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

Sergey Gorelick,Roger Pocock,Monica Pia Caggiano,Alex De Marco,James C Whisstock,Travis K Johnson,Genevieve Buckley,Gediminas Gervinskas,Ava Handley

doi:10.7554/elife.45919

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.

Highlights

Cryo-electron tomography is currently the principal method for investigating the structure of proteins and protein complexes directly in their native environment (Beck and Baumeister, 2016)
To ensure that existing focused ion beam microscope (FIB)/SEM systems in the field can be retrofitted with the solution proposed here, we chose a design that would be applicable to as many systems as possible regardless of the SEM working distance and column shape
The LM does not image the sample at the coincidence point but 49 mm away from it along the X axis (Figure 2). We developed it to fit on a standard ThermoFisher FIB/SEM chamber, with minor modifications it can be adapted to other systems

Summary

Introduction

Cryo-electron tomography (cryo-ET) is currently the principal method for investigating the structure of proteins and protein complexes directly in their native environment (Beck and Baumeister, 2016). It is not possible to image a cell in regions thicker than 500 nm using a conventional 300 keV transmission electron microscope (Frank, 1996). To overcome the issue of cell thickness, the most common and successful approach is to use a cryo-focused ion beam microscope (FIB) to thin the sample and produce flat electron-transparent lamellas of approximately 300 nm thick (Marko et al, 2006; Marko et al, 2007) (Hsieh et al, 2014; Rigort et al, 2010). Considering that every step from FIB milling to cryo-ET imaging is time-

Methods

Results

Conclusion