TY - JOUR
T1 - Multi-resolution correlative focused ion beam scanning electron microscopy
T2 - Applications to cell biology
AU - Narayan, Kedar
AU - Danielson, Cindy M.
AU - Lagarec