A targeted 3D EM and correlative microscopy method using SEM array tomography

Burel, Agnès; Lavault, Marie Thérèse; Chevalier, Clément; Gnaegi, Helmut; Prigent, Sylvain; Mucciolo, Antonio; Dutertre, Stéphanie; Humbel, Bruno B.M.; Guillaudeux, Thierry; Kolotuev, Irina

doi:10.1242/dev.160879

Cited by 60 publications

(64 citation statements)

References 64 publications

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“…However, these structures are difficult to visualize using standard approaches because their appearance depends on the angle of sectioning and the developmental stage of the embryo. We used a method called positional correlative anatomy EM to provide greater control over these variables (Kolotuev, 2014;Burel et al, 2018). Briefly, a highpressure frozen sample is flat-embedded at the surface of a resin block, a region of interest is identified at high magnification and the sample is then oriented for ultramicrotome sectioning with respect to known anatomical features.…”

Section: Dyf-7 Forms Fibrils In Vivo and In Vitromentioning

confidence: 99%

Morphogenesis of neurons and glia within an epithelium

et al. 2019

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To sense the outside world, some neurons protrude across epithelia, the cellular barriers that line every surface of our bodies. To study the morphogenesis of such neurons, we examined the C. elegans amphid, in which dendrites protrude through a glial channel at the nose. During development, amphid dendrites extend by attaching to the nose via DYF-7, a type of protein typically found in epithelial apical ECM. Here, we show that amphid neurons and glia exhibit epithelial properties, including tight junctions and apical-basal polarity, and develop in a manner resembling other epithelia. We find that DYF-7 is a fibril-forming apical ECM component that promotes formation of the tube-shaped glial channel, reminiscent of roles for apical ECM in other narrow epithelial tubes. We also identify a requirement for FRM-2, a homolog of EPBL15/moe/Yurt that promotes epithelial integrity in other systems. Finally, we show that other environmentally exposed neurons share a requirement for DYF-7. Together, our results suggest that these neurons and glia can be viewed as part of an epithelium continuous with the skin, and are shaped by mechanisms shared with other epithelia.

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Section: Dyf-7 Forms Fibrils In Vivo and In Vitromentioning

confidence: 99%

Morphogenesis of neurons and glia within an epithelium

et al. 2019

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“…One simply has to choose which process works best for them, or devise their own strategy. 1 Gay and Anderson (1954) ; 2 Westfall and Healy (1962) ; 3 Fahrenbach Wolf (1984) ; 4 Galey and Nilsson (1966) ; 5 Mironov et al (2008) ; 6 Anderson and Brenner (1971) ; 7 Rowley and Moran (1975) ; 8 Abad (1988) ; 9 Wells (1974) ; 10 Mironov et al (2008) ; 11 Stevens et al (1980) ; 12 Hall (1995) ; 13 Schalek et al (2012) ; 14 Micheva and Smith (2007) ; 15 Burel et al (2018) ; 16 Leica Microsystems, Germany.…”

Section: Step-by-step Description Of Methods and Considerationsmentioning

confidence: 99%

A Pipeline for Volume Electron Microscopy of the Caenorhabditis elegans Nervous System

Mulcahy

Witvliet

Holmyard

et al. 2018

Front. Neural Circuits

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The “connectome,” a comprehensive wiring diagram of synaptic connectivity, is achieved through volume electron microscopy (vEM) analysis of an entire nervous system and all associated non-neuronal tissues. White et al. (1986) pioneered the fully manual reconstruction of a connectome using Caenorhabditis elegans. Recent advances in vEM allow mapping new C. elegans connectomes with increased throughput, and reduced subjectivity. Current vEM studies aim to not only fill the remaining gaps in the original connectome, but also address fundamental questions including how the connectome changes during development, the nature of individuality, sexual dimorphism, and how genetic and environmental factors regulate connectivity. Here we describe our current vEM pipeline and projected improvements for the study of the C. elegans nervous system and beyond.

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“…Furthermore, in our implementation of LASSO, we used silicon/silicon-nitride substrates. In their current state, these substrates could be amenable to automated multi-parameter analysis by combining ssTEM with other analysis methods such as electrophysiology [ 31 ], array tomography [ 34 , 35 ], and genetic analysis [ 36 ]. Alternatively, these substrates could used with other imaging modalities, such as multi-beam scanning electron microscopy (SEM) [ 37 ], scanning transmission electron microscopy (STEM) [ 38 ], or x-ray microscopy [ 39 ], to provide imaging at multiple resolutions.…”

Section: Resultsmentioning

confidence: 99%

Large-scale neuroanatomy using LASSO: Loop-based Automated Serial Sectioning Operation

et al. 2018

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Serial section transmission electron microscopy (ssTEM) is the most promising tool for investigating the three-dimensional anatomy of the brain with nanometer resolution. Yet as the field progresses to larger volumes of brain tissue, new methods for high-yield, low-cost, and high-throughput serial sectioning are required. Here, we introduce LASSO (Loop-based Automated Serial Sectioning Operation), in which serial sections are processed in “batches.” Batches are quantized groups of individual sections that, in LASSO, are cut with a diamond knife, picked up from an attached waterboat, and placed onto microfabricated TEM substrates using rapid, accurate, and repeatable robotic tools. Additionally, we introduce mathematical models for ssTEM with respect to yield, throughput, and cost to access ssTEM scalability. To validate the method experimentally, we processed 729 serial sections of human brain tissue (~40 nm x 1 mm x 1 mm). Section yield was 727/729 (99.7%). Sections were placed accurately and repeatably (x-direction: -20 ± 110 μm (1 s.d.), y-direction: 60 ± 150 μm (1 s.d.)) with a mean cycle time of 43 s ± 12 s (1 s.d.). High-magnification (2.5 nm/px) TEM imaging was conducted to measure the image quality. We report no significant distortion, information loss, or substrate-derived artifacts in the TEM images. Quantitatively, the edge spread function across vesicle edges and image contrast were comparable, suggesting that LASSO does not negatively affect image quality. In total, LASSO compares favorably with traditional serial sectioning methods with respect to throughput, yield, and cost for large-scale experiments, and represents a flexible, scalable, and accessible technology platform to enable the next generation of neuroanatomical studies.

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A targeted 3D EM and correlative microscopy method using SEM array tomography

Cited by 60 publications

References 64 publications

Morphogenesis of neurons and glia within an epithelium

Morphogenesis of neurons and glia within an epithelium

A Pipeline for Volume Electron Microscopy of the Caenorhabditis elegans Nervous System

Large-scale neuroanatomy using LASSO: Loop-based Automated Serial Sectioning Operation

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