prior information about the overall topological structure of the GRN can guide inference techniques toward better results. In addition to gene expression data, recently biological information from heterogeneous data sources have been integrated by GRN inference methods as well. The objective of this chapter is to present an overview of GRN inference models and techniques with focus on incorporation of prior information such as, global and local topological features and integration of several heterogeneous data sources.
... Through the portal, users can acquire images from our JEM-4000EX Intermediate High Voltage Electron Microscope (IVEM) and our new JEM-3100EF energy filtering IVEM and perform all of the steps and computations necessary for end-to-end electron tomography. For managing and mining cell level microscopic information, we have created the Cell Centered Database or CCDB, a web-accessible database for 3D reconstructions derived from light and electron microscopy, including electron tomography 4 . The CCDB (www.ncmir.ucsd.edu/CCDB) ...
Informatics Resources for Electron Tomography - Volume 9 Issue S02 - Maryann E. Martone, Shenglan Zhang, Steven Peltier, Abel W. Lin, David Lee, Tomas Molina, Amarnath Gupta, Mark H. Ellisman
... Entries include light and electron microscopy imaging, as 67 well as correlated datasets. The resource includes datasets previously available as the Cell 68 Centered Database (CCDB), an online repository of high-resolution, often 3D, light and electron 69 microscopy data, including many electron tomograms [22][23][24]. 70 ...
Three-dimensional electron microscopy techniques like electron tomography provide valuable insights into cellular structures, and present significant challenges for data storage and dissemination. Here we explored a novel method to publicly release more than 11,000 such datasets, more than 30 TB in total, collected by our group. Our method, based on a peer-to-peer file sharing network built around a blockchain ledger, offers a distributed solution to data storage. In addition, we offer a user-friendly browser-based interface, https://etdb.caltech.edu, for anyone interested to explore and download our data. We discuss the relative advantages and disadvantages of this system and provide tools for other groups to mine our data and/or use the same approach to share their own imaging datasets.
... Entries include light and electron microscopy imaging, as 67 well as correlated datasets. The resource includes datasets previously available as the Cell 68 Centered Database (CCDB), an online repository of high-resolution, often 3D, light and electron 69 microscopy data, including many electron tomograms [22][23][24]. 70 ...
Three-dimensional electron microscopy techniques like electron tomography provide valuable insights into cellular structures, and present significant challenges for data storage and dissemination. Here we explored a novel method to publicly release more than 11,000 such datasets, more than 30 TB in total, collected by our group. Our method, based on a peer-to-peer file sharing network built around a blockchain ledger, offers a distributed solution to data storage. In addition, we offer a user-friendly browser-based interface, https://etdb.caltech.edu, for anyone interested to explore and download our data. We discuss the relative advantages and disadvantages of this system and provide tools for other groups to mine our data and/or use the same approach to share their own imaging datasets.
... Moreover, it is possible to combine different TEM images of the same specimen to create a 3D tomography representation which usually requires a very timeintensive manual segmentation (Section 3.1). Central repositories, such as the Cell-centered database, provide access to a number of microscopic images and including some segmentations [27]. Another relevant topic in this context is the coloring of the cell and its components. ...
The structural modeling and representation of cells is a complex task as different microscopic, spectroscopic and other information resources have to be combined to achieve a three-dimensional representation with high accuracy. Moreover, to provide an appropriate spatial representation of the cell, a stereoscopic 3D (S3D) visualization is favorable. In this work, a structural cell model is created by combining information from various light microscopic and electron microscopic images as well as from publication-related data. At the mesoscopic level each cell component is presented with special structural and visual properties; at the molecular level a cell membrane composition and the underlying modeling method are discussed; and structural information is correlated with those at the functional level (represented by simplified energy-producing metabolic pathways).
The organism used as an example is the unicellular Chlamydomonas reinhardtii , which might be important in future alternative energy production processes. Based on the 3D model, an educative S3D animation was created which was shown at conferences. The complete workflow was accomplished by using the open source 3D modeling software Blender.
The discussed project including the animation is available from: http://Cm5.CELLmicrocosmos.org
... The images show hippocampal CA1 molecular layer protoplasmic astrocytes loaded with a fluorescent dye (Lucifer yellow). The images are from the Cell Centered Database [6,123,124] at the National Center for Microscopy and Imaging Research (http://ccdb.ucsd.edu/ index.shtm) ...
Astrocytes are abundant within mature neural circuits and are involved in brain disorders. Here, we summarize our current understanding of astrocytes and Huntington’s disease (HD), with a focus on correlative and causative dysfunctions of ion homeostasis, calcium signaling, and neurotransmitter clearance, as well as on the use of transplanted astrocytes to produce therapeutic benefit in mouse models of HD. Overall, the data suggest that astrocyte dysfunction is an important contributor to the onset and progression of some HD symptoms in mice. Additional exploration of astrocytes in HD mouse models and humans is needed and may provide new therapeutic opportunities to explore in conjunction with neuronal rescue and repair strategies.
... Another class of biological data can be obtained in the form of images, which try to represent in a pictorial way the cells and molecules in various biological phenomena of interest. Currently there are some biological imaging databases such as Allen Brain Atlas (Lein et al, 2007), Cell Centered Database (Martone et al, 2002) and FlyEx Database (Poustelnikova, Pisarev, Blagov, Samsonova, & Reinitz, 2004). There are several recent initiatives where biological information is incorporated in the inference/prediction methods. ...
The inference of Gene Regulatory Networks (GRNs) is a very challenging problem which has attracted increasing attention since the development of high-throughput sequencing and gene expression measurement technologies. Many models and algorithms have been developed to identify GRNs using mainly gene expression profile as data source. As the gene expression data usually has limited number of samples and inherent noise, the integration of gene expression with several other sources of information can be vital for accurately inferring GRNs. For instance, some prior information about the overall topological structure of the GRN can guide inference techniques toward better results. In addition to gene expression data, recently biological information from heterogeneous data sources have been integrated by GRN inference methods as well. The objective of this chapter is to present an overview of GRN inference models and techniques with focus on incorporation of prior information such as, global and local topological features and integration of several heterogeneous data sources.
... The experimental dataset consists of a freeze-substituted Ver-oE6 cultured cell infected with the SARS-CoV (The Cell Centered Database, project P2005, microscopy product 6021, [14,15]). It includes 131 TEM images acquired with a 1°angular step at the range [ À 65°, þ65°] using single tilt projection geometry. ...
3D image reconstruction with electron tomography holds problems due to the severely limited range of projection angles and low signal to noise ratio of the acquired projection images. The maximum a posteriori (MAP) reconstruction methods have been successful in compensating for the missing information and suppressing noise with their intrinsic regularization techniques. There are two major problems in MAP reconstruction methods: (1) selection of the regularization parameter that controls the balance between the data fidelity and the prior information, and (2) long computation time. One aim of this study is to provide an adaptive solution to the regularization parameter selection problem without having additional knowledge about the imaging environment and the sample. The other aim is to realize the reconstruction using sequences of resolution levels to shorten the computation time. The reconstructions were analyzed in terms of accuracy and computational efficiency using a simulated biological phantom and publically available experimental datasets of electron tomography. The numerical and visual evaluations of the experiments show that the adaptive multiresolution method can provide more accurate results than the weighted back projection (WBP), simultaneous iterative reconstruction technique (SIRT), and sequential MAP expectation maximization (sMAPEM) method. The method is superior to sMAPEM also in terms of computation time and usability since it can reconstruct 3D images significantly faster without requiring any parameter to be set by the user.
... Moreover, scenarios 1, 2, 4 and 5 represent tours based on pre-tour configurations, whereas 3 and 6 represent ontour presentations, where the stereoscopic effect has to be interactively optimized (because no tour points containing eye distance information exist). The six scenarios were based on three different cell models: two interpretative models of an animal cell and a plant cell which were modeled in context of the CELLmicrocosmos project, and one 3D-microscopy-based model of a neuron cell downloaded from the Cell-centered database (CCDB-ID 77503) [23], [24]. Each cell model contains a number of cell components, such as the nucleus, cell membrane, mitochondrion, etc. ...
Although large-scale stereoscopic 3D environments like CAVEs are a favorable location for group presentations, the perspective projection and stereoscopic optimization usually follows a navigator-centric approach. Therefore, these presentations are usually accompanied by strong side-effects, such as motion sickness which is often caused by a disturbed stereoscopic vision. The reason is that the stereoscopic visualization is usually optimized for the only head-tracked person in the CAVE – the navigator – ignoring the needs of the real target group – the audience.
To overcome this misconception, this work proposes an alternative to the head tracking-based stereoscopic effect optimization. By using an interactive virtual overview map in 3D, the pre-tour and on-tour configuration of the stereoscopic effect is provided, partly utilizing our previously published interactive projection plane approach. This Stereoscopic Space Map is visualized by the zSpace 200®, whereas the virtual world is shown on a panoramic 330° CAVE2TM.
A pilot expert study with eight participants was conducted using pre-configured tours through 3D models. The comparison of the manual and automatic stereoscopic adjustment showed that the proposed approach is an appropriate alternative to the nowadays commonly used head tracking-based stereoscopic adjustment.
Read the freely available paper here:
http://ist.publisher.ingentaconnect.com/contentone/ist/ei/2016/00002016/00000005/art00006
See the video here:
https://www.youtube.com/watch?v=W5Xl6WC-dl0
... The datasets used for the experiments in this work were collected from the Cell Centered Database (CCDB) supported by the National Center for Microscopy and Imaging Research (NCMIR) (Martone et al., 2008(Martone et al., , 2003(Martone et al., , 2002. We chose eight TEM datasets which include tomograms having diversity in image contrast and variety in mitochondrial membrane and crista characteristics. ...
