PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy
- Monash University, Australia
Abstract
Cryo-Electron Tomography (cryo-ET) is emerging as a revolutionary method for resolving the structure of macromolecular complexes in situ. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-Focused Ion Beam (FIB) milling and correlative light microscopy to ensure that the event of interest is present in the lamella. Here, we present an integrated cryo-FIB and light microscope setup called the Photon Ion Electron microscope (PIE-scope) that enables direct and rapid isolation of cellular regions containing protein complexes of interest. Specifically, we demonstrate the versatility of PIE-scope by preparing targeted cryo-lamellae from subcellular compartments of neurons from transgenic Caenorhabditis elegans and Drosophila melanogaster expressing fluorescent proteins. We designed PIE-scope to enable retrofitting of existing microscopes, which will increase the throughput and accuracy on projects requiring correlative microscopy to target protein complexes. This new approach will make cryo-correlative workflow safer and more accessible.
Data availability
All significant data are included in this manuscript. The mechanical drawings for all of the components, the bill of material, and the control software have been placed on the laboratory webpage (www.demarco-lab.com/resources) and on Zenodo (10.5281/zenodo.3260173).
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PIE-scope, integrated cryo-correlative light and FIB/SEM microscopyZenodo, 10.5281/zenodo.3260173.
Article and author information
Author details
Funding
ARC centre of excellence in advanced molecular imaging (CE140100011)
- Sergey Gorelick
- Genevieve Buckley
- James C Whisstock
- Alex de Marco
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Sriram Subramaniam, University of British Columbia, Canada
Version history
- Received: February 8, 2019
- Accepted: June 26, 2019
- Accepted Manuscript published: July 1, 2019 (version 1)
- Version of Record published: July 4, 2019 (version 2)
Copyright
© 2019, Gorelick et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy
eLife 8:e45919.
https://doi.org/10.7554/eLife.45919
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