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

eLife

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[1] 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;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;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;Department of Anatomy and Developmental Biology, Biomedicine Discovery Institute, Monash University, Clayton, Australia;Ramaciotti Center for Cryo-Electron Microscopy, Monash University, Clayton, Australia;School of Biological Sciences, Monash University, Clayton, Australia;

关键词: cryo-FIB;    cryo-CLEM;    cryo-LM;    targeted cryo-FIB;    correlative microscopy;    in situ structural biology;    C. elegans;    D. melanogaster;    S. cerevisiae;   

DOI  :  10.7554/eLife.45919

来源: publisher

【 摘 要 】

10.7554/eLife.45919.001Cryo-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.

【 授权许可】

CC BY