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

Area of Science:

• Structural Biology
• Cellular Imaging
• Microscopy Technology

Background:

• Cryo-electron tomography (cryo-ET) is a powerful technique for in situ structural analysis of macromolecular complexes.
• Current cryo-ET sample preparation is labor-intensive, often requiring cryo-focused ion beam (FIB) milling and correlative light microscopy (CLM).

Purpose of the Study:

• To develop an integrated system for streamlined and efficient cryo-ET sample preparation.
• To improve the accuracy and accessibility of cryo-correlative workflows for targeting protein complexes.

Main Methods:

• Development of the Photon Ion Electron microscope (PIE-scope), integrating cryo-FIB milling and light microscopy.
• Demonstration of PIE-scope for targeted cryo-lamella preparation from neurons in transgenic *Caenorhabditis elegans* and *Drosophila melanogaster*.

Main Results:

• The PIE-scope enables direct and rapid isolation of cellular regions containing protein complexes of interest.
• Successfully prepared targeted cryo-lamellae from subcellular compartments in model organisms.
• The PIE-scope design allows retrofitting of existing microscopes to enhance throughput and accuracy.

Conclusions:

• The PIE-scope significantly simplifies and accelerates the sample preparation process for in situ cryo-ET.
• This integrated approach enhances the safety and accessibility of cryo-correlative workflows.
• PIE-scope is a versatile tool for structural biology research, particularly for targeting specific protein complexes within cells.