Assembly and Imaging set up of PIE-Scope.

Area of Science:

• Structural Biology
• Cell Biology
• Microscopy

Background:

• Cryo-electron tomography (cryo-ET) visualizes macromolecular complexes within their cellular context.
• Current cryo-ET sample preparation, involving cryo-Focused Ion Beam (FIB) milling and correlative light microscopy, is time-consuming.
• Efficient isolation of target regions is crucial for successful in situ structural studies.

Purpose of the Study:

• To present an integrated cryo-FIB and light microscope system, the Photon Ion Electron microscope (PIE-scope).
• To enable direct and rapid isolation of cellular regions containing specific protein complexes for cryo-ET.
• To streamline the workflow for in situ structural biology.

Main Methods:

• Development of the Photon Ion Electron microscope (PIE-scope), integrating cryo-FIB milling and light microscopy.
• Retrofitting the PIE-scope onto existing microscope platforms, specifically ThermoFisher DualBeams with adaptable modifications.
• Demonstration of rapid isolation of cellular regions of interest.

Main Results:

• The PIE-scope facilitates direct and rapid isolation of cellular regions containing protein complexes.
• The integrated setup simplifies the labor-intensive sample preparation process for cryo-ET.
• The system is designed for adaptability to various existing microscope setups.

Conclusions:

• The PIE-scope offers a significant advancement in cryo-ET sample preparation.
• This technology accelerates the process of identifying and isolating cellular targets for structural analysis.
• The PIE-scope has the potential to broaden the accessibility of in situ structural studies.