ARC Centre of Excellence in Advanced Molecular Imaging, Clayton, AustraliaBiomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia
David Dierickx
ARC Centre of Excellence in Advanced Molecular Imaging, Clayton, AustraliaBiomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia
Genevieve Buckley
ARC Centre of Excellence in Advanced Molecular Imaging, Clayton, AustraliaBiomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia
James Whisstock
ARC Centre of Excellence in Advanced Molecular Imaging, Clayton, AustraliaBiomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia, EMBL Australia, Monash University, Clayton, Australia
Alex Marco
ARC Centre of Excellence in Advanced Molecular Imaging, Clayton, AustraliaBiomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia
Cryo-Electron Tomography (cryo-ET) is a method that enables resolving the structure of macromolecular complexes directly in the cellular environment. 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. The PIE-scope can be retrofitted on existing microscopes, although the drawings we provide are meant to work on ThermoFisher DualBeams with small mechanical modifications those can be adapted on other brands.