Recent studies reveal that mitochondria take substantial responsibility in cellular functions that are closely related to aging diseases caused by degeneration of neurons. These studies emphasize that the membrane and crista morphology of a mitochondrion should receive attention in order to investigate the link between mitochondrial function and its physical structure. Electron microscope tomography (EMT) allows analysis of the inner structures of mitochondria by providing highly detailed visual data from large volumes. Computerized segmentation of mitochondria with minimum manual effort is essential to accelerate the study of mitochondrial structure/function relationships. In this work, we improved and extended our previous attempts to detect and segment mitochondria from transmission electron microcopy (TEM) images. A parabolic arc model was utilized to extract membrane structures. Then, curve energy based active contours were employed to obtain roughly outlined candidate mitochondrial regions. Finally, a validation process was applied to obtain the final segmentation data. 3D extension of the algorithm is also presented in this paper. Our method achieved an average F-score performance of 0.84. Average Dice Similarity Coefficient and boundary error were measured as 0.87 and 14 nm respectively.
... By reconstructing precise volumetric models of mitochondria associated both with rods and cones, Perkins inferred the energy requirements of the two different cell types and suggested that cones have greater energy consumption compared to light adapted rods (Perkins, Ellisman et al. 2003). As further illustration, Martone et al. have developed a sophisticated database of image and segmentation data useful for cross-correlated quantitative studies of segmented features (Martone, Gupta et al. 2002). ...
Crowd sourced strategies have the potential to increase the throughput of tasks historically constrained by the performance of individual experts. A critical open question is how to configure crowd-based mechanisms, such as online micro-task markets, to accomplish work normally done by experts. In the context of one kind of expert work, feature extraction from electron microscope images, this thesis describes three experiments conducted with Amazon???s Mechanical Turk to explore the feasibility of crowdsourcing for tasks that traditionally rely on experts.
The first experiment combined the output from learning algorithms with judgments made by non-experts to see whether the crowd could efficiently and accurately detect the best algorithmic performance for image segmentation. Image segmentation is an important but rate limiting step in analyzing biological imagery. Current best practice relies on extracting features by hand. Results showed that crowd workers were able to match the results of expert workers in 87.5% of the cases given the same task and that they did so with very little training. The second experiment used crowd responses to progressively refine task instructions. Results showed that crowd workers were able to consistently add information to the instructions and produced results the crowd perceived as more clear by an average of 8.7%. Finally, the third experiment mapped images to abstract representations to see whether the crowd could efficiently and accurately identify target structures. Results showed that crowd workers were able to find 100% of known structures with an 82% decrease in false positives compared to conventional automated image processing.
This thesis makes a number of contributions. First, the work demonstrates that tasks previously performed by highly-trained experts, such as image extraction, can be accomplished by non-experts in less time and with comparable accuracy when organized through a micro-task market. Second, the work shows that engaging crowd workers to reflect on the description of tasks can be used to have them refine tasks to produce increased engagement by subsequent crowd workers. Finally, the work shows that abstract representations perform nearly as well as actual images in terms of using a crowd of non-experts to locate targeted features.
... The CCDB project was started in 1998 under the auspices of the Human Brain Project to provide a venue for sharing and mining, cellular and sub cellular data derived from light and electron microscopy, including correlated imaging. It was one of the first web databases devoted to the then emerging technique of electron tomography [18,19]. The data are released together with application-specific validation performed for each antibody, including immunohistochemisty, Western blot analysis and, for a large fraction, a protein array assay and immunofluorescent based confocal microscopy. ...
... The CCDB project was started in 1998 under the auspices of the Human Brain Project to provide a venue for sharing and mining, cellular and sub cellular data derived from light and electron microscopy, including correlated imaging. It was one of the first web databases devoted to the then emerging technique of electron tomography [18,19]. The data are released together with application-specific validation performed for each antibody, including immunohistochemisty, Western blot analysis and, for a large fraction, a protein array assay and immunofluorescent based confocal microscopy. ...
The collaboration of public health education and information technology has made patient care safer and more reliable than before. Nurses and doctors use handheld computers to record a patient's medical history and check that they are administering the correct treatment. Fortunately Public Health Informatics (PHI) is the intersecting point of technology and public health. Therefore, the inclusion of online medical and epidemiology databases in the course curriculum of budding medical professionals and postgraduate students would be beneficial in enhancing the quality of health care, extensive epidemiological research, health education, health policies, health planning and consumer satisfaction as well. The purpose of this article is to discuss and provide introduction of various databases which have huge information and it could be used to enhance the public health education.
... Many databases for structural data have been described (e.g. the BioImage database (Carazo et al., 1999;Lindek et al., 1999), EMEN2 (Ludtke et al., 2003;Rees et al., 2013), the Carragher Lab database (Fellmann et al., 2002), IMIRS (Dai et al., 2003), E-MSD (Boutselakis et al., 2003), PDB (Berman et al., 2003;Bernstein et al., 1977), and the EMDB Lawson et al., 2011;Tagari et al., 2002)), and we first explored whether an existing program might meet our needs. We installed and tested the Cell Centered Database developed at UC San Diego, a sophisticated and complex tool designed to house microscopy data of many different types in a distributed fashion across disk drives spread across the world (Martone et al., 2002;Martone et al., 2008). We found, however, that we preferred something simpler, local, and more customizable, so we designed and developed our own database, which we call the Caltech Tomography Database. ...
Here we describe the Caltech Tomography Database and automatic image processing pipeline, designed to process, store, display, and distribute electron tomographic data including tilt-series, sample information, data collection parameters, 3D reconstructions, correlated light microscope images, snapshots, segmentations, movies, and other associated files. Tilt-series are typically uploaded automatically during collection to a user's "Inbox" and processed automatically, but can also be entered and processed in batches via scripts or file-by-file through an internet interface. As with the video website YouTube, each tilt-series is represented on the browsing page with a link to the full record, a thumbnail image and a video icon that delivers a movie of the tomogram in a pop-out window. Annotation tools allow users to add notes and snapshots. The database is fully searchable, and sets of tilt-series can be selected and re-processed, edited, or downloaded to a personal workstation. The results of further processing and snapshots of key results can be recorded in the database, automatically linked to the appropriate tilt-series. While the database is password-protected for local browsing and searching, datasets can be made public and individual files can be shared with collaborators over the Internet. Together these tools facilitate high-throughput tomography work by both individuals and groups.
Copyright © 2015. Published by Elsevier Inc.
... We tested the proposed method on ten slices of eight different datasets from the Cell Centered Database (CCDB) supported by the National Center for Microscopy and Imaging Research (NCMIR) [17][18][19] . The datasets show some mitochondria which appear in a variety of shape, size and cristae characteristics Figure 3 illustrates the curve fitting results for a sample slice. ...
Mitochondria are sub-cellular components which are mainly responsible for synthesis of adenosine tri-phosphate (ATP) and involved in the regulation of several cellular activities such as apoptosis. The relation between some common diseases of aging and morphological structure of mitochondria is gaining strength by an increasing number of studies. Electron microscope tomography (EMT) provides high-resolution images of the 3D structure and internal arrangement of mitochondria. Studies that aim to reveal the correlation between mitochondrial structure and its function require the aid of special software tools for manual segmentation of mitochondria from EMT images. Automated detection and segmentation of mitochondria is a challenging problem due to the variety of mitochondrial structures, the presence of noise, artifacts and other sub-cellular structures. Segmentation methods reported in the literature require human interaction to initialize the algorithms. In our previous study, we focused on 2D detection and segmentation of mitochondria using an ellipse detection method. In this study, we propose a new approach for automatic detection of mitochondria from EMT images. First, a preprocessing step was applied in order to reduce the effect of nonmitochondrial sub-cellular structures. Then, a curve fitting approach was presented using a Hessian-based ridge detector to extract membrane-like structures and a curve-growing scheme. Finally, an automatic algorithm was employed to detect mitochondria which are represented by a subset of the detected curves. The results show that the proposed method is more robust in detection of mitochondria in consecutive EMT slices as compared with our previous automatic method.
... The OME provides a data model of common specification for storing details of microscope setup and image acquisition. Cell-Centered Database (CCDB) [64,65] provides a data model to capture image analysis output, image data, and information on the specimen preparation and imaging conditions that generated the image data. DICOM Working Group 26 is developing a DICOM based standard for storing microscopy images [66]. ...
... The reconstructed single-axis electron tomographic dataset was created by (Capani et al., 2001) and downloaded from the Cell Centered Database (http://ccdb.ucsd.edu/index.shtm, microscopy product ID: 22), (Martone et al., 2002). In this tomographic experiment, images were typically obtained over a range of ±60°at a magnification of 30,000Â, with the final pixel size of 2 nm. ...
The limited specimen tilting range that is typically available in electron tomography gives rise to a region in the Fourier space of the reconstructed object where experimental data are unavailable - the missing wedge. Since this region is sharply delimited from the area of available data, the reconstructed signal is typically hampered by convolution with its impulse response, which gives rise to the well-known missing wedge artefacts in 3D reconstructions. Despite the recent progress in the field of reconstruction and regularization techniques, the missing wedge artefacts remain untreated in most current reconstruction workflows in structural biology. Therefore we have designed a simple Fourier angular filter that effectively suppresses the ray artefacts in the single-axis tilting projection acquisition scheme, making single-axis tomographic reconstructions easier to interpret in particular at low signal-to-noise ratio in acquired projections. The proposed filter can be easily incorporated into current electron tomographic reconstruction schemes.
... These data sets are often stored in data banks and made available through web portals. 29,30 These web sites often use thumbnail images of the data sets as previews for users to distinguish different data sets without having to download them. It is not practical for the operators of such systems to manually rotate each data set to create good views for the snapshot. ...
We propose a new algorithm for automatic viewpoint selection for volume
data sets. While most previous algorithms depend on information
theoretic frameworks, our algorithm solely focuses on the data itself
without off-line rendering steps, and finds a view direction which shows
the data set's features well. The algorithm consists of two main steps:
feature selection and viewpoint selection. The feature selection step is
an extension of the 2D Harris interest point detection algorithm. This
step selects corner and/or high-intensity points as features, which
captures the overall structures and local details. The second step,
viewpoint selection, takes this set and finds a direction that lays out
those points in a way that the variance of projected points is
maximized, which can be formulated as a Principal Component Analysis
(PCA) problem. The PCA solution guarantees that surfaces with detected
corner points are less likely to be degenerative, and it minimizes
occlusion between them. Our entire algorithm takes less than a second,
which allows it to be integrated into real-time volume rendering
applications where users can modify the volume with transfer functions,
because the optimized viewpoint depends on the transfer function.
