Journal
JOURNAL OF STRUCTURAL BIOLOGY
Volume 192, Issue 2, Pages 262-269Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.jsb.2015.07.012
Keywords
Cryo-SEM; Cryo-FIB; Cryo-TEM; Cryo-planing; Cryo-FLM; Correlative microscopy; Sample preparation
Funding
- EMBO [ALTF 1131-2011]
- HFSP [LT000685/2012-L]
- Weizmann Institute Women in Science Program
- FEI Company Eindhoven
Ask authors/readers for more resources
Cryo-electron tomography provides 3D views of cellular architecture with molecular resolution. A principal limitation of cryo-transmission electron microscopy performed on cells or tissues is the accessible specimen thickness. Recently it has been shown that cryo-focused ion beam milling of plunge-frozen eukaryotic cells can produce homogeneously thin, distortion free lamellas for cryo-electron tomography. Multicellular organisms and tissue cannot be properly vitrified and thinned using this technique because they are considerably thicker. High pressure freezing is therefore necessary to provide optimal preservation. Here, we describe a workflow for preparing lamellas from Caenorhabditis elegans worms using cryo-FIB applied to high pressure frozen samples. We employ cryo-planing followed by correlative cryo-fluorescence microscopy to navigate this large multicellular volume and to localize specific targets within. To produce vitreous lamellas amenable to cryo-TEM observations at these targeted locations, we have developed a dedicated lift-out procedure at cryogenic temperature. (C) 2015 Elsevier Inc. All rights reserved.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available