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

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19 Citations (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

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