... Nevertheless, advances in information technology such as the increase in mass storage and the improving rapidity of database access have accelerated the construction of databases holding large files. For example, the Cell Centered Database (CCDB) houses several types of image data including electron micrographs from various kinds of organisms including plants (Martone et al. 2002, Martone et al. 2007, Orloff et al. 2013. A specifically organellar example is the Chloroplast Function Database II, which is dedicated to chloroplast morphology and its function, and this also collects electron micrographs (Myouga et al. 2013). ...
The Plant Organelles Database 2 (PODB2), which was first launched in 2006 as PODB, provides static image and movie data of
plant organelles, protocols for plant organelle research and external links to relevant websites. PODB2 has facilitated plant
organellar research and the understanding of plant organelle dynamics. To provide comprehensive information on plant organelles
in more detail, PODB2 was updated to PODB3 (http://podb.nibb.ac.jp/Organellome/). PODB3 contains two additional components: the electron micrograph database and the perceptive organelles database. Through
the electron micrograph database, users can examine the subcellular and/or suborganellar structures in various organs of wild-type
and mutant plants. The perceptive organelles database provides information on organelle dynamics in response to external stimuli.
In addition to the extra components, the user interface for access has been enhanced in PODB3. The data in PODB3 are directly
submitted by plant researchers and can be freely downloaded for use in further analysis. PODB3 contains all the information
included in PODB2, and the volume of data and protocols deposited in PODB3 continue to grow steadily. We welcome contributions
of data from all plant researchers to enhance the utility and comprehensiveness of PODB3.
... 411 triangulated surface reconstructions of spine dendrites were produced by manual contouring of tomographic reconstructions of neurons and curated at the Cell-Centered DataBase at https://ccdb.ucsd.edu/CCDB/index.shtml ( [11], [12]). The reconstructed spines were aligned with a standard coordinate system with respect to the smallest Wild Type (WT) spine via similitude matching (scale or noscale, rotation, translation) of 14 landmarks suitably placed on each spine. ...
In this article, we model the metric distances of den- drite spines of mice based on various morphometric (shape and size related) measures, condition, and type of the spines. The question of interest is how the met- ric distances differ with respect to various morphomet- ric measures, two different conditions and six spine types which are based on pre-assigned shape catego- ries. Large Deformation Diffeomorphic Metric Map- ping algorithm is one of the tools to measure mor- phometric differences in dendrite spines with respect to a template spine. We use Principal Component Analysis (PCA) on continuous numerical morphomet- ric measures to obtain uncorrelated morphometric features, since PCA provides the orthogonal directions with most variation. We demonstrate that for the raw scores (i.e., values not adjusted for scale) metric dis- tances differ significantly by size and type of spines, and size and length at each spine type level. Since size (or scale) dominates the other variables in vari- ance and spine type is based on shape, differences in metric distances due to other variables might be masked. Hence, we adjust metric distances and other morphometric measures for scale. We demonstrate that after adjusting for scale, metric distances differ most significantly by shape (i.e., spine type), and then by length and size for each spine type. In the presence of other morphometric measures, the condition seems not to be significant in explaining variation in metric dis- tances, hence we need to analyze spines for each shape type separately. Although the methodology used here is applied on morphometric measures of mouse spines, it is also valid for morphometric measures of other organs or tissues and other metric dis-
... We also compared the fidelity of the skeletonization process using both NEMO and ImageJ and the morphological and Sholl parameters output by NEMO and NeuronMorpho with ImageJ on four representative images of Purkinje neurons from different sources [respectively a bright field image, a fluorescence micrograph, an organotypic slice and a confocal image (Martone et al., 2002)]. The process was faster with NEMO due to the semi-automated tracing of neurite pathways (Figure 4). ...
Morphometric analysis of neurons and brain tissue is relevant to the study of neuron circuitry development during the first phases of brain growth or for probing the link between microstructural morphology and degenerative diseases. As neural imaging techniques become ever more sophisticated, so does the amount and complexity of data generated. The NEuronMOrphological analysis tool NEMO was purposely developed to handle and process large numbers of optical microscopy image files of neurons in culture or slices in order to automatically run batch routines, store data and apply multivariate classification and feature extraction using 3-way principal component analysis (PCA). Here we describe the software's main features, underlining the differences between NEMO and other commercial and non-commercial image processing tools, and show an example of how NEMO can be used to classify neurons from wild-type mice and from animal models of autism.
... Eighteen datasets resulting from this publication have been released to the public through the CCDB. Our large scale mosaics can be browsed or downloaded through the Cell Centered Database (CCDB;Martone et al., 2002Martone et al., , 2008) at http://ccdb.ucsd.edu by selecting project ID P20002. ...
Pannexin1 (Panx1) channels release cytosolic ATP in response to signaling pathways. Panx1 is highly expressed in the central nervous system. We used four antibodies with different Panx1 anti-peptide epitopes to analyze four regions of rat brain. These antibodies labeled the same bands in Western blots and had highly similar patterns of immunofluorescence in tissue culture cells expressing Panx1, but Western blots of brain lysates from Panx1 knockout and control mice showed different banding patterns. Localizations of Panx1 in brain slices were generated using automated wide field mosaic confocal microscopy for imaging large regions of interest while retaining maximum resolution for examining cell populations and compartments. We compared Panx1 expression over the cerebellum, hippocampus with adjacent cortex, thalamus, and olfactory bulb. While Panx1 localizes to the same neuronal cell types, subcellular localizations differ. Two antibodies with epitopes against the intracellular loop and one against the carboxy terminus preferentially labeled cell bodies, while an antibody raised against an N-terminal peptide highlighted neuronal processes more than cell bodies. These labeling patterns may be a reflection of different cellular and subcellular localizations of full-length and/or modified Panx1 channels where each antibody is highlighting unique or differentially accessible Panx1 populations. However, we cannot rule out that one or more of these antibodies have specificity issues. All data associated with experiments from these four antibodies are presented in a manner that allows them to be compared and our claims thoroughly evaluated, rather than eliminating results that were questionable. Each antibody is given a unique identifier through the NIF Antibody Registry that can be used to track usage of individual antibodies across papers and all image and metadata are made available in the public repository, the Cell Centered Database, for on-line viewing, and download.
... Computation time is almost constant as a function of the number of objects. MOGAC was applied to cell tracking in a microscopy image acquired from the Cell Centered Database (CCDB) [17]. The image was first automatically segmented with Medical Image Processing And Visualization (MIPAV) [18] through gray-level morphology and thresholding techniques (Fig. 3a). ...
An emerging topic is to build image segmentation systems that can segment hundreds to thousands of objects (i.e. cell segmentation\tracking, full brain parcellation, full body segmentation, etc.). Multi-object Level Set Methods (MLSM) perform this task with the benefit of sub-pixel precision. However, current implementations of MLSM are not as computationally or memory efficient as their region growing and graph cut counterparts which lack sub-pixel precision. To address this performance gap, we present a novel parallel implementation of MLSM that leverages the sparse properties of the algorithm to minimize its memory footprint for multiple objects. The new method, Multi-Object Geodesic Active Contours (MOGAC), can represent N objects with just two functions: a label mask image and unsigned distance field. The time complexity of the algorithm is shown to be O((M (power)d)/P) for M (power)d pixels and P processing units in dimension d = {2,3}, independent of the number of objects. Results are presented for 2D and 3D image segmentation problems.
Understanding functions of astrocytes can be greatly enhanced by building and simulating computational models that capture their morphological details. Novel computational tools enable utilization of existing morphological data of astrocytes and building models that have appropriate level of details for specific simulation purposes. In addition to analyzing existing computational tools for constructing, transforming, and assessing astrocyte morphologies, we present here the CellRemorph toolkit implemented as an add-on for Blender, a 3D modeling platform increasingly recognized for its utility for manipulating 3D biological data. To our knowledge, CellRemorph is the first toolkit for transforming astrocyte morphologies from polygonal surface meshes into adjustable surface point clouds and vice versa, precisely selecting nanoprocesses, and slicing morphologies into segments with equal surface areas or volumes. CellRemorph is an open-source toolkit under the GNU General Public License and easily accessible via an intuitive graphical user interface. CellRemorph will be a valuable addition to other Blender add-ons, providing novel functionality that facilitates the creation of realistic astrocyte morphologies for different types of morphologically detailed simulations elucidating the role of astrocytes both in health and disease.
Integrating spatially localized molecular networks into virtual cell environments is an approach which is only provided by a very small number of tools. As this task requires the combination of a set of Biotechnology/Bioinformatics-related information sources, it can be seen as an appropriate example for Integrative Bioinformatics research. Here, we want to show new immersive perspectives for cytological pathway integration by combining recent explorative technologies with the software CELLmicrocosmos 4 PathwayIntegration. A mesoscopic-localized metabolic pathway—i.e. the citrate cycle and the glycolysis—is localized based on database entries onto an abstract cell environment of Arabidopsis thaliana. The created cell model is used in three different contexts providing different degrees of immersion: (1) Web-based 2D exploration of 3D Scenarios (using Gather.town), (2) Exploration and Annotation in a VR Design Application (using Gravity Sketch), and (3) Large-Scale VR Visualization and Navigation (using the CAVE2 and zSpace). All these examples promise to be very useful in the context of Integrative Bioinformatics-related education as well as communication.
Neuronal firing and neuron-to-neuron synaptic wiring are currently widely described as orchestrated by astrocytes—elaborately ramified glial cells tiling the cortical and hippocampal space into non-overlapping domains, each covering hundreds of individual dendrites and hundreds thousands synapses. A key component to astrocytic signaling is the dynamics of cytosolic Ca2+ which displays multiscale spatiotemporal patterns from short confined elemental Ca2+ events (puffs) to Ca2+ waves expanding through many cells. Here, we synthesize the current understanding of astrocyte morphology, coupling local synaptic activity to astrocytic Ca2+ in perisynaptic astrocytic processes and morphology-defined mechanisms of Ca2+ regulation in a distributed model. To this end, we build simplified realistic data-driven spatial network templates and compile model equations as defined by local cell morphology. The input to the model is spatially uncorrelated stochastic synaptic activity. The proposed modeling approach is validated by statistics of simulated Ca2+ transients at a single cell level. In multicellular templates we observe regular sequences of cell entrainment in Ca2+ waves, as a result of interplay between stochastic input and morphology variability between individual astrocytes. Our approach adds spatial dimension to the existing astrocyte models by employment of realistic morphology while retaining enough flexibility and scalability to be embedded in multiscale heterocellular models of neural tissue. We conclude that the proposed approach provides a useful description of neuron-driven Ca2+-activity in the astrocyte syncytium.
