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Automated Detection of Synapses in Serial Section Transmission Electron Microscopy Image Stacks  [PDF]
Anna Kreshuk, Ullrich Koethe, Elizabeth Pax, Davi D. Bock, Fred A. Hamprecht
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0087351
Abstract: We describe a method for fully automated detection of chemical synapses in serial electron microscopy images with highly anisotropic axial and lateral resolution, such as images taken on transmission electron microscopes. Our pipeline starts from classification of the pixels based on 3D pixel features, which is followed by segmentation with an Ising model MRF and another classification step, based on object-level features. Classifiers are learned on sparse user labels; a fully annotated data subvolume is not required for training. The algorithm was validated on a set of 238 synapses in 20 serial 7197×7351 pixel images (4.5×4.5×45 nm resolution) of mouse visual cortex, manually labeled by three independent human annotators and additionally re-verified by an expert neuroscientist. The error rate of the algorithm (12% false negative, 7% false positive detections) is better than state-of-the-art, even though, unlike the state-of-the-art method, our algorithm does not require a prior segmentation of the image volume into cells. The software is based on the ilastik learning and segmentation toolkit and the vigra image processing library and is freely available on our website, along with the test data and gold standard annotations (http://www.ilastik.org/synapse-detection?/sstem).
An Integrated Micro- and Macroarchitectural Analysis of the Drosophila Brain by Computer-Assisted Serial Section Electron Microscopy  [PDF]
Albert Cardona,Stephan Saalfeld,Stephan Preibisch,Benjamin Schmid,Anchi Cheng,Jim Pulokas,Pavel Tomancak,Volker Hartenstein
PLOS Biology , 2012, DOI: 10.1371/journal.pbio.1000502
Abstract: The analysis of microcircuitry (the connectivity at the level of individual neuronal processes and synapses), which is indispensable for our understanding of brain function, is based on serial transmission electron microscopy (TEM) or one of its modern variants. Due to technical limitations, most previous studies that used serial TEM recorded relatively small stacks of individual neurons. As a result, our knowledge of microcircuitry in any nervous system is very limited. We applied the software package TrakEM2 to reconstruct neuronal microcircuitry from TEM sections of a small brain, the early larval brain of Drosophila melanogaster. TrakEM2 enables us to embed the analysis of the TEM image volumes at the microcircuit level into a light microscopically derived neuro-anatomical framework, by registering confocal stacks containing sparsely labeled neural structures with the TEM image volume. We imaged two sets of serial TEM sections of the Drosophila first instar larval brain neuropile and one ventral nerve cord segment, and here report our first results pertaining to Drosophila brain microcircuitry. Terminal neurites fall into a small number of generic classes termed globular, varicose, axiform, and dendritiform. Globular and varicose neurites have large diameter segments that carry almost exclusively presynaptic sites. Dendritiform neurites are thin, highly branched processes that are almost exclusively postsynaptic. Due to the high branching density of dendritiform fibers and the fact that synapses are polyadic, neurites are highly interconnected even within small neuropile volumes. We describe the network motifs most frequently encountered in the Drosophila neuropile. Our study introduces an approach towards a comprehensive anatomical reconstruction of neuronal microcircuitry and delivers microcircuitry comparisons between vertebrate and insect neuropile.
An Integrated Micro- and Macroarchitectural Analysis of the Drosophila Brain by Computer-Assisted Serial Section Electron Microscopy  [PDF]
Albert Cardona,Stephan Saalfeld,Stephan Preibisch,Benjamin Schmid,Anchi Cheng,Jim Pulokas,Pavel Tomancak,Volker Hartenstein
PLOS Biology , 2010, DOI: 10.1371/journal.pbio.1000502
Abstract: The analysis of microcircuitry (the connectivity at the level of individual neuronal processes and synapses), which is indispensable for our understanding of brain function, is based on serial transmission electron microscopy (TEM) or one of its modern variants. Due to technical limitations, most previous studies that used serial TEM recorded relatively small stacks of individual neurons. As a result, our knowledge of microcircuitry in any nervous system is very limited. We applied the software package TrakEM2 to reconstruct neuronal microcircuitry from TEM sections of a small brain, the early larval brain of Drosophila melanogaster. TrakEM2 enables us to embed the analysis of the TEM image volumes at the microcircuit level into a light microscopically derived neuro-anatomical framework, by registering confocal stacks containing sparsely labeled neural structures with the TEM image volume. We imaged two sets of serial TEM sections of the Drosophila first instar larval brain neuropile and one ventral nerve cord segment, and here report our first results pertaining to Drosophila brain microcircuitry. Terminal neurites fall into a small number of generic classes termed globular, varicose, axiform, and dendritiform. Globular and varicose neurites have large diameter segments that carry almost exclusively presynaptic sites. Dendritiform neurites are thin, highly branched processes that are almost exclusively postsynaptic. Due to the high branching density of dendritiform fibers and the fact that synapses are polyadic, neurites are highly interconnected even within small neuropile volumes. We describe the network motifs most frequently encountered in the Drosophila neuropile. Our study introduces an approach towards a comprehensive anatomical reconstruction of neuronal microcircuitry and delivers microcircuitry comparisons between vertebrate and insect neuropile.
