How to Target Fluorescent Structures in 3D for Cryo-FIB Milling

Cryo-confocal microscopy and its benefits for cryo-electron tomography

From super-resolution cryo-confocal 3D towards cryo-FIB lamella Cryo-Confocal-3D-Lamella-Targeting.jpg

In this article we show that cryo-confocal microscopy is an essential component in cryo-workflows to assess the quality and target distribution of vitrified samples on EM grids. The highly resolved confocal data recorded under cryo-conditions enable the identification of structures of interest under fluorescence in 3D. Furthermore, the 3D volume serves as reference for the correlative approach to retrieve the target structures in the FIB-SEM for milling and subsequent electron tomography in the cryo-TEM to achieve sub-nanometer resolution of the targets.

The Coral Cryo-workflow with the new confocal platform STELLARIS and the Coral Cryo-software enables even unexperienced users to create grid overviews, super-resolved 3D images and precisely defined coordinate markers to ensure a solid basis for the subsequent FIB milling and cryo-electron tomography.

Key Learnings

  • Cell culture on EM grids
  • Micropatterning
  • Plunge freezing
  • Storage, loading and transfer under cryo-genic conditions
  • Check of quality and target distribution
  • Mark landmarks, lamellae points and bead centers
  • How to correlate and retrieve lamellae positions
  • How do I benefit from the Coral Cryo-workflow?
  • Cryo-Transmission Electron Microscopy

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