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J Struct Biol. 2014 Mar;185(3):278-84. doi: 10.1016/j.jsb.2013.11.008. Epub 2013 Dec 1.

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

Author information

1
Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
2
Department of Cell and Molecular Biology, Northwestern University, Chicago, IL 60611, USA.
3
Fibics Incorporated, 556 Booth St., Suite 200, Ottawa, Ontario K1A 0G1, Canada.
4
National Library of Medicine, NIH, Bethesda, MD 20892, USA.
5
Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA. Electronic address: ss1@nih.gov.

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.

KEYWORDS:

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

PMID:
24300554
PMCID:
PMC3943650
DOI:
10.1016/j.jsb.2013.11.008
[Indexed for MEDLINE]
Free PMC Article

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