Multi-resolution correlative focused ion beam scanning electron microscopy: Applications to cell biology

Kedar Narayan, Cindy M. Danielson, Ken Lagarec, Bradley C. Lowekamp, Phil Coffman, Alexandre Laquerre, Michael W. Phaneuf, Thomas J. Hope, Sriram Subramaniam*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

57 Scopus citations

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 ~3nm. 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 109 in a single automated experimental run.

Original languageEnglish (US)
Pages (from-to)278-284
Number of pages7
JournalJournal of Structural Biology
Volume185
Issue number3
DOIs
StatePublished - Mar 2014

Keywords

  • 3D imaging of bacteria
  • Correlative microscopy
  • FIB-SEM
  • HIV-1 core
  • Ion abrasion scanning electron microscopy
  • Three-dimensional electron microscopy
  • Tomography

ASJC Scopus subject areas

  • Structural Biology

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