Correlative 3D imaging of whole mammalian cells with light and electron microscopy

Gavin E. Murphy, Kedar Narayan, Bradley C. Lowekamp, Lisa M. Hartnell, Jurgen A W Heymann, Jing Fu, Sriram Subramaniam

Research output: Contribution to journalArticle

Abstract

We report methodological advances that extend the current capabilities of ion-abrasion scanning electron microscopy (IA-SEM), also known as focused ion beam scanning electron microscopy, a newly emerging technology for high resolution imaging of large biological specimens in 3D. We establish protocols that enable the routine generation of 3D image stacks of entire plastic-embedded mammalian cells by IA-SEM at resolutions of ∼10-20 nm at high contrast and with minimal artifacts from the focused ion beam. We build on these advances by describing a detailed approach for carrying out correlative live confocal microscopy and IA-SEM on the same cells. Finally, we demonstrate that by combining correlative imaging with newly developed tools for automated image processing, small 100 nm-sized entities such as HIV-1 or gold beads can be localized in SEM image stacks of whole mammalian cells. We anticipate that these methods will add to the arsenal of tools available for investigating mechanisms underlying host-pathogen interactions, and more generally, the 3D subcellular architecture of mammalian cells and tissues.

Original languageEnglish (US)
Pages (from-to)268-278
Number of pages11
JournalJournal of Structural Biology
Volume176
Issue number3
DOIs
StatePublished - Dec 2011
Externally publishedYes

Fingerprint

Electron Microscopy
Electron Scanning Microscopy
Ions
Light
Host-Pathogen Interactions
Confocal Microscopy
Gold
Artifacts
Plastics
HIV-1
Technology

Keywords

  • Correlative light electron microscopy
  • Focused ion beam scanning electron microscopy
  • HIV
  • Ion-abrasion scanning electron microscopy
  • T cells

ASJC Scopus subject areas

  • Structural Biology

Cite this

Murphy, G. E., Narayan, K., Lowekamp, B. C., Hartnell, L. M., Heymann, J. A. W., Fu, J., & Subramaniam, S. (2011). Correlative 3D imaging of whole mammalian cells with light and electron microscopy. Journal of Structural Biology, 176(3), 268-278. https://doi.org/10.1016/j.jsb.2011.08.013

Correlative 3D imaging of whole mammalian cells with light and electron microscopy. / Murphy, Gavin E.; Narayan, Kedar; Lowekamp, Bradley C.; Hartnell, Lisa M.; Heymann, Jurgen A W; Fu, Jing; Subramaniam, Sriram.

In: Journal of Structural Biology, Vol. 176, No. 3, 12.2011, p. 268-278.

Research output: Contribution to journalArticle

Murphy, GE, Narayan, K, Lowekamp, BC, Hartnell, LM, Heymann, JAW, Fu, J & Subramaniam, S 2011, 'Correlative 3D imaging of whole mammalian cells with light and electron microscopy', Journal of Structural Biology, vol. 176, no. 3, pp. 268-278. https://doi.org/10.1016/j.jsb.2011.08.013
Murphy GE, Narayan K, Lowekamp BC, Hartnell LM, Heymann JAW, Fu J et al. Correlative 3D imaging of whole mammalian cells with light and electron microscopy. Journal of Structural Biology. 2011 Dec;176(3):268-278. https://doi.org/10.1016/j.jsb.2011.08.013
Murphy, Gavin E. ; Narayan, Kedar ; Lowekamp, Bradley C. ; Hartnell, Lisa M. ; Heymann, Jurgen A W ; Fu, Jing ; Subramaniam, Sriram. / Correlative 3D imaging of whole mammalian cells with light and electron microscopy. In: Journal of Structural Biology. 2011 ; Vol. 176, No. 3. pp. 268-278.
@article{4b7d225b54134281935e17b120207846,
title = "Correlative 3D imaging of whole mammalian cells with light and electron microscopy",
abstract = "We report methodological advances that extend the current capabilities of ion-abrasion scanning electron microscopy (IA-SEM), also known as focused ion beam scanning electron microscopy, a newly emerging technology for high resolution imaging of large biological specimens in 3D. We establish protocols that enable the routine generation of 3D image stacks of entire plastic-embedded mammalian cells by IA-SEM at resolutions of ∼10-20 nm at high contrast and with minimal artifacts from the focused ion beam. We build on these advances by describing a detailed approach for carrying out correlative live confocal microscopy and IA-SEM on the same cells. Finally, we demonstrate that by combining correlative imaging with newly developed tools for automated image processing, small 100 nm-sized entities such as HIV-1 or gold beads can be localized in SEM image stacks of whole mammalian cells. We anticipate that these methods will add to the arsenal of tools available for investigating mechanisms underlying host-pathogen interactions, and more generally, the 3D subcellular architecture of mammalian cells and tissues.",
keywords = "Correlative light electron microscopy, Focused ion beam scanning electron microscopy, HIV, Ion-abrasion scanning electron microscopy, T cells",
author = "Murphy, {Gavin E.} and Kedar Narayan and Lowekamp, {Bradley C.} and Hartnell, {Lisa M.} and Heymann, {Jurgen A W} and Jing Fu and Sriram Subramaniam",
year = "2011",
month = "12",
doi = "10.1016/j.jsb.2011.08.013",
language = "English (US)",
volume = "176",
pages = "268--278",
journal = "Journal of Structural Biology",
issn = "1047-8477",
publisher = "Academic Press Inc.",
number = "3",

