PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy
Sergey Gorelick,
Genevieve Buckley,
Gediminas Gervinskas,
Travis K Johnson,
Ava Handley,
Monica Pia Caggiano,
James C Whisstock,
Roger Pocock,
Alex de Marco
Affiliations
Sergey Gorelick
ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Australia; Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Australia
Genevieve Buckley
ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Australia; Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Australia
Gediminas Gervinskas
Ramaciotti Center for Cryo-Electron Microscopy, Monash University, Clayton, Australia
Travis K Johnson
School of Biological Sciences, Monash University, Clayton, Australia
Ava Handley
Department of Anatomy and Developmental Biology, Biomedicine Discovery Institute, Monash University, Clayton, Australia
Monica Pia Caggiano
ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Australia; Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Australia
James C Whisstock
ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Australia; Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Australia; University of Warwick, Coventry, United Kingdom; EMBL Australia, Clayton, Australia
ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Australia; Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Australia; University of Warwick, Coventry, United Kingdom
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.
