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En bloc preparation of Drosophila brains enables high-throughput FIB-SEM connectomics
Deriving the detailed synaptic connections of an entire nervous system is the unrealized goal of the nascent field of connectomics. For the fruit fly Drosophila, in particular, we need to dissect the brain, connectives, and ventral nerve cord as a single continuous unit, fix and stain it, and undert...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2022
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9801301/ https://www.ncbi.nlm.nih.gov/pubmed/36589862 http://dx.doi.org/10.3389/fncir.2022.917251 |
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author | Lu, Zhiyuan Xu, C. Shan Hayworth, Kenneth J. Pang, Song Shinomiya, Kazunori Plaza, Stephen M. Scheffer, Louis K. Rubin, Gerald M. Hess, Harald F. Rivlin, Patricia K. Meinertzhagen, Ian A. |
author_facet | Lu, Zhiyuan Xu, C. Shan Hayworth, Kenneth J. Pang, Song Shinomiya, Kazunori Plaza, Stephen M. Scheffer, Louis K. Rubin, Gerald M. Hess, Harald F. Rivlin, Patricia K. Meinertzhagen, Ian A. |
author_sort | Lu, Zhiyuan |
collection | PubMed |
description | Deriving the detailed synaptic connections of an entire nervous system is the unrealized goal of the nascent field of connectomics. For the fruit fly Drosophila, in particular, we need to dissect the brain, connectives, and ventral nerve cord as a single continuous unit, fix and stain it, and undertake automated segmentation of neuron membranes. To achieve this, we designed a protocol using progressive lowering of temperature dehydration (PLT), a technique routinely used to preserve cellular structure and antigenicity. We combined PLT with low temperature en bloc staining (LTS) and recover fixed neurons as round profiles with darkly stained synapses, suitable for machine segmentation and automatic synapse detection. Here we report three different PLT-LTS methods designed to meet the requirements for FIB-SEM imaging of the Drosophila brain. These requirements include: good preservation of ultrastructural detail, high level of en bloc staining, artifact-free microdissection, and smooth hot-knife cutting to reduce the brain to dimensions suited to FIB-SEM. In addition to PLT-LTS, we designed a jig to microdissect and pre-fix the fly’s delicate brain and central nervous system. Collectively these methods optimize morphological preservation, allow us to image the brain usually at 8 nm per voxel, and simultaneously speed the formerly slow rate of FIB-SEM imaging. |
format | Online Article Text |
id | pubmed-9801301 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-98013012022-12-31 En bloc preparation of Drosophila brains enables high-throughput FIB-SEM connectomics Lu, Zhiyuan Xu, C. Shan Hayworth, Kenneth J. Pang, Song Shinomiya, Kazunori Plaza, Stephen M. Scheffer, Louis K. Rubin, Gerald M. Hess, Harald F. Rivlin, Patricia K. Meinertzhagen, Ian A. Front Neural Circuits Neural Circuits Deriving the detailed synaptic connections of an entire nervous system is the unrealized goal of the nascent field of connectomics. For the fruit fly Drosophila, in particular, we need to dissect the brain, connectives, and ventral nerve cord as a single continuous unit, fix and stain it, and undertake automated segmentation of neuron membranes. To achieve this, we designed a protocol using progressive lowering of temperature dehydration (PLT), a technique routinely used to preserve cellular structure and antigenicity. We combined PLT with low temperature en bloc staining (LTS) and recover fixed neurons as round profiles with darkly stained synapses, suitable for machine segmentation and automatic synapse detection. Here we report three different PLT-LTS methods designed to meet the requirements for FIB-SEM imaging of the Drosophila brain. These requirements include: good preservation of ultrastructural detail, high level of en bloc staining, artifact-free microdissection, and smooth hot-knife cutting to reduce the brain to dimensions suited to FIB-SEM. In addition to PLT-LTS, we designed a jig to microdissect and pre-fix the fly’s delicate brain and central nervous system. Collectively these methods optimize morphological preservation, allow us to image the brain usually at 8 nm per voxel, and simultaneously speed the formerly slow rate of FIB-SEM imaging. Frontiers Media S.A. 2022-12-16 /pmc/articles/PMC9801301/ /pubmed/36589862 http://dx.doi.org/10.3389/fncir.2022.917251 Text en Copyright © 2022 Lu, Xu, Hayworth, Pang, Shinomiya, Plaza, Scheffer, Rubin, Hess, Rivlin and Meinertzhagen. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Neural Circuits Lu, Zhiyuan Xu, C. Shan Hayworth, Kenneth J. Pang, Song Shinomiya, Kazunori Plaza, Stephen M. Scheffer, Louis K. Rubin, Gerald M. Hess, Harald F. Rivlin, Patricia K. Meinertzhagen, Ian A. En bloc preparation of Drosophila brains enables high-throughput FIB-SEM connectomics |
title | En bloc preparation of Drosophila brains enables high-throughput FIB-SEM connectomics |
title_full | En bloc preparation of Drosophila brains enables high-throughput FIB-SEM connectomics |
title_fullStr | En bloc preparation of Drosophila brains enables high-throughput FIB-SEM connectomics |
title_full_unstemmed | En bloc preparation of Drosophila brains enables high-throughput FIB-SEM connectomics |
title_short | En bloc preparation of Drosophila brains enables high-throughput FIB-SEM connectomics |
title_sort | en bloc preparation of drosophila brains enables high-throughput fib-sem connectomics |
topic | Neural Circuits |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9801301/ https://www.ncbi.nlm.nih.gov/pubmed/36589862 http://dx.doi.org/10.3389/fncir.2022.917251 |
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