title: Multi-resolution correlative focused ion beam scanning electron microscopy: applications to cell biology.
abstract: Efficient correlative imaging of small targets within large fields is a central problem in cell biology. Here, we demonstrate a series of technical advances in focused ion beam scanning electron microscopy (FIB-SEM) to address this issue. We report increases in the speed, robustness and automation of the process, and achieve consistent z slice thickness of ∼3 nm. We introduce "keyframe imaging" as a new approach to simultaneously image large fields of view and obtain high-resolution 3D images of targeted sub-volumes. We demonstrate application of these advances to image post-fusion cytoplasmic intermediates of the HIV core. Using fluorescently labeled cell membranes, proteins and HIV cores, we first produce a "target map" of an HIV infected cell by fluorescence microscopy. We then generate a correlated 3D EM volume of the entire cell as well as high-resolution 3D images of individual HIV cores, achieving correlative imaging across a volume scale of 10(9) in a single automated experimental run.
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
authors.forename: Kedar
authors.initials: K
authors.lastname: Narayan
authors.affiliation: Department of Cell and Molecular Biology, Northwestern University, Chicago, IL 60611, USA.
authors.forename: Cindy M
authors.initials: CM
authors.lastname: Danielson
authors.affiliation: Fibics Incorporated, 556 Booth St., Suite 200, Ottawa, Ontario K1A 0G1, Canada.
authors.forename: Ken
authors.initials: K
authors.lastname: Lagarec
authors.affiliation: National Library of Medicine, NIH, Bethesda, MD 20892, USA.
authors.forename: Bradley C
authors.initials: BC
authors.lastname: Lowekamp
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
authors.forename: Phil
authors.initials: P
authors.lastname: Coffman
authors.affiliation: Fibics Incorporated, 556 Booth St., Suite 200, Ottawa, Ontario K1A 0G1, Canada.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Laquerre
authors.affiliation: Fibics Incorporated, 556 Booth St., Suite 200, Ottawa, Ontario K1A 0G1, Canada.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Phaneuf
authors.affiliation: Department of Cell and Molecular Biology, Northwestern University, Chicago, IL 60611, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Hope
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA. Electronic address: ss1@nih.gov.
authors.forename: Sriram
authors.initials: S
authors.lastname: Subramaniam
mesh_terms: D003585:Cell Biology; D002460:Cell Line; D006801:Humans; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D008856:Microscopy, Fluorescence
keywords: 3D imaging of bacteria; Correlative microscopy; FIB-SEM; HIV-1 core; Ion abrasion scanning electron microscopy; Three-dimensional electron microscopy; Tomography