Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells

• Type of Material: Article
• Authors: Cheri M Hampton, Emory University; Joshua D Strauss, Emory University; Zunlong Ke, Georgia Institute of Technology; Rebecca S Dillard, Emory University; Jason Hammonds, Emory University; Eric Alonas, Georgia Institute of Technology; Tanay M Desai, Emory University; Mariana Marin, Emory University; Rachel E Storms, Emory University; Fredrick Leon, Emory University; Gregory Melikian, Emory University; Philip J. Santangelo, Emory University; Paul Spearman, Emory University; Elizabeth R. Wright, Emory University
• Language: English
• Date: 2017-01-01
• Publisher: Nature Publishing Group
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
• Final Published Version (URL): http://dx.doi.org/10.1038/nprot.2016.168
• Title of Journal or Parent Work: Nature Protocols
• ISSN: 1754-2189
• Volume: 12
• Issue: 1
• Start Page: 150
• End Page: 167
• Grant/Funding Information: This work was supported in part by grants from Emory University, Children’s Healthcare of Atlanta, and the Georgia Research Alliance to E.R.W.; a grant from the Center for AIDS Research at Emory University (P30 AI050409); a grant from the James B. Pendleton Charitable Trust to E.R.W. and P.W.S.; public health service grants R01GM104540, R21AI101775, and R01GM104540-03S1 from the NIH to E.R.W.; NSF grant 0923395 to E.R.W.; public health service grant R01GM114561 from the NIH to E.R.W. and P.J.S.; public health service grant R01AI058828 from the NIH to P.W.S.; public health service grants R01GM054787 and R01AI053668 from the NIH to G.B.M.; public health service grant R01GM094198 from the NIH to P.J.S.; and public health service grant F32GM112517 from the NIH to J.D.S.
• Keywords: LOCALIZATION; IMAGE; Biochemistry & Molecular Biology; Life Sciences & Biomedicine; ARCHITECTURE; CRYOTOMOGRAPHY; Science & Technology; CRYO-ELECTRON-MICROSCOPY; VISUALIZATION; LIGHT-MICROSCOPY; VITREOUS SECTIONS; Biochemical Research Methods; CRYOMICROSCOPY; TYPE-5 ADENOVIRUS
• Research Categories: Health Sciences, Epidemiology; Biology, Microbiology; Biology, Virology

Abstract

Correlative light and electron microscopy (CLEM) combines spatiotemporal information from fluorescence light microscopy (fLM) with high-resolution structural data from cryo-electron tomography (cryo-ET). These technologies provide opportunities to bridge knowledge gaps between cell and structural biology. Here we describe our protocol for correlated cryo-fLM, cryo-electron microscopy (cryo-EM), and cryo-ET (i.e., cryo-CLEM) of virus-infected or transfected mammalian cells. Mammalian-derived cells are cultured on EM substrates, using optimized conditions that ensure that the cells are spread thinly across the substrate and are not physically disrupted. The cells are then screened by fLM and vitrified before acquisition of cryo-fLM and cryo-ET images, which is followed by data processing. A complete session from grid preparation through data collection and processing takes 5-15 d for an individual experienced in cryo-EM.