We propose a method to reconstruct the optical properties of a scattering medium with subwavelength resolution. The method is based on the solution to the inverse scattering problem with internal sources. Applications to photoactivated localization microscopy are described.
Bioimage informatics is a field wherein high-throughput image informatics methods are used to solve challenging scientific problems related to biology and medicine. When the image datasets become larger and more complicated, many conventional image analysis approaches are no longer applicable. Here, we discuss two critical challenges of large-scale bioimage informatics applications, namely, data accessibility and adaptive data analysis. We highlight case studies to show that these challenges can be tackled based on distributed image computing as well as machine learning of image examples in a multidimensional environment.
Many aspects of skeletal morphological research in bryozoans have involved destructive methods, such as thin sectioning of Palaeozoic fossils or removal of basal walls to view internal structures in cheilostomes. Two relatively new technologies allow non-destructive visualisation of internal zooidal skeletal structures. Tomography is shown to be very effective for cheilostome zooid cavities as it allows resolution down to 1 μm, which can resolve features such as the morphological evidence of origins of frontal shields in ascophoran cheilostomes. It also generates a three-dimensional reconstruction of the whole structure, which can resolve complex internal structures like those of Siphonicytara, or how multilaminar colonies develop the communication between layers. The Synchrotron is best suited to image the internal structures of Palaeozoic bryozoans that have mineral-filled zooidal cavities, as it can better resolve differences in composition.
In most vertebrate neurons, action potentials are initiated in the axon initial segment (AIS), a specialized region of the axon containing a high density of voltage-gated sodium and potassium channels. It has recently been proposed that neurons use plasticity of AIS length and/or location to regulate their intrinsic excitability. Here we quantify the impact of neuron morphology on AIS plasticity using computational models of simplified and realistic somatodendritic morphologies. In small neurons (e.g., dentate granule neurons), excitability was highest when the AIS was of intermediate length and located adjacent to the soma. Conversely, neurons having larger dendritic trees (e.g., pyramidal neurons) were most excitable when the AIS was longer and/or located away from the soma. For any given somatodendritic morphology, increasing dendritic membrane capacitance and/or conductance favored a longer and more distally located AIS. Overall, changes to AIS length, with corresponding changes in total sodium conductance, were far more effective in regulating neuron excitability than were changes in AIS location, while dendritic capacitance had a larger impact on AIS performance than did dendritic conductance. The somatodendritic influence on AIS performance reflects modest soma-to-AIS voltage attenuation combined with neuron size-dependent changes in AIS input resistance, effective membrane time constant, and isolation from somatodendritic capacitance. We conclude that the impact of AIS plasticity on neuron excitability will depend largely on somatodendritic morphology, and that, in some neurons, a shorter or more distally located AIS may promote, rather than limit, action potential generation.
The launch of the US BRAIN and European Human Brain Projects coincides with growing international efforts toward transparency and increased access to publicly funded research in the neurosciences. The need for data-sharing standards and neuroinformatics infrastructure is more pressing than ever. However, 'big science' efforts are not the only drivers of data-sharing needs, as neuroscientists across the full spectrum of research grapple with the overwhelming volume of data being generated daily and a scientific environment that is increasingly focused on collaboration. In this commentary, we consider the issue of sharing of the richly diverse and heterogeneous small data sets produced by individual neuroscientists, so-called long-tail data. We consider the utility of these data, the diversity of repositories and options available for sharing such data, and emerging best practices. We provide use cases in which aggregating and mining diverse long-tail data convert numerous small data sources into big data for improved knowledge about neuroscience-related disorders.
Between technological breakthrough and new computational approaches, the amount of biological data is increasing explosively. Till to 2007, there are 968 biological databases. To provide biologists central and uniform access to all kinds of data stored in biological databases becomes a critical research To minimize disruption of current operation, maintain local autonomy and handle heterogeneities, federation databases has been proposed as an ideal solution. This paper looks into this situation and reports on our experience with trying multiple approaches for biological database integration. It discusses the trade-offs among performance, support for heterogeneity, robustness and scalability. Of significance, is the discovery that the most flexible approach --Web Services performs very competitively.
Integrative Bioinformatics combines a number of different disciplines related to biology as well as informatics. One major target of this research area is the creation of a virtual cell. Naturally, this topic is accompanied by a vast amount of problems which arise due to the fact that a large number of highly specific disciplines have to be addressed. In this publication a subproblem will be discussed, functional cell modeling. Beginning with a virtual cell environment which provides cell components featuring different subcompartmental layers, protein-related networks will be localized and visualized. For the localization, a data warehouse will be accessed. Special interactive techniques will be applied to the semiautomatic analysis of localization entries found in databases. Finally, different visualization approaches will be shown, and 2D and 3D network visualization will be discussed, as well as quantitative illustrations using charts.
Cell visualization is an important area of scientific and educational visualization. There is already a number of astonishing animations illustrating the structural and functional properties of biological cells available in the Internet. However, these visualizations usually do not take advantage of three-dimensional (3-D) stereoscopic techniques. The stereoscopic visualization of the microcosmos cell—invisible to the human eye—bears high potential for educational as well as scientific approaches. Using open source tools it will be shown that it is possible to generate statically rendered as well as interactive stereoscopic cell visualizations. First, the 3-D modeling software Blender in conjunction with Schneider’s stereoscopic camera plug-in will be used to generate a stereoscopic cell animation. While static renderings have an advantage in that the stereoscopic effect can be optimized for spectators, interactive stereoscopic visualizations always have to adjust and optimize the stereoscopic effect for users who can freely navigate through space. Cell visualization is paradigmatic for this problem because the scale differences from the mesoscopic to the molecular level account for a factor of 100,000. Therefore, two stereoscopic approaches of the CELLmicrocosmos project will be introduced, which address the stereoscopic scaling problem. The stereoscopic quality was positively evaluated by 20 students.
Electron tomography (ET) is an emerging technology for the three dimensional imaging of cellular ultrastructure. In combination with other techniques, it can provide three dimensional reconstructions of protein assemblies, correlate 3D structures with functional investigations at the light microscope level and provide structural information which extends the findings of genomics and molecular biology.
Realistic physical details are essential for the task of modeling over many spatial scales. While the electron microscope resolution can be as low as a fraction of a nm, a typical 3D reconstruction may just cover 1/1015 of the volume of an optical microscope reconstruction. In order to bridge the gap between those two approaches, the available spatial range of an ET reconstruction has been expanded by various techniques. Large sensor arrays and wide-field camera assemblies have increased the field dimensions by a factor of ten over the past decade, and new techniques for serial tomography and montaging make possible the assembly of many three-dimensional reconstructions.
The number of tomographic volumes necessary to incorporate an average cell down to the protein assembly level is of the order 104, and given the imaging and algorithm requirements, the computational problem lays well in the exascale range. Tomographic reconstruction can be made parallel to a very high degree, and their associated algorithms can be mapped to the simplified processors comprising, for example, a graphics processor unit. Programming this on a GPU board yields a large speedup, but we expect that many more orders of magnitude improvement in computational capabilities will still be required in the coming decade. Exascale computing will raise a new set of problems, associated with component energy requirements (cost per operation and costs of data transfer) and heat dissipation issues. As energy per operation is driven down, reliability decreases, which in turn raises difficult problems in validation of computer models (is the algorithmic approach faithful to physical reality), and verification of codes (is the computation reliably correct and replicable). Leaving aside the hardware issues, many of these problems will require new mathematical and algorithmic approaches, including, potentially, a re-evaluation of the Turing model of computation.
Automatic viewpoint selection algorithms try to optimize the view of a data set to best show its features. They are often based on information theoretic frameworks. Although many algorithms have shown useful results, they often take several seconds to produce a result because they render the scene from a variety of viewpoints and analyze the result. In this article, we propose a new algorithm for volume data sets that dramatically reduces the running time. Our entire algorithm takes less than a second, which allows it to be integrated into real-time volume-rendering applications. The interactive performance is achieved by solving a maximization problem with a small sample of the data set, instead of rendering it from a variety of directions. We compare performance results of our algorithm to state-of-the-art approaches and show that our algorithm achieves comparable results for the resulting viewpoints. Furthermore, we apply our algorithm to multichannel volume data sets.
The Biomedical Informatics Research Network is wide breadth project sponsored by the American National Institutes of Health (NIH) to promote the use of modern telecommunication for data exchange and collaboration in brain research. The project is attempting to buid a database and network infrastructure in which neuroscientists will post, query, and analyze raw data, processed data, and the results of the analysis. The project is divided into parts, which analyze mouse brain data and human brain data, respectively. In this phase of the project, the data are essentially anatomical, while in a future phase we foresee the introduction of functional data. One important source of raw data, both for the mouse and the human brains are magnetic resonance images (MRI), which provide dense volumetric information of the density of the brain or (in the case of functional MRI), of the brain activity. In the case of the brain mouse, these data are supplemented with images of slices of brains and other histological measure. One important technical problem that we are facing in BIRN is that of managing these volumetric data, processing them (possibly using tools available only remotely), storing the results of the analyses, and making them available to all the institutions participating in the project. This paper describes the problems posed by the BIRN project, the importance of image data in these activities, and the challenges they pose. We will describe the shared environment that we are creating, and the facilities for storing, querying, remotely processing, and sharing the image data that constitute the bulk of the brain data that scientists are producing.