Variations of the Neutralino Elastic Cross-Section with CP Violating Phases  [PDF]
Toby Falk,Andrew Ferstl,Keith A. Olive
Physics , 1999, DOI: 10.1016/S0927-6505(99)00125-5
Abstract: We analyze the neutralino-nucleus elastic cross-section in the MSSM, including contributions from CP-violating phases, using the four-fermi neutralino-quark interaction. Over a wide range of the MSSM parameter space we show the variations in the cross-sections due to these phases. We further concentrate on the regions which are consistent with constraints from the electric dipole moment of the electron, neutron and mercury atom. In the regions we examine in detail, we find suppressions by up to a factor of two, while enhancements in the cross-sections are no greater than $\sim$ 10%.
Combining Confocal Laser Scanning Microscopy with Serial Section Reconstruction in the Study of Adult Neurogenesis  [PDF]
Federico Luzzati
Frontiers in Neuroscience , 2011, DOI: 10.3389/fnins.2011.00070
Abstract: Current advances in imaging techniques have extended the possibility of visualizing small structures within large volumes of both fixed and live specimens without sectioning. These techniques have contributed valuable information to study neuronal plasticity in the adult brain. However, technical limits still hamper the use of these approaches to investigate neurogenic regions located far from the ventricular surface such as parenchymal neurogenic niches, or the scattered neuroblasts induced by brain lesions. Here, we present a method to combine confocal laser scanning microscopy (CLSM) and serial section reconstruction in order to reconstruct large volumes of brain tissue at cellular resolution. In this method a series of thick sections are imaged with CLSM and the resulting stacks of images are registered and 3D reconstructed. This approach is based on existing freeware software and can be performed on ordinary laboratory personal computers. By using this technique we have investigated the morphology and spatial organization of a group of doublecortin (DCX)+ neuroblasts located in the lateral striatum of the late post-natal guinea pig. The 3D study unraveled a complex network of long and poorly ramified cell processes, often fascicled and mostly oriented along the internal capsule fiber bundles. These data support CLSM serial section reconstruction as a reliable alternative to the whole mount approaches to analyze cyto-architectural features of adult germinative niches.
THREE DIMENSIONAL RECONSTRUCTION OF THE SERIAL SECTION MICROGRAPH OF PULMONARY ALVEOLUS
肺泡显微切片的三维重建

GUO Honghui,PENG Qunsheng,LI Jie,
郭红晖
,彭群生,李捷

软件学报 , 1997,
Abstract: A method of reconstruction of the pulmonary alveolus based on the serial microtomies is presented. It includes eliminating the noises, adjusting the gray level, correcting the geometric distortion and resuming the morphology. An Exclusive -Or Index registration method based on pyramid structure is proposed to accelerate the displacement correction. Experirnents show that this method is fit for the reconstruction of many other tlssues.
Serial Section Scanning Electron Microscopy (S3EM) on Silicon Wafers for Ultra-Structural Volume Imaging of Cells and Tissues  [PDF]
Heinz Horstmann, Christoph K?rber, Kurt S?tzler, Daniel Aydin, Thomas Kuner
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0035172
Abstract: High resolution, three-dimensional (3D) representations of cellular ultrastructure are essential for structure function studies in all areas of cell biology. While limited subcellular volumes have been routinely examined using serial section transmission electron microscopy (ssTEM), complete ultrastructural reconstructions of large volumes, entire cells or even tissue are difficult to achieve using ssTEM. Here, we introduce a novel approach combining serial sectioning of tissue with scanning electron microscopy (SEM) using a conductive silicon wafer as a support. Ribbons containing hundreds of 35 nm thick sections can be generated and imaged on the wafer at a lateral pixel resolution of 3.7 nm by recording the backscattered electrons with the in-lens detector of the SEM. The resulting electron micrographs are qualitatively comparable to those obtained by conventional TEM. S3EM images of the same region of interest in consecutive sections can be used for 3D reconstructions of large structures. We demonstrate the potential of this approach by reconstructing a 31.7 μm3 volume of a calyx of Held presynaptic terminal. The approach introduced here, Serial Section SEM (S3EM), for the first time provides the possibility to obtain 3D ultrastructure of large volumes with high resolution and to selectively and repetitively home in on structures of interest. S3EM accelerates process duration, is amenable to full automation and can be implemented with standard instrumentation.
