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Updated: Jan 15, 2026

Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography
Published on: October 25, 2021
Exploring shaped focused ion beams for lamella preparation
Johann Brenner1, Jürgen M Plitzko2, Sven Klumpe2
1Research Group CryoEM Technology, Max Planck Institute of Biochemistry, Martinsried, Germany; Research Group Mechanisms of Cellular Quality Control, Max Planck Institute of Biophysics, Frankfurt am Main, Germany.
Abstract:
Focused ion beams (FIB) are widely used instruments in transmission electron microscopy (TEM) sample preparation across scientific disciplines. Generally, site-specific ablation of material is achieved by scanning a highly focused probe across a selected area, leading to the removal of material. However, the geometries of TEM lamellae milled with the FIB are usually highly non-isometric, with their thickness generally being orders of magnitude smaller than their width and length. Here, we explore a changed probe shape for milling. Instead of using an ion beam with the standard, Gaussian-like probe, we characterize the use of the stigmator as quasi-cylindrical lens to create a highly astigmatic probe that we term 'ion knife'. Using the ion knife allows for material removal by spreading the current over a larger area and changes the dimension of the probe as observed in spot burn cross-sections. To allow for rapid alignment of parameters in beam shaping, we demonstrate a method to approximate the shapes of our probes by imaging. Finally, exploring shaped probes in cryogenic lamella preparation, we demonstrate the feasibility of cellular lamella milling and sectioning of cryo-lift-out volumes with the ion knife.
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