We quantitatively analyzed the contrast degradation and blur of 20-nm gold nanoparticles adsorbed on the top of amorphous silicon films of thicknesses of 0.54, 1.09, 1.63 and 2.2 μm in bright-field transmission electron microscope (TEM) images taken at accelerating voltages of 0.5, 1, 2 and 3 MeV. The thickness dependence of the transmission was well explained and consistent with our calculations. The blur function, derived by assuming that the TEM image of a thick specimen can be reproduced by convolving the TEM image of a very thin specimen with it, was found to be expressed by a two-dimensional Lorentzian function. Considering the two characteristics of the Lorentzian function, a sharp peak around the center and a long tail, we concluded that, for TEM observations of thick specimens, the image contrast is degraded predominantly by inelastic scattering and the image is blurred predominantly by multiple elastic scattering.
Image mining is the application of computer-based techniques that extract and exploit information from large image sets to support human users in generating knowledge from these sources. This review focuses on biomedical applications of this technique, in particular automated imaging at the cellular level. Due to increasing automation and the availability of integrated instruments, biomedical users are becoming increasingly confronted with the problem of analyzing such data. Image database applications need to combine data management, image analysis and visual data mining. The main point of such a system is a software layer that represents objects within an image and the ability to use a large spectrum of quantitative and symbolic object features. Image analysis needs to be adapted to each particular experiment; therefore, 'end user programming' will be desired to make the technology more widely applicable.
The cell: an image library-CCDB (CIL-CCDB) (http://www.cellimagelibrary.org) is a searchable database and archive of cellular images. As a repository for microscopy data, it accepts all forms of cell
imaging from light and electron microscopy, including multi-dimensional images, Z- and time stacks in a broad variety of raw-data
formats, as well as movies and animations. The software design of CIL-CCDB was intentionally designed to allow easy incorporation
of new technologies and image formats as they are developed. Currently, CIL-CCDB contains over 9250 images from 358 different
species. Images are evaluated for quality and annotated with terms from 14 different ontologies in 16 different fields as
well as a basic description and technical details. Since its public launch on 9 August 2010, it has been designed to serve
as not only an archive but also an active site for researchers and educators.
Electron microscopy (EM) is the standard method for imaging cellular structures with nanometer resolution, but existing genetic tags are inactive in most cellular compartments or require light and can be difficult to use. Here we report the development of 'APEX', a genetically encodable EM tag that is active in all cellular compartments and does not require light. APEX is a monomeric 28-kDa peroxidase that withstands strong EM fixation to give excellent ultrastructural preservation. We demonstrate the utility of APEX for high-resolution EM imaging of a variety of mammalian organelles and specific proteins using a simple and robust labeling procedure. We also fused APEX to the N or C terminus of the mitochondrial calcium uniporter (MCU), a recently identified channel whose topology is disputed. These fusions give EM contrast exclusively in the mitochondrial matrix, suggesting that both the N and C termini of MCU face the matrix. Because APEX staining is not dependent on light activation, APEX should make EM imaging of any cellular protein straightforward, regardless of the size or thickness of the specimen.
Protoplasmic astrocytes are increasingly thought to interact extensively with neuronal elements in the brain and to influence their activity. Recent reports have also begun to suggest that physiologically, and perhaps functionally, diverse forms of these cells may be present in the CNS. Our current understanding of astrocyte form and distribution is based predominately on studies that used the astrocytic marker glial fibrillary acidic protein (GFAP) and on studies using metal-impregnation techniques. The prevalent opinion, based on studies using these methods, is that astrocytic processes overlap extensively and primarily share the underlying neuropil. However, both of these techniques have serious shortcomings for visualizing the interactions among these structurally complex cells. In the present study, intracellular injection combined with immunohistochemistry for GFAP show that GFAP delineates only ∼15% of the total volume of the astrocyte. As a result, GFAP-based images have led to incorrect conclusions regarding the interaction of processes of neighboring astrocytes. To investigate these interactions in detail, groups of adjacent protoplasmic astrocytes in the CA1 stratum radiatum were injected with fluorescent intracellular tracers of distinctive emissive wavelengths and analyzed using three-dimensional (3D) confocal analysis and electron microscopy. Our findings show that protoplasmic astrocytes establish primarily exclusive territories. The knowledge of how the complex morphology of protoplasmic astrocytes affects their 3D relationships with other astrocytes, oligodendroglia, neurons, and vasculature of the brain should have important implications for our understanding of nervous system function.
High voltage electron microscopy and computer axial tomography have been used to study the 3-D structure of trans-Golgi cisternae and trans-Golgi networks (TGNs) in NRK cells. Both structures were specifically labeled by photoconversion of a fluorescent analogue of ceramide using a modification of the techique of Pagano et al. (J. Cell Biol. 1991. 113: 1267-1279). Regions of the Golgi ribbon in fixed, stained cells were cut in 250-nm sections and analyzed by tilt series microscopy and subsequent tomographic reconstruction. Resolution of the reconstructions ranged from 6 to 10 nm. The size and structure of the TGN varied considerably throughout the Golgi ribbon; all reconstructions were made from regions with pronounced TGN. Most regions analyzed contained multiple (2-4) Golgi cisternae that stain with ceramide. These "peel off" from the closely stacked cisternae and are continuous at their ends with tubules that contribute to the TGN. Most vesicular profiles visualized in the TGN are connected to TGN tubules. The budding of vesicles appears to occur synchronously along the length of a TGN tubule. Two distinct coats were visualized on budding vesicles: clathrin cages and a novel, lace-like structure. Individual TGN tubules produce vesicles of only one coat type. These observations lead to the following predictions: (a) sorting of molecules must occur prior to the formation of TGN tubules; (b) vesicle formation takes place almost synchronously along a given TGN tubule; and (c) lace-like coats form an exocytic vesicles.
Presents a QT database designed for evaluation of algorithms that
detect waveform boundaries in the ECG. The database consists of 105
fifteen-minute excerpts of two-channel ECG Holter recordings, chosen to
include a broad variety of QRS and ST-T morphologies. Waveform
boundaries for a subset of beats in these recordings have been manually
determined by expert annotators using an interactive graphic display to
view both signals simultaneously and to insert the annotations. Examples
of each morphology were included in this subset of annotated beats; at
least 30 beats in each record, 3622 beats in all, were manually
annotated in the database. In 11 records, two independent sets of
annotations have been included, to allow inter-observer variability
studies. The QT Database is available on a CD-ROM in the format
previously used for the MIT-BIH Arrhythmia Database and the European
ST-T Database, from which some of the recordings in the QT Database have
been obtained
High voltage electron microscopy and computer axial tomography have been used to study the 3-D structure of trans-Golgi cisternae and trans-Golgi networks (TGNs) in NRK cells. Both structures were specifically labeled by photoconversion of a fluorescent analogue of ceramide using a modification of the techique of Pagano et al. (J. Cell Biol. 1991. 113: 1267-1279). Regions of the Golgi ribbon in fixed, stained cells were cut in 250-nm sections and analyzed by tilt series microscopy and subsequent tomographic reconstruction. Resolution of the reconstructions ranged from 6 to 10 nm. The size and structure of the TGN varied considerably throughout the Golgi ribbon; all reconstructions were made from regions with pronounced TGN. Most regions analyzed contained multiple (2-4) Golgi cisternae that stain with ceramide. These "peel off" from the closely stacked cisternae and are continuous at their ends with tubules that contribute to the TGN. Most vesicular profiles visualized in the TGN are connected to TGN tubules. The budding of vesicles appears to occur synchronously along the length of a TGN tubule. Two distinct coats were visualized on budding vesicles: clathrin cages and a novel, lace-like structure. Individual TGN tubules produce vesicles of only one coat type. These observations lead to the following predictions: (a) sorting of molecules must occur prior to the formation of TGN tubules; (b) vesicle formation takes place almost synchronously along a given TGN tubule; and (c) lace-like coats form an exocytic vesicles.
Nowadays it is possible to unravel complex information at all levels of cellular organization by obtaining multi-dimensional
image information. at the macromolecular level, three-dimensional (3D) electron microscopy, together with other techniques,
is able to reach resolutions at the nanometer or subnanometer level. The information is delivered in the form of 3D volumes
containing samples of a given function, for example, the electron density distribution within a given macromolecule. The same
situation happens at the cellular level with the new forms of light microscopy, particularly confocal microscopy, all of which
produce biological 3D volume information. Furthermore, it is possible to record sequences of images over time (videos), as
well as sequences of volumes, bringing key information on the dynamics of living biological systems. It is in this context
that work on bioimage started two years ago, and that its first version is now presented here. In essence, Bioimage is a database
specifically designed to contain multi-dimensional images, perform queries and interactively work with the resulting multi-dimensional
information on the World Wide Web, as well as accomplish the required cross-database links. Two sister home pages of bioimage
can be accessed at http://www.bioimage.org and http://www-embl.bioimage.org
We used electron tomography to map the three-dimensional architecture of the ribbon-class afferent synapses in frog saccular hair cells. The synaptic body (SB) at each synapse was nearly spherical (468 +/- 65 nm diameter; mean +/- SD) and was covered by a monolayer of synaptic vesicles (34.3 nm diameter; 8.8% coefficient of variation), many of them tethered to it by approximately 20-nm-long filaments, at an average density of 55% of close-packed (376 +/- 133 vesicles per SB). These vesicles could support approximately 900 msec of exocytosis at the reported maximal rate, which the cells can sustain for at least 2 sec, suggesting that replenishment of vesicles on the SB is not rate limiting. Consistent with this interpretation, prolonged K+ depolarization did not deplete vesicles on the SB. The monolayer of SB-associated vesicles remained after cell lysis in the presence of 4 mM Ca2+, indicating that the association is tight and Ca2+-resistant. The space between the SB and the plasma membrane contained numerous vesicles, many of which ( approximately 32 per synapse) were in contact with the plasma membrane. This number of docked vesicles could support maximal exocytosis for at most approximately 70 msec. Additional docked vesicles were seen within a few hundred nanometers of the synapse and occasionally at greater distances. The presence of omega profiles on the plasma membrane around active zones, in the same locations as coated pits and coated vesicles labeled with an extracellular marker, suggests that local membrane recycling may contribute to the three- to 14-fold greater abundance of vesicles in the cytoplasm (not associated with the SB) near synapses than in nonsynaptic regions.