3D Pathology Volumetric Technique: A Method for Calculating Breast Tumour Volume from Whole-Mount Serial Section Images  [PDF]
G. M. Clarke,M. Murray,C. M. B. Holloway,K. Liu,J. T. Zubovits,M. J. Yaffe
International Journal of Breast Cancer , 2012, DOI: 10.1155/2012/691205
Abstract: Tumour size, most commonly measured by maximum linear extent, remains a strong predictor of survival in breast cancer. Tumour volume, proportional to the number of tumour cells, may be a more accurate surrogate for size. We describe a novel “3D pathology volumetric technique” for lumpectomies and compare it with 2D measurements. Volume renderings and total tumour volume are computed from digitized whole-mount serial sections using custom software tools. Results are presented for two lumpectomy specimens selected for tumour features which may challenge accurate measurement of tumour burden with conventional, sampling-based pathology: (1) an infiltrative pattern admixed with normal breast elements; (2) a localized invasive mass separated from the in situ component by benign tissue. Spatial relationships between key features (tumour foci, close or involved margins) are clearly visualized in volume renderings. Invasive tumour burden can be underestimated using conventional pathology, compared to the volumetric technique (infiltrative pattern: 30% underestimation; localized mass: 3% underestimation for invasive tumour, 44% for in situ component). Tumour volume approximated from 2D measurements (i.e., maximum linear extent), assuming elliptical geometry, was seen to overestimate volume compared to the 3D volumetric calculation (by a factor of 7x for the infiltrative pattern; 1.5x for the localized invasive mass). 1. Introduction Tumour size is a commonly used predictor of survival in breast cancer and correlates strongly with lymph node involvement [1–5]. Tumour size is included in the American Joint Committee on Cancer/Union for International Cancer Control (AJCC/UICC) Cancer Staging Manual [6] and is represented by the maximum linear extent of disease. Here we describe a novel methodology for measuring tumour volume in lumpectomies, referred to as the 3D pathology volumetric technique. For proof of concept, we demonstrate the technique using two lumpectomy specimens, comparing volumes as measured from serial whole-mount sections versus simulated conventional, sampling-based pathology. Serial, whole-mount sections are produced utilizing “3D pathology” techniques and then digitized [7]. Conformational distortion is minimized by first encapsulating the fresh tissue sample in a buoyant, density-matching gel. Automatic microwave processing is employed to accelerate processing of the large tissue samples. Quantitative analysis is performed on the large image dataset (30–70?GB) using custom software tools to create volume renderings and estimate volumes of both in
3D Reconstruction of VZV Infected Cell Nuclei and PML Nuclear Cages by Serial Section Array Scanning Electron Microscopy and Electron Tomography  [PDF]
Mike Reichelt ,Lydia Joubert,John Perrino,Ai Leen Koh,Ibanri Phanwar,Ann M. Arvin
PLOS Pathogens , 2012, DOI: 10.1371/journal.ppat.1002740
Abstract: Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) in vitro and in human dorsal root ganglia and skin xenografts in vivo. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell level.
Individual flexor tendon identification within the carpal tunnel: A semi-automated analysis method for serial cross-section magnetic resonance images
Nicole M Kunze, Jessica E Goetz, Daniel R Thedens, et al
Orthopedic Research and Reviews , 2009, DOI: http://dx.doi.org/10.2147/ORR.S7386
Abstract: dividual flexor tendon identification within the carpal tunnel: A semi-automated analysis method for serial cross-section magnetic resonance images Original Research (4608) Total Article Views Authors: Nicole M Kunze, Jessica E Goetz, Daniel R Thedens, et al Published Date December 2009 Volume 2009:1(Default) Pages 31 - 42 DOI: http://dx.doi.org/10.2147/ORR.S7386 Nicole M Kunze1, Jessica E Goetz2, Daniel R Thedens3, Thomas E Baer2, Ericka A Lawler2, Thomas D Brown2 1Department of Biomedical Engineering, 2Department of Orthopaedics and Rehabilitation, 3Department of Radiology, University of Iowa, Iowa City, IA, USA Abstract: Carpal tunnel syndrome is commonly viewed as resulting from chronic mechanical insult of the median nerve by adjacent anatomical structures. Both the median nerve and its surrounding soft tissue structures are well visualized on magnetic resonance (MR) images of the wrist and hand. Addressing nerve damage from impingement of flexor digitorum tendons co-occupying the tunnel is attractive, but to date has been restricted by a lack of means for making individual identifications of the respective tendons. In this image analysis work, we have developed a region-growing method to positively identify each individual digital flexor tendon within the carpal tunnel by tracking it from a more distal MR section where the respective tendon identities are unambiguous. Illustratively, the new method was applied to MRI scans from four different subjects in a variety of hand poses. Conventional shape measures yielded less discriminatory information than did evaluations of individual tendon location and arrangement. This new method of rapid identification of individual tendons will facilitate analysis of tendon/nerve interactions within the tunnel, thereby providing better information about mechanical insult of the median nerve.
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