Skip to main content
bioRxiv
  • Home
  • About
  • Submit
  • ALERTS / RSS
Advanced Search
New Results

En bloc preparation of Drosophila brains enables high-throughput FIB-SEM connectomics

View ORCID ProfileZhiyuan Lu, View ORCID ProfileC. Shan Xu, View ORCID ProfileKenneth J. Hayworth, Patricia Rivlin, Stephen M. Plaza, View ORCID ProfileLouis Scheffer, View ORCID ProfileGerald M. Rubin, View ORCID ProfileHarald F. Hess, View ORCID ProfileIan A. Meinertzhagen
doi: https://doi.org/10.1101/855130
Zhiyuan Lu
1Department of Psychology and Neuroscience, Life Sciences Centre, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4R2
2Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn VA 20147, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Zhiyuan Lu
C. Shan Xu
2Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn VA 20147, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for C. Shan Xu
Kenneth J. Hayworth
2Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn VA 20147, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Kenneth J. Hayworth
Patricia Rivlin
2Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn VA 20147, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Stephen M. Plaza
2Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn VA 20147, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Louis Scheffer
2Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn VA 20147, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Louis Scheffer
Gerald M. Rubin
2Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn VA 20147, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Gerald M. Rubin
Harald F. Hess
2Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn VA 20147, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Harald F. Hess
Ian A. Meinertzhagen
1Department of Psychology and Neuroscience, Life Sciences Centre, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4R2
2Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn VA 20147, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Ian A. Meinertzhagen
  • For correspondence: iam@dal.ca
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Preview PDF
Loading

Abstract

Deriving the detailed synaptic connections of the entire nervous system has been a long term but unrealized goal of the nascent field of connectomics. For Drosophila, in particular, three sample preparation problems must be solved before the requisite imaging and analysis can even begin. The first is dissecting the brain, connectives, and ventral nerve cord (roughly comparable to the brain, neck, and spinal cord of vertebrates) as a single contiguous unit. Second is fixing and staining the resulting specimen, too large for previous techniques such as flash freezing, so as to permit the necessary automated segmentation of neuron membranes. Finally the contrast must be sufficient to support synapse detection at imaging speeds that enable the entire connectome to be collected. To address these issues, we report three major novel methods to dissect, fix, dehydrate and stain this tiny but complex nervous system in its entirety; together they enable us to uncover a Focused Ion-Beam Scanning Electron Microscopy (FIB-SEM) connectome of the entire Drosophila brain. They reliably recover fixed neurons as round profiles with darkly stained synapses, suitable for machine segmentation and automatic synapse detection, for which only minimal human intervention is required. Our advanced procedures use: a custom-made jig to microdissect both regions of the central nervous system, dorsal and ventral, with their connectives; fixation and Durcupan embedment, followed by a special hot-knife slicing protocol to reduce the brain to dimensions suited to FIB; contrast enhancement by heavy metals; together with a progressive lowering of temperature protocol for dehydration. Collectively these optimize the brain’s morphological preservation, imaging it at a usual resolution of 8nm per voxel while simultaneously speeding the formerly slow rate of FIB-SEM. With these methods we could recently obtain a FIB-SEM image stack of the Drosophila brain eight times faster than hitherto, at approximately the same rate as, but without the requirement to cut, nor imperfections in, EM serial sections.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
Back to top
PreviousNext
Posted November 27, 2019.
Download PDF
Email

Thank you for your interest in spreading the word about bioRxiv.

NOTE: Your email address is requested solely to identify you as the sender of this article.

Enter multiple addresses on separate lines or separate them with commas.
En bloc preparation of Drosophila brains enables high-throughput FIB-SEM connectomics
(Your Name) has forwarded a page to you from bioRxiv
(Your Name) thought you would like to see this page from the bioRxiv website.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
En bloc preparation of Drosophila brains enables high-throughput FIB-SEM connectomics
Zhiyuan Lu, C. Shan Xu, Kenneth J. Hayworth, Patricia Rivlin, Stephen M. Plaza, Louis Scheffer, Gerald M. Rubin, Harald F. Hess, Ian A. Meinertzhagen
bioRxiv 855130; doi: https://doi.org/10.1101/855130
Digg logo Reddit logo Twitter logo Facebook logo Google logo LinkedIn logo Mendeley logo
Citation Tools
En bloc preparation of Drosophila brains enables high-throughput FIB-SEM connectomics
Zhiyuan Lu, C. Shan Xu, Kenneth J. Hayworth, Patricia Rivlin, Stephen M. Plaza, Louis Scheffer, Gerald M. Rubin, Harald F. Hess, Ian A. Meinertzhagen
bioRxiv 855130; doi: https://doi.org/10.1101/855130

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Subject Area

  • Neuroscience
Subject Areas
All Articles
  • Animal Behavior and Cognition (3701)
  • Biochemistry (7825)
  • Bioengineering (5698)
  • Bioinformatics (21348)
  • Biophysics (10606)
  • Cancer Biology (8210)
  • Cell Biology (11978)
  • Clinical Trials (138)
  • Developmental Biology (6790)
  • Ecology (10427)
  • Epidemiology (2065)
  • Evolutionary Biology (13912)
  • Genetics (9733)
  • Genomics (13111)
  • Immunology (8175)
  • Microbiology (20074)
  • Molecular Biology (7881)
  • Neuroscience (43182)
  • Paleontology (321)
  • Pathology (1285)
  • Pharmacology and Toxicology (2268)
  • Physiology (3364)
  • Plant Biology (7258)
  • Scientific Communication and Education (1317)
  • Synthetic Biology (2012)
  • Systems Biology (5551)
  • Zoology (1135)