Nicotinic acetylcholine receptors containing alpha7 subunits are widely distributed in the vertebrate nervous system. In the chick ciliary ganglion such receptors generate large synaptic currents but appear to be excluded from postsynaptic densities on the cells. We show here that alpha7-containing receptors are concentrated on somatic spines in close proximity to putative sites of presynaptic transmitter release. Intermediate voltage electron microscopy on thick sections, together with tomographic reconstruction, permitted three-dimensional analysis of finger-like projections emanating from cell bodies. The projections were identified as spines based on their morphology, cytoskeletal content, and proximity to presynaptic elements. Both in situ and after ganglionic dissociation, the spines were grouped on the cell surface and tightly folded into mats. Immunogold labeling of receptors containing alpha7 subunits showed them to be preferentially concentrated on the somatic spines. Postsynaptic densities were present in vivo both on the soma near spines and occasionally on the spines themselves. Synaptic vesicle-filled projections from the presynaptic calyx were interdigitated among the spines. Moreover, the synaptic vesicles often abutted the membrane and sometimes included Omega profiles as if caught in an exocytotic event, even when no postsynaptic densities were juxtaposed on the spine. The results suggest several mechanisms for delivering transmitter to alpha7-containing receptors, and they support new ideas about synaptic signaling via spines. They also indicate that neurons must have specific mechanisms for targeting alpha7-containing receptors to desired locations.
Three-dimensional reconstructions of portions of the Golgi complex from cryofixed, freeze-substituted normal rat kidney cells have been made by dual-axis, high-voltage EM tomography at approximately 7-nm resolution. The reconstruction shown here ( approximately 1 x 1 x 4 microm3) contains two stacks of seven cisternae separated by a noncompact region across which bridges connect some cisternae at equivalent levels, but none at nonequivalent levels. The rest of the noncompact region is filled with both vesicles and polymorphic membranous elements. All cisternae are fenestrated and display coated buds. They all have about the same surface area, but they differ in volume by as much as 50%. The trans-most cisterna produces exclusively clathrin-coated buds, whereas the others display only nonclathrin coated buds. This finding challenges traditional views of where sorting occurs within the Golgi complex. Tubules with budding profiles extend from the margins of both cis and trans cisternae. They pass beyond neighboring cisternae, suggesting that these tubules contribute to traffic to and/or from the Golgi. Vesicle-filled "wells" open to both the cis and lateral sides of the stacks. The stacks of cisternae are positioned between two types of ER, cis and trans. The cis ER lies adjacent to the ER-Golgi intermediate compartment, which consists of discrete polymorphic membranous elements layered in front of the cis-most Golgi cisterna. The extensive trans ER forms close contacts with the two trans-most cisternae; this apposition may permit direct transfer of lipids between ER and Golgi membranes. Within 0.2 microm of the cisternae studied, there are 394 vesicles (8 clathrin coated, 190 nonclathrin coated, and 196 noncoated), indicating considerable vesicular traffic in this Golgi region. Our data place structural constraints on models of trafficking to, through, and from the Golgi complex.
The Protein Data Bank (PDB; http://www.rcsb.org/pdb/ ) is the single worldwide archive of structural data of biological macromolecules. This paper describes the goals of the PDB, the systems in place for data deposition and access, how to obtain further information, and near-term plans for the future development of the resource.
Motivation:
Scientific data pertaining to GABA receptors, which are of medical importance, are widely scattered throughout numerous heterogeneous Internet resources. This situation has made the integrated acquisition of such data difficult and substantially time consuming even for researchers who are Internet aficionados. Thus, there exists a genuine need for the development of Internet applications, such as GABAagent, which provide efficient and timely access to concise and integrated information.
Results:
We report here the establishment of a novel server (GABAagent) which has been written in Perl script, and which is freely accessible through the Internet. GABAagent is designed to assist researchers in retrieving focused and integrated information related to GABA receptors from various public domain databases. GABAagent relies on server-side flat-file databases that have been created through data mining from Internet sources such as the PubMed, DDBJ, SWISS-PROT and TrEMBL, in addition to the many World Wide Web (Web) sites which are accessible through Excite (E-Web). These warehouse databases are regularly updated and contain among other things, information concerning: (i) GABA receptor publications, (ii) DNA and protein sequences and (iii) the contents of related E-Web sites along with their addresses. Our system also provides hard links to the above-mentioned Web sites and E-Web sites; the feature which adds to it the character of virtual federation type of database. The current version of GABAagent provides two user-friendly services. The first is a search engine possessing intelligent query reformulation support (GABAengine), the second an elaborate email alert service was designed into the system (GABAalert). The GABAengine allows the user to search server-side databases exclusively for GABA receptor-related queries. Whereas, GABAalert allows the user, by means of subscription, to receive immediate and/or monthly updates automatically.
Availability:
GABAagent is freely accessible at the following Web address http://www.ust.hk/gaba.
Electron tomography is the only technique available that allows us to visualize the three-dimensional structure of unfixed and unstained cells currently with a resolution of 6-8 nm, but with the prospect to reach 2-4 nm. This raises the possibility of detecting and identifying specific macromolecular complexes within their cellular context by virtue of their structural signature. Templates derived from the high-resolution structure of the molecule under scrutiny are used to search the reconstructed volume. Here we outline and test a computationally feasible two-step procedure: In a first step, mean-curvature motion is used for segmentation, yielding subvolumes that contain with a high probability macromolecules in the expected size range. Subsequently, the particles contained in the subvolumes are identified by cross-correlation, using a set of three-dimensional templates. With simulated and real tomographic data we demonstrate that such an approach is feasible and we explore the detection limits. Even structurally similar particles, such as the thermosome, GroEL, and the 20S proteasome can be identified with high fidelity. This opens up exciting prospects for mapping the territorial distribution of macromolecules and for analyzing molecular interactions in situ.
The paper provides an overview of neuroinformatics research at Yale University being performed as part of the national Human Brain Project. This research is exploring the integration of multidisciplinary sensory data, using the olfactory system as a model domain. The neuroinformatics activities fall into three main areas: 1) building databases and related tools that support experimental olfactory research at Yale and can also serve as resources for the field as a whole, 2) using computer models (molecular models and neuronal models) to help understand data being collected experimentally and to help guide further laboratory experiments, 3) performing basic neuroinformatics research to develop new informatics technologies, including a flexible data model (EAV/CR, entity-attribute-value with classes and relationships) designed to facilitate the integration of diverse heterogeneous data within a single unifying framework.
Synaptic ribbons, the organelles identified in electron micrographs of the sensory synapses involved in vision, hearing, and balance, have long been hypothesized to play an important role in regulating presynaptic function because they associate with synaptic vesicles at the active zone. Their physiology and molecular composition have, however, remained largely unknown. Recently, a series of elegant studies spurred by technical innovation have finally begun to shed light on the ultrastructure and function of ribbon synapses. Electrical capacitance measurements have provided sub-millisecond resolution of exocytosis, evanescent-wave microscopy has filmed the fusion of single 30 nm synaptic vesicles, electron tomography has revealed the 3D architecture of the synapse, and molecular cloning has begun to identify the proteins that make up ribbons. These results are consistent with the ribbon serving as a vesicle "conveyor belt" to resupply the active zone, and with the suggestion that ribbon and conventional chemical synapses have much in common.
Recombinant proteins containing tetracysteine tags can be successively labeled in living cells with different colors of biarsenical fluorophores so that older and younger protein molecules can be sharply distinguished by both fluorescence and electron microscopy. Here we used this approach to show that newly synthesized connexin43 was transported predominantly in 100- to 150-nanometer vesicles to the plasma membrane and incorporated at the periphery of existing gap junctions, whereas older connexins were removed from the center of the plaques into pleiomorphic vesicles of widely varying sizes. Selective imaging by correlated optical and electron microscopy of protein molecules of known ages will clarify fundamental processes of protein trafficking in situ.
These are comments written by the Governing Council of the Organization for Human Brain Mapping (OHBM), the primary international
organization dedicated to neuroimaging research. The purpose of these comments is to identify and frame issues concerning
data sharing within the neuroimaging community. Data sharing has become an important issue in most fields of science. The
neuroimaging community is no exception, and it clearly perceives potential benefits in such efforts, as have been realized
in other fields such as genomics. At the same time, such efforts can be costly (both in time and expense), and there are important
factors that differentiate brain imaging from other fields and that pose specific challenges to the generation of useful neuroimaging
databases. These include the rapid pace of change in brain imaging technologies; the complexity of the variables that must
be specified to meaningfully interpret the results (such as the method of image acquisition, behavioral design, and subject
characteristics); and concerns about participant confidentiality. These issues are outlined with the goal of framing and promoting
a public discussion of the benefits and risks of data sharing, which can inform the field of neuroimaging as well as others
that face similar challenges.
Three-dimensional (3D) protein localization at the electron microscopic level is of great benefit when determining the precise distribution of a given protein relative to complex subcellular structures. We have developed several 3D ultrastructural labeling approaches for use with higher voltage electron microscopy. High voltage electron microscopy allows for the use of much thicker sections than conventional transmission electron microscopy. Many of our staining protocols are based on the photooxidation of diaminobenzidine (DAB) by fluorescent markers. As a fluorescent dye is excited in the presence of DAB, the reactive oxygen generated gradually oxidizes DAB into an insoluble polymer which can be rendered electron dense. We previously demonstrated that photooxidation by the fluorophore eosin can be used for high resolution immunolocalization. Because of its small size, eosin-conjugated reagents penetrate well into tissue slices, providing labeling through a reasonable depth of tissue. When combined with high voltage electron microscopy using thick sections, the 3D distribution of a variety of proteins can be visualized. Analysis and interpretation of these 3D labeling patterns is further facilitated by the use of electron tomography, which allows for the derivation of 3D structure from a series of images taken at different orientations. In this review, several examples of 3D protein localization using these technologies are presented.
The Protein Data Bank (PDB; http://www.rcsb.org/pdb/ ) is the single worldwide archive of structural data of biological macromolecules. This paper describes the goals of the PDB, the systems in place for data deposition and access, how to obtain further information, and near-term plans for the future development of the resource.
