2021Bieber FIBET

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Bieber, A.; Capitanio, C.; Wilfling, F.; Plitzko, J. & Erdmann, P. S. Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography. Journal of visualized experiments: JoVE, 2021


Cryo-electron tomography (cryo-ET) has become the method of choice for investigating cellular ultrastructure and molecular complexes in their native, frozen-hydrated state. However, cryo-ET requires that samples are thin enough to not scatter or block the incident electron beam. For thick cellular samples, this can be achieved by cryo-focused ion beam (FIB) milling. This protocol describes how to target specific cellular sites during FIB milling using a 3D-correlative approach, which combines three-dimensional fluorescence microscopy data with information from the FIB-scanning electron microscope. Using this technique, rare cellular events and structures can be targeted with high accuracy and visualized at molecular resolution using cryo-transmission electron microscopy (cryo-TEM).




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