title: Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells.
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.
authors.affiliation: Division of Infectious Diseases, Department of Pediatrics, Emory University School of Medicine, Children's Healthcare of Atlanta, Atlanta, Georgia, USA.
authors.forename: Cheri M
authors.identifier: http://orcid.org/0000-0003-0069-8712
authors.initials: CM
authors.lastname: Hampton
authors.affiliation: Division of Infectious Diseases, Department of Pediatrics, Emory University School of Medicine, Children's Healthcare of Atlanta, Atlanta, Georgia, USA.
authors.forename: Joshua D
authors.initials: JD
authors.lastname: Strauss
authors.affiliation: School of Biology, Georgia Institute of Technology, Atlanta, Georgia, USA.
authors.forename: Zunlong
authors.initials: Z
authors.lastname: Ke
authors.affiliation: Division of Infectious Diseases, Department of Pediatrics, Emory University School of Medicine, Children's Healthcare of Atlanta, Atlanta, Georgia, USA.
authors.forename: Rebecca S
authors.initials: RS
authors.lastname: Dillard
authors.affiliation: Division of Infectious Diseases, Department of Pediatrics, Emory University School of Medicine, Children's Healthcare of Atlanta, Atlanta, Georgia, USA.
authors.forename: Jason E
authors.initials: JE
authors.lastname: Hammonds
authors.affiliation: Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Alonas
authors.affiliation: Division of Infectious Diseases, Department of Pediatrics, Emory University School of Medicine, Children's Healthcare of Atlanta, Atlanta, Georgia, USA.
authors.forename: Tanay M
authors.initials: TM
authors.lastname: Desai
authors.affiliation: Division of Infectious Diseases, Department of Pediatrics, Emory University School of Medicine, Children's Healthcare of Atlanta, Atlanta, Georgia, USA.
authors.forename: Mariana
authors.initials: M
authors.lastname: Marin
authors.affiliation: Division of Infectious Diseases, Department of Pediatrics, Emory University School of Medicine, Children's Healthcare of Atlanta, Atlanta, Georgia, USA.
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Storms
authors.affiliation: Division of Infectious Diseases, Department of Pediatrics, Emory University School of Medicine, Children's Healthcare of Atlanta, Atlanta, Georgia, USA.
authors.forename: Fredrick
authors.initials: F
authors.lastname: Leon
authors.affiliation: Division of Infectious Diseases, Department of Pediatrics, Emory University School of Medicine, Children's Healthcare of Atlanta, Atlanta, Georgia, USA.
authors.forename: Gregory B
authors.initials: GB
authors.lastname: Melikyan
authors.affiliation: Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Santangelo
authors.affiliation: Division of Infectious Diseases, Department of Pediatrics, Emory University School of Medicine, Children's Healthcare of Atlanta, Atlanta, Georgia, USA.
authors.forename: Paul W
authors.initials: PW
authors.lastname: Spearman
authors.affiliation: Division of Infectious Diseases, Department of Pediatrics, Emory University School of Medicine, Children's Healthcare of Atlanta, Atlanta, Georgia, USA.|Robert P. Apkarian Integrated Electron Microscopy Core, Emory University, Atlanta, Georgia, USA.
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Wright
mesh_terms: D002460:Cell Line; D020285:Cryoelectron Microscopy / Q000379:methods*; D015497:HIV-1 / Q000502:physiology*; D018259:Herpesvirus 1, Human / Q000502:physiology*; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D014162:Transfection