}

TY - JOUR

T1 - Correlative 3D imaging of whole mammalian cells with light and electron microscopy

AU - Murphy, Gavin E.

AU - Narayan, Kedar

AU - Lowekamp, Bradley C.

AU - Hartnell, Lisa M.

AU - Heymann, Jurgen A W

AU - Fu, Jing

AU - Subramaniam, Sriram

PY - 2011/12

Y1 - 2011/12

N2 - We report methodological advances that extend the current capabilities of ion-abrasion scanning electron microscopy (IA-SEM), also known as focused ion beam scanning electron microscopy, a newly emerging technology for high resolution imaging of large biological specimens in 3D. We establish protocols that enable the routine generation of 3D image stacks of entire plastic-embedded mammalian cells by IA-SEM at resolutions of ∼10-20 nm at high contrast and with minimal artifacts from the focused ion beam. We build on these advances by describing a detailed approach for carrying out correlative live confocal microscopy and IA-SEM on the same cells. Finally, we demonstrate that by combining correlative imaging with newly developed tools for automated image processing, small 100 nm-sized entities such as HIV-1 or gold beads can be localized in SEM image stacks of whole mammalian cells. We anticipate that these methods will add to the arsenal of tools available for investigating mechanisms underlying host-pathogen interactions, and more generally, the 3D subcellular architecture of mammalian cells and tissues.

AB - We report methodological advances that extend the current capabilities of ion-abrasion scanning electron microscopy (IA-SEM), also known as focused ion beam scanning electron microscopy, a newly emerging technology for high resolution imaging of large biological specimens in 3D. We establish protocols that enable the routine generation of 3D image stacks of entire plastic-embedded mammalian cells by IA-SEM at resolutions of ∼10-20 nm at high contrast and with minimal artifacts from the focused ion beam. We build on these advances by describing a detailed approach for carrying out correlative live confocal microscopy and IA-SEM on the same cells. Finally, we demonstrate that by combining correlative imaging with newly developed tools for automated image processing, small 100 nm-sized entities such as HIV-1 or gold beads can be localized in SEM image stacks of whole mammalian cells. We anticipate that these methods will add to the arsenal of tools available for investigating mechanisms underlying host-pathogen interactions, and more generally, the 3D subcellular architecture of mammalian cells and tissues.

KW - Correlative light electron microscopy

KW - Focused ion beam scanning electron microscopy

KW - HIV

KW - Ion-abrasion scanning electron microscopy

KW - T cells

UR - http://www.scopus.com/inward/record.url?scp=80455143874&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=80455143874&partnerID=8YFLogxK

U2 - 10.1016/j.jsb.2011.08.013

DO - 10.1016/j.jsb.2011.08.013

M3 - Article

VL - 176

SP - 268

EP - 278

JO - Journal of Structural Biology

JF - Journal of Structural Biology

SN - 1047-8477

IS - 3

ER -