PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy

Research output: Contribution to journalArticleResearchpeer-review

1 Citation (Scopus)

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.

Original languageEnglish
Article numbere45919
Number of pages15
JournaleLife
Volume8
DOIs
Publication statusPublished - 1 Jul 2019

Keywords

  • C. elegans
  • correlative microscopy
  • cryo-CLEM
  • cryo-FIB
  • cryo-LM
  • D. melanogaster
  • in situ structural biology
  • molecular biophysics
  • S. cerevisiae
  • structural biology
  • targeted cryo-FIB

Cite this

@article{da2475e192f14b418b5b2163c4eb80b9,
title = "PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy",
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.",
keywords = "C. elegans, correlative microscopy, cryo-CLEM, cryo-FIB, cryo-LM, D. melanogaster, in situ structural biology, molecular biophysics, S. cerevisiae, structural biology, targeted cryo-FIB",
author = "Sergey Gorelick and Genevieve Buckley and Gediminas Gervinskas and Johnson, {Travis K} and Ava Handley and Caggiano, {Monica Pia} and Whisstock, {James C} and Roger Pocock and {de Marco}, Alex",
year = "2019",
month = "7",
day = "1",
doi = "10.7554/eLife.45919",
language = "English",
volume = "8",
journal = "eLife",
issn = "2050-084X",
publisher = "eLife Sciences Publications, Ltd",

}

PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy. / Gorelick, Sergey; Buckley, Genevieve; Gervinskas, Gediminas; Johnson, Travis K; Handley, Ava; Caggiano, Monica Pia; Whisstock, James C; Pocock, Roger; de Marco, Alex.

In: eLife, Vol. 8, e45919, 01.07.2019.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy

AU - Gorelick, Sergey

AU - Buckley, Genevieve

AU - Gervinskas, Gediminas

AU - Johnson, Travis K

AU - Handley, Ava

AU - Caggiano, Monica Pia

AU - Whisstock, James C

AU - Pocock, Roger

AU - de Marco, Alex

PY - 2019/7/1

Y1 - 2019/7/1

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KW - in situ structural biology

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KW - S. cerevisiae

KW - structural biology

KW - targeted cryo-FIB

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