![]() The main limit of TEM to study synaptic architecture is the limited resolution in the z-dimension, e.g., its section thickness typically ranging between 40 and 100 nm for ultra-thin sections. ![]() Transmission Electron Microscopy (TEM) is the standard technique that provides high resolution images of cells and tissues down to the nm-scale in the lateral x- and y-dimensions. Due to the small size of synaptic components such as synaptic vesicles far below the resolution limit of diffraction limited light microscopy, high resolution techniques studying the ultrastructure of synapses are essential in order to acquire a broad understanding of important neurological functions like exocytosis and synaptic recycling. Studying the ultrastructural architecture of chemical synapses is central to the understanding of synaptic function. Furthermore, the precise chemical signal transduction in the peripheral nervous system through neuromuscular junctions is essential for precise coordination of the locomotor system. The accurate interconnection and signal transmission of chemical synapses is crucial for the processing of stimuli in the brain, which is incisive for all mental processes, ranging from simple reflexes to control of complex behavioral traits and learning. This is a PLOS Computational Biology Software paper. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.ĭata Availability: All relevant data are within the paper and its Supporting Information files.įunding: This work was supported by: DFG (Deutsche Forschungsgemeinschaft) grant SFB688/A2 and Universitätsbund Würzburg AZ14-48. Received: AugAccepted: DecemPublished: January 5, 2017Ĭopyright: © 2017 Kaltdorf et al. PLoS Comput Biol 13(1):Įditor: Eva Dyer, Northwestern University, UNITED STATES Both automatic and semi-automatic modes are explained including a tutorial.Ĭitation: Kaltdorf KV, Schulze K, Helmprobst F, Kollmannsberger P, Dandekar T, Stigloher C (2017) FIJI Macro 3D ART VeSElecT: 3D Automated Reconstruction Tool for Vesicle Structures of Electron Tomograms. 3D ART VeSElecT shows small error rates and its speed gain can be up to 68 times faster in comparison to manual annotation. We furthermore show gain in segmentation and morphological filtering compared to learning based methods and a large time gain compared to manual segmentation. Detailed evaluation considered performance (detected vesicles) and specificity (true vesicles) as well as precision and recall. Results are collected in color using the RoiManager plugin including the possibility of manual removal of non-matching confounder vesicles. 3D ART VeSElecT removes interfering components, detects vesicles by 3D segmentation and calculates vesicle volume and diameter (spherical approximation, inner/outer diameter). Semi-automated cell selection was integrated. Preprocessing and contrast enhancement work on image-stacks of 1nm/pixel in x and y direction. This flexible arrangement allows in particular reducing false positives by an optional manual revision step. Automated vesicle registration and characterization was implemented in Fiji as two macros (registration and measurement). elegans septin mutant unc-59(e261) on vesicle pool formation and vesicle size. We confirm the functionality of our macro by applying our 3D ART VeSElecT on zebrafish NMJ showing smaller vesicles in 8 dpf embryos then 4 dpf, which was validated by manual reconstruction of the vesicle pool. We demonstrate development-specific and mutant-specific changes in synaptic vesicle pools in both models. We apply this tool to quantify vesicle properties (i) in embryonic Danio rerio 4 and 8 days past fertilization (dpf) and (ii) to compare Caenorhabditis elegans N2 neuromuscular junctions (NMJ) wild-type and its septin mutant ( unc-59(e261)). We describe here the Fiji macro 3D ART VeSElecT which we developed to study synaptic vesicles in electron tomograms. Automatic image reconstruction is critical to cope with steadily increasing data from advanced microscopy.
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