Array Tomography: A New Tool for Imaging the Molecular Architecture and Ultrastructure of Neural Circuits

Micheva, Kristina D.; Smith, Stephen J.

doi:10.1016/j.neuron.2007.06.014

Cited by 745 publications

(649 citation statements)

References 35 publications

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“…At the TEM level, these problems are particularly acute and the production of ultrathin sections is a timeconsuming and less efficient procedure. However, physical ultrathin sectioning underlies various forms of array tomography: for example, ribbons of sections can be deposited on suitable substrates and viewed by SEM (Micheva and Smith 2007;Wacker and Schroeder 2013) or uncoated block-faces of TEM sections can be viewed by using SEM in backscattering mode (Denk and Horstmann 2004). Both SEM and TEM can be used in conjunction with array tomography and ICC (Micheva et al 2010;Kay et al 2013).…”

Section: Discussionmentioning

confidence: 99%

Quantitative immunocytochemistry at the ultrastructural level: a stereology-based approach to molecular nanomorphomics

Mayhew

2014

Cell Tissue Res

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Biological systems span multiple levels of structural organisation from the macroscopic, via the microscopic, to the nanoscale. Therefore, comprehensive investigation of systems biology requires application of imaging modalities that reveal structure at multiple resolution scales. Nanomorphomics is the part of morphomics devoted to the systematic study of functional morphology at the nanoscale and an important element of its achievement is the combination of immunolabelling and transmission electron microscopy (TEM). The ultimate goal of quantitative immunocytochemistry is to estimate numbers of target molecules (usually peptides, proteins or protein complexes) in biological systems and to map their spatial distributions within them. Immunogold cytochemistry utilises target-specific affinity markers (primary antibodies) and visualisation aids (e.g., colloidal gold particles or silver-enhanced nanogold particles) to detect and localise target molecules at high resolution in intact cells and tissues. In the case of post-embedding labelling of ultrathin sections for TEM, targets are localised as a countable digital readout by using colloidal gold particles. The readout comprises a spatial distribution of gold particles across the section and within the context of biological ultrastructure. The observed distribution across structural compartments (whether volume- or surface-occupying) represents both specific and non-specific labelling; an assessment by eye alone as to whether the distribution is random or non-random is not always possible. This review presents a coherent set of quantitative methods for testing whether target molecules exhibit preferential and specific labelling of compartments and for mapping the same targets in two or more groups of cells as their TEM immunogold-labelling patterns alter after experimental manipulation. The set also includes methods for quantifying colocalisation in multiple-labelling experiments and mapping absolute numbers of colloidal gold particles across compartments at specific positions within cells having a point-like inclusion (e.g., centrosome, nucleolus) and a definable vertical axis. Although developed for quantifying colloidal gold particles, the same methods can in principle be used to quantify other electron-dense punctate nanoparticles, including quantum dots.

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Section: Discussionmentioning

confidence: 99%

Quantitative immunocytochemistry at the ultrastructural level: a stereology-based approach to molecular nanomorphomics

Mayhew

2014

Cell Tissue Res

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“…We have applied our elastic alignment method to two outstanding ssTEM datasets (Supplementary Table 1 The light microscopy dataset has been generated using Array Tomography 15 by Forrest Collman, Nick…”

Section: Imaged Datasetsmentioning

confidence: 99%

Elastic volume reconstruction from series of ultra-thin microscopy sections

et al. 2012

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Anatomy of large biological specimens is often reconstructed from serially sectioned volumes imaged by high-resolution microscopy. We developed a method to reassemble a continuous volume from such large section series that explicitly minimizes artificial deformation by applying a global elastic constraint. We demonstrate our method on a series of transmission electron microscopy sections covering the entire 558-cell Caenorhabditis elegans embryo and a segment of the Drosophila melanogaster larval ventral nerve cord. DOI: https://doi.org/10. 1038/nmeth.2072 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-127852 Accepted Version Originally published at: Saalfeld, Stephan; Fetter, Richard; Cardona, Albert; Tomancak, Pavel (2012). Elastic volume reconstruction from series of ultra-thin microscopy sections. Nature Methods, 9(7):717-720. DOI: https://doi.org/10.1038/nmeth.2072 Elastic Volume Reconstruction from Series of Ultra-thin Microscopy SectionsStephan The downside of the method is that by physically cutting a block into sections the continuity between sections is lost and individual sections are deformed. To recover the imaged volume and extract biologically interesting information such as the reconstruction of neuronal circuits 2,3,5 , sections need to be aligned and distortion must be removed.Alignment can be achieved by maximizing the overlap of similar image content between adjacent sections. However, there are two unknowns that change image content across the section series: specimen's shape and independent section distortion introduced during preparation. Naively warping one section into another would compensate for the shape of the specimen and by that introduce artificial deformation. Our method exploits the fact that the biological specimen's shape typically changes smoothly across sections whereas the independent distortion in each section is random and uncorrelated with neighboring sections. We align all sections not only to their direct neighbors in the series but to all sections in a local neighborhood by modelling sections as two-dimensional elastic sheets that penalize non-rigid deformation ( Fig. 1a and Supplementary Fig. 1).All sections are treated as moving targets in a template-free global alignment. The elastic constraint is implemented as a spring-connected particle system where each section is represented as a triangular spring-mesh ( Fig. 1b and Supplementary Video 1 and Online methods).For each vertex of the spring-mesh, we search for the corresponding location in other sections by pairwise block-matching using normalized cross-correlation (NCC). To that end, we explore all translation vectors in an immediate neighborhood which requires sections to be in approximate alignment (Fig. 1c,d). We estimate this approximate alignment using automatically extracted landmark correspondences from invariant local image features as described previously 7,8 . Originally proposed for robust object recognition under partial occlus...

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“…In the array tomography technique developed by Smith's team 7 , for example, a resin-embedded sample is continually sliced by a diamond knife, with the sections sequentially collected on an adhesive surface, enabling them to be arrayed on a slide. These arrays can then be subjected to multiple rounds of immunofluorescence staining and, ultimately, prepared for SEM imaging.…”

Section: Filling In the Detailsmentioning

confidence: 99%