Here, we present a pipeline for 3D-correlative focused ion beam milling on guiding the preparation of cellular samples for cryo-electron tomography. The 3D position of fluorescently tagged proteins of interest is first determined by cryo-fluorescence microscopy, and then targeted for milling. The protocol is suitable for mammalian, yeast, and bacterial cells.
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. J. Vis. Exp. (176), e62886, doi:10.3791/62886 (2021).