This chapter discusses the advantages and limitations of the electron tomography technique as applied to mitochondrial research and also outlines the important steps in a typical tomographic investigation, with special attention to optimization of specimen preparation. Some of the biological applications of transmission electron microscopy (TEM) are investigations into the internal organization of the mitochondrion. The two dimensionality of conventional TEM imaging is imposed by the physics of the interaction of electrons with the specimen. Most structural information in electron micrographs is provided by electrons scattered elastically (without energy loss to the specimen) only once. However, for incident electrons of a given energy, as specimen thickness increases, the fraction of singly elastically scattered electrons drops and the fractions of singly inelastically and multiply elastically and inelastically scattered electrons increase, reducing image contrast greatly. Moreover, tomography of thick, plastic section specimens has the potential to become a routine tool for the three-dimensional (3D) analysis of biological structure. Furthermore, advances in methodology are leading to the extension of this 3D imaging technique to frozen-hydrated specimens.
Motor actions of myosin were directly visualized by electron tomography of insect flight muscle quick-frozen during contraction. In 3D images, active crossbridges are usually single myosin heads, bound preferentially to actin target zones sited midway between troponins. Active attached bridges (∼30% of all heads) depart markedly in axial and azimuthal angles from Rayment's rigor acto-S1 model, one-third requiring motor domain (MD) tilting on actin, and two-thirds keeping rigor contact with actin while the light chain domain (LCD) tilts axially from ∼105° to ∼70°. The results suggest the MD tilts and slews on actin from weak to strong binding, followed by swinging of the LCD through an ∼35° axial angle, giving an ∼13 nm interaction distance and an ∼4-6 nm working stroke.
Both light and electron microscopy have experienced exciting advances in the past few years, driven by the development of new imaging technologies and the ready availability of powerful computers and associated algorithms for visualization, analysis, and image restoration. The concomitant development of molecular genetic methods for engineering fluorescent molecules into proteins and for targeting indicator dyes to subcellular compartments has led to unprecedented opportunities for the study of dynamic processes and for correlating physiology and structure. Although the effective resolution of light microscopy has been increased through the use of optical sectioning and computational deblussing of images, the resolution of the light microscope is still not sufficient to address Inany issues of cell and tissue structure and protein localization. Electron microscopy has also undergone a quiet revolution, in particular high voltage electron microscopy, where the technique of electron tomography allows 3D reconstruction of relatively large structures such as neuronal spiny dendrites without the need for serial sectioning. In combination with light microscopy, high voltage EM and electron tomography provide a relatively straightforward method to bridge the resolution gap between light and electron microscopy. In this review, we discuss correlated 3D light and electron microscopy imaging of cells and tissues, including the adaptation of fluorescent dyes for electron microscopy imaging. (The J Histotechnol 23:261, 2000)
The Protein Data Bank (PDB; http://www.rcsb.org/pdb/ ) is the single worldwide archive of structural data of biological macromolecules. This paper describes the goals of the PDB, the systems in place for data deposition and access, how to obtain further information, and near-term plans for the future development of the resource.
Dendritic spines differ considerably in their size, shape, and internal organization between brain regions. We examined the actin cytoskeleton in dendritic spines in hippocampus (areas CA1, CA3, and dentate gyrus), neostriatum, and cerebellum at both light and electron microscopic levels by using a novel high-resolution photoconversion method based in the high affinity of phalloidin for filamentous (F)-actin. In all brain regions, labeling was strongest in the heads of dendritic spines, diminishing in the spine neck. The number of labeled spines varied by region. Compared with the cerebellar molecular layer and area CA3, where nearly every dendritic spine was labeled, less than half the spines were labeled in CA1, dentate gyrus, and neostriatum. Serial section reconstructions of spines in these areas indicated that phalloidin labeling was restricted to the largest and most morphologically diverse dendritic spines. The resolution of the photoconversion technique allowed us to examine the localization and organization of actin filaments in the spine. The most intense staining for actin was found in the postsynaptic density and associated with the spines internal membrane system. In mushroom-shaped spines, F-actin staining was particularly strong between the lamellae of the spine apparatus. Three-dimensional reconstruction of labeled spines by using electron tomography showed that the labeled dense material was in continuity with the postsynaptic density. These results highlight differences in the actin cytoskeleton between different spine populations and provide novel information on the organization of the actin cytoskeleton in vivo. J. Comp. Neurol. 435:156–170, 2001. © 2001 Wiley-Liss, Inc.
We present a method for combining single axis tomography and serial sectioning techniques to derive a three-dimensional reconstruction of large structures at electron microscopic resolution. This serial-tomography method allows the use of sufficiently thin sections to achieve adequate resolution with electron tomography, yet enables the generation of large reconstructions with considerably fewer sections than would be required using a serial thin section reconstruction technique. Serial thick sections (1-2 μm) are cut through the structure of interest, tomographic volume reconstructions are obtained for each section from a single axis tilt series, and the resulting series of volumes are then aligned and combined to form a single large volume. The serial-tomography method is illustrated with several samples, including red blood cells, the Golgi apparatus, and a spiny dendrite of a cortical pyramidal neuron. In some of these samples, the reconstruction is compared to correlated light microscopic views. The resulting large volume reconstructions appear to represent accurately the size and shape of objects such as red blood cells and spiny dendrites. The continuity of complex, tortuous structures such as the Golgi apparatus is also maintained across serial volumes. These examples demonstrate that it is possible to align and link a series of tomographic volumes accurately and that serial-tomography is a useful method for reconstructing relatively large structures without resorting to large numbers of Serial thin sections.
A method based on Fourier transforms is described for obtaining a 3-D reconstruction from a paracrystalline object with static disorder. The method is derived from the standard methods used in 3-D reconstruction of 2-D crystals except that all of the Fourier coefficients are used and not just the sampled data from the periodic lattice. Thus, not only is the spatially ordered part of the structure visualized in 3-D, but also the spatially disordered part. Application of the method to 3-D reconstructions of insect flight muscle is described as well as prospects for extension of the method to radiation-sensitive specimens.
The ability for remote microscope operation via a network connection was added recently to the ultrahigh voltage electron microscope (UHVEM) in Osaka University, and used successfully for the observation of thick biological samples across the Pacific Ocean by researchers at the National Center for Microscopy and Imaging Research (NCMIR) at the University of California San Diego. High-quality images at video rate were transferred by a satellite link and control signals were transmitted by an ISDN connecting the workstations at both sites. Most microscope functions operated from the console of the UHVEM were replicated on the graphical user interface of the remote workstation. By clicking on icons or in boxes in the display window with a mouse, the researcher could operate the UHVEM from the remote-site. The total delay time for sending images and returning control signals was about 0.7 s, which did not interfere significantly with the smooth operation of the instrument. Researchers at the remote site were able to record images on film in the microscope which were later sent to San Diego.
We have developed a computer software package, IMOD, as a tool for analyzing and viewing three-dimensional biological image data. IMOD is useful for studying and modeling data from tomographic, serial section, and optical section reconstructions. The software allows image data to be visualized by several different methods. Models of the image data can be visualized by volume or contour surface rendering and can yield quantitative information.
Cited By (since 1996): 71, Export Date: 3 May 2012, Source: Scopus
The structure of neuronal mitochondria from chick and rat was examined using electron microscope tomography of chemically fixed tissue embedded in plastic and sliced in approximately 500 nm-thick sections. Three-dimensional reconstructions of representative mitochondria were made from single-axis tilt series acquired with an intermediate voltage electron microscope (400 kV). The tilt increment was either 1 degree or 2 degrees ranging from -60 degrees to +60 degrees. The mitochondrial ultrastructure was similar across species and neuronal regions. The outer and inner membranes were each approximately 7 nm thick. The inner boundary membrane was found to lie close to the outer membrane, with a total thickness across both membranes of approximately 22 nm. We discovered that the inner membrane invaginates to form cristae only through narrow, tubular openings, which we call crista junctions. Sometimes the cristae remain tubular throughout their length, but often multiple tubular cristae merge to form lamellar compartments. Punctate regions, approximately 14 nm in diameter, were observed in which the inner and outer membranes appeared in contact (total thickness of both membranes approximately 14 nm). These contact sites are known to a play a key role in the transport of proteins into the mitochondrion. It has been hypothesized that contact sites may be proximal to crista junctions to facilitate transport of proteins destined for the cristae. However, our statistical analyses indicated that contact sites are randomly located with respect to these junctions. In addition, a close association was observed between endoplasmic reticulum membranes and the outer mitochondrial membrane, consistent with the reported mechanism of transport of certain lipids into the mitochondrion.
Three-dimensional data representing biological structures can be derived using several methods, including serial section reconstruction, optical sectioning, and tomography. The investigation, comprehension, and communication of structural relationships to others is greatly facilitated by computer-based visualization procedures. We describe SYNU, a suite of programs developed for interactive investigation of three-dimensional structure and for the production of high-quality three-dimensional images and animations. We illustrate the capabilities of SYNU in applications to biological data obtained by confocal light microscopy, serial section, and high-resolution electron microscopy from investigations at the cellular, subcellular, and molecular levels.
High-voltage electron microscopy (HVEM) and axial tomography were used to reconstruct the three-dimensional structure of dendrites of intracellularly stained neostriatal spiny neurons. Neurons were stained using iontophoretic injection of horseradish peroxidase from an intracellular micropipet. Thick sections were cut on a vibratome, reacted with diaminobenzidine, and embedded in plastic. High-voltage electron microscopy was used to obtain high-resolution images of neuronal processes in 2- to 3-micron plastic sections cut from those blocks. Series of high-voltage electron micrographs were taken at 2 degrees increments of specimen tilt over a range of at least +/- 60 degrees, and three-dimensional reconstructions were generated from the series using an R-weighted backprojection method. An interactive procedure was used to draw the dendritic profiles from slices through the reconstructed volume and to measure volumes and surface areas of the dendrites. Values obtained in this manner matched previous findings using reconstruction from serial thin sections. The HVEM-tomography method offers an alternative to the serial-thin-section method for the quantitative analysis of neuronal shape.
