259 METHODS IN CELL BIOLOGY, VOL 111 Copyright 2012, Elsevier Inc. All rights reserved. 0091-679X http://dx.doi.org/10.1016/B978-0-12-416026-2.00014-5 CHAPTER 14 Integrative Approaches for Cellular Cryo-electron Tomography: Correlative Imaging and Focused Ion Beam Micromachining Alexander Rigort, Elizabeth Villa, Felix J.B. Bäuerlein, Benjamin D. Engel and Jürgen M. Plitzko Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany Abstract I. Introduction II. Rationale III. Methods A. Cryo-correlative Microscopy B. Cryo-fluorescence Instrumentation C. Cryo-focused Ion Beam Milling D. Cryo-focused Ion Beam Instrumentation E. Experimental Workflow for Preparing Thin Cellular Specimens F. Cryo-electron Microscopy and Tomography G. Quantitative Analysis of Data from Cellular Tomograms IV. Instrumentation and Materials A. Preparation and Vitrification of Cells B. Cryo-fluorescence Correlative Microscopy C. Cryo-focused Ion Beam Milling D. Cryo-electron Microscopy and Tomography V. Discussion and Outlook Acknowledgments References Abstract The application of cryo-electron tomography to cells and tissues is commonly referred to as “cellular tomography,” and enables visualization of the supramolecular