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Updated: Nov 6, 2025

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A streamlined workflow for automated cryo focused ion beam milling.

Sebastian Tacke1, Philipp Erdmann2, Zhexin Wang1

  • 1Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, Germany.

Journal of Structural Biology
|May 10, 2021
PubMed
Summary

New hardware and software improve cryo-electron tomography (cryo-ET) sample preparation. This enhanced cryo-focused ion beam (cryo-FIB) milling reduces contamination and increases throughput for high-resolution cellular imaging.

Keywords:
AutomationContaminationCryo shieldCryo shutterElectron tomographyFocused ion beam

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Area of Science:

  • Cellular and Molecular Imaging
  • Structural Biology
  • Microscopy Techniques

Background:

  • Cryo-electron tomography (cryo-ET) enables high-resolution in situ imaging of cellular structures and proteins.
  • Preparing biological specimens for cryo-ET often involves focused ion beam (FIB) milling under cryogenic conditions (cryo-FIB).
  • Current cryo-FIB milling is hindered by contamination and tedious manual processes, impacting result quality and throughput.

Purpose of the Study:

  • To present novel hardware and software solutions for overcoming limitations in cryo-FIB milling.
  • To reduce ice contamination during sample transfer and milling.
  • To streamline the cryo-FIB workflow and enhance sample handling.

Main Methods:

  • Development and integration of a new glove box and high vacuum cryo transfer system.
  • Installation of a stage heater, cryo-shield, and cryo-shutter within the FIB milling microscope.
  • Implementation and testing of automated software for key cryo-FIB milling steps.

Main Results:

  • Significant reduction in ice contamination during sample transfer and milling processes.
  • Simplified and improved handling of biological specimens.
  • Successful automation of critical milling steps, leading to higher quality and throughput.
  • Demonstration of high-quality, high-throughput cryo-FIB milling capabilities.

Conclusions:

  • The developed hardware and software significantly improve cryo-FIB milling for cryo-ET.
  • These advancements mitigate contamination and streamline sample preparation.
  • The innovations enable new, previously infeasible experiments in cellular and structural biology.