After a general introduction to three-dimensional electron microscopy and particularly to electron tomography (ET), the perspectives of applying ET to native (frozen-hydrated) cellular structures are discussed. In ET, a set of 2-D images of an object is recorded at different viewing directions and is then used for calculating a 3-D image. ET at a resolution of 2-5 nm would allow the 3-D organization of structural cellular components to be studied and would provide important information about spatial relationships and interactions. The question of whether it is a realistic long-term goal to visualize or--by sophisticated pattern recognition methods--identify macromolecules in cells frozen in toto or in frozen sections of cells is addressed. Because of the radiation sensitivity of biological specimens, a prerequisite of application of ET is the automation of the imaging process. Technical aspects of automated ET as realized in Martinsried and experiences are presented, and limitations of the technique are identified, both theoretically and experimentally. Possible improvements of instrumentation to overcome at least part of the limitations are discussed in some detail. Those means include increasing the accelerating voltage into the intermediate voltage range (300 to 500 kV), energy filtering, the use of a field emission gun, and a liquid-helium-cooled specimen stage. Two additional sections deal with ET of isolated macromolecules and of macromolecular structures in situ, and one section is devoted to possible methods for the detection of structures in volume data.
Electron tomography is an extremely useful method for deriving three-dimensional structure from electron microscope images. The application of this technique to the reconstruction of large, complex structures such as mitochondria is described in conjunction with several tools for segmentation, measurement, classification, and visualization. In addition, the use of massively parallel computers to perform the tomographic reconstruction efficiently using R-weighted backprojection or iterative techniques is described.
We used EM tomography to examine the fine structure of the apparently amorphous electron dense material that is seen at active zones of axon terminals when viewed by conventional 2D electron microscopy. Serial 1-nm optical slices from 3D reconstructions of individual thin tissue sections reveal that the material is composed of an interconnecting network of elongate components directly linked to synaptic vesicles and the presynaptic membrane. Each vesicle at the active zone that lies adjacent to the presynaptic plasma membrane has several such connections. Information provided by reconstruction data may be useful in generating experiments aimed at understanding the mechanisms involved in the docking of synaptic vesicles and their exocytosis during synaptic transmission.
The Internet is a powerful communication medium increasingly exploited by business and science alike, especially in structural biology and bioinformatics. The traditional presentation of static two-dimensional images of real-world objects on the limited medium of paper can now be shown interactively in three dimensions. Many facets of this new capability have already been developed, particularly in the form of VRML (virtual reality modeling language), but there is a need to extend this capability for visualizing scientific data. Here we introduce a real-time isosurfacing node for VRML, based on the marching cube approach, allowing interactive isosurfacing. A second node does three-dimensional (3D) texture-based volume-rendering for a variety of representations. The use of computers in the microscopic and structural biosciences is extensive, and many scientific file formats exist. To overcome the problem of accessing such data from VRML and other tools, we implemented extensions to SGI's IFL (image format library). IFL is a file format abstraction layer defining communication between a program and a data file. These technologies are developed in support of the BioImage project, aiming to establish a database prototype for multidimensional microscopic data with the ability to view the data within a 3D interactive environment.
The BioImage database is a new scientific database for multidimensional microscopic images of biological specimens, which is available through the World Wide Web (WWW). The development of this database has followed an iterative approach, in which requirements and functionality have been revised and extended. The complexity and innovative use of the data meant that technical and biological expertise has been crucial in the initial design of the data model. A controlled vocabulary was introduced to ensure data consistency. Pointers are used to reference information stored in other databases. The data model was built using InfoModeler as a database design tool. The database management system is the Informix Dynamic Server with Universal Data Option. This object-relational system allows the handling of complex data using features such as collection types, inheritance, and user-defined data types. Informix datablades are used to provide additional functionality: the Web Integration Option enables WWW access to the database; the Video Foundation Blade provides functionality for video handling.
The BioImage Database Project collects and structures multidimensional data sets recorded by various microscopic techniques relevant to modern life sciences. It provides, as precisely as possible, the circumstances in which the sample was prepared and the data were recorded. It grants access to the actual data and maintains links between related data sets. In order to promote the interdisciplinary approach of modern science, it offers a large set of key words, which covers essentially all aspects of microscopy. Nonspecialists can, therefore, access and retrieve significant information recorded and submitted by specialists in other areas. A key issue of the undertaking is to exploit the available technology and to provide a well-defined yet flexible structure for dealing with data. Its pivotal element is, therefore, a modern object relational database that structures the metadata and ameliorates the provision of a complete service. The BioImage database can be accessed through the Internet.
Progress in neuroscience might be faster if researchers shared their
results in a network of databases. But the technical challenges are huge,
and reaching a consensus on what to archive won't be easy, says Marina Chicurel.
The database reported here is derived using the Combinatorial Extension (CE) algorithm which compares pairs of protein polypeptide
chains and provides a list of structurally similar proteins along with their structure alignments. Using CE, structure–structure
alignments can provide insights into biological function. When a protein of known function is shown to be structurally similar
to a protein of unknown function, a relationship might be inferred; a relationship not necessarily detectable from sequence
comparison alone. Establishing structure–structure relationships in this way is of great importance as we enter an era of
structural genomics where there is a likelihood of an increasing number of structures with unknown functions being determined.
Thus the CE database is an example of a useful tool in the annotation of protein structures of unknown function. Comparisons
can be performed on the complete PDB or on a structurally representative subset of proteins. The source protein(s) can be
from the PDB (updated monthly) or uploaded by the user. CE provides sequence alignments resulting from structural alignments
and Cartesian coordinates for the aligned structures, which may be analyzed using the supplied Compare3D Java applet, or downloaded
for further local analysis. Searches can be run from the CE web site, http://cl.sdsc.edu/ce.html, or the database and software downloaded from the site for local use.
Active zone material at the nervous system's synapses is situated next
to synaptic vesicles that are docked at the presynaptic plasma membrane, and
calcium channels that are anchored in the membrane. Here we use electron microscope
tomography to show the arrangement and associations of structural components
of this compact organelle at a model synapse, the frog's neuromuscular junction.
Our findings indicate that the active zone material helps to dock the vesicles
and anchor the channels, and that its architecture provides both a particular
spatial relationship and a structural linkage between them. The structural
linkage may include proteins that mediate the calcium-triggered exocytosis
of neurotransmitter by the synaptic vesicles during synaptic transmission.
Cited By (since 1996): 7, Export Date: 3 May 2012, Source: Scopus
NEURON is a simulation environment for models of individual neurons and networks of neurons that are closely linked to experimental data. NEURON provides tools for conveniently constructing, exercising, and managing models, so that special expertise in numerical methods or programming is not required for its productive use. This article describes two tools that address the problem of how to achieve computational efficiency and accuracy.
Accurate data on the three-dimensional architecture of the Golgi is prerequisite for evaluating the mechanisms of transit through this organelle. Here we detail the structure of the Golgi ribbon within part of an insulin-secreting cell in three dimensions at approximately 6 nm resolution. Rapid freezing, freeze-substitution and electron tomography were employed. The Golgi in this region is composed of seven cisternae. The cis-most element is structurally intermediate between the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) and the cis-most cisterna characterized in three dimensions at high resolution in a normal rat kidney cell [Ladinsky, Mastronarde, McIntosh, Howell and Staehelin (1999) J. Cell Biol. 144, 1135-1149]. There are three trans-cisternae that demonstrate morphological and functional variation. The membrane surface areas and volumes of these elements decrease from cis to trans. The two trans-most cisternae are dissociated from the stack and are fragmented by tubulation. ER closely adheres to and inserts between individual trans-cisternae. Many of the 2119 small, clathrin-negative vesicles that are in close proximity to the Golgi fill the region where trans-cisternae have moved out of register with the ribbon. These data provide evidence that cisternal progression/maturation, trafficking via membrane tubules and vesicle-mediated transport act in concert in the same region of the Golgi ribbon, and suggest an important role for the ER in regulating membrane dynamics at the trans-Golgi.
We have implemented a pair of database projects, one serving cortical electrophysiology and the other invertebrate neurones and recordings. The design for each combines aspects of two proven schemes for information interchange. The journal article metaphor determined the type, scope, organization and quantity of data to comprise each submission. Sequence databases encouraged intuitive tools for data viewing, capture, and direct submission by authors. Neurophysiology required transcending these models with new datatypes. Time-series, histogram and bivariate datatypes, including illustration-like wrappers, were selected by their utility to the community of investigators. As interpretation of neurophysiological recordings depends on context supplied by metadata attributes, searches are via visual interfaces to sets of controlled-vocabulary metadata trees. Neurones, for example, can be specified by metadata describing functional and anatomical characteristics. Permanence is advanced by data model and data formats largely independent of contemporary technology or implementation, including Java and the XML standard. All user tools, including dynamic data viewers that serve as a virtual oscilloscope, are Java-based, free, multiplatform, and distributed by our application servers to any contemporary networked computer. Copyright is retained by submitters; viewer displays are dynamic and do not violate copyright of related journal figures. Panels of neurophysiologists view and test schemas and tools, enhancing community support.
Faster than ever, neuroscience is generating vast amounts of data that await cross-referencing, comparison, integration and interpretation in the endeavour to unravel the mechanisms of the brain. The complex, diverse and distributed nature of these data requires the development of advanced neuroinformatics methodologies for databases and associated tools that are now beginning to emerge. This paper presents an overview of current issues in the representation, integration and analysis of neuroscience data from molecular to brain systems levels, including issues of implementation, standardization, management, quality control, copyright, confidentiality and acceptance. Particular emphasis is given to integrative neuroinformatics approaches for exploring structure-function relationships in the brain.
Sharing scientific data containing complex information requires new concepts and new technology. NEUROGENERATOR is a database generator for the neuroimaging community. A database generator is a database that generates new databases. The scientists submit raw PET and fMRI data to NEUROGENERATOR, which then processes the data in a uniform way to create databases of homogeneous data suitable for data sharing, met-analysis and modelling the human brain at the systems level. These databases are then distributed to the scientists.
Two new methods, single-particle cryo-electron microscopy reconstruction and electron tomography, are increasingly used to visualize molecular machines in vitro and in the cellular context, respectively. Current efforts focus on the development of methods capable of visualizing molecular signatures in the cell, and first progress in this direction has now been made.