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
C2 - 21907806
AN - SCOPUS:80455143874
SN - 1047-8477
VL - 176
SP - 268
EP - 278
JO - Journal of Structural Biology
JF - Journal of Structural Biology
IS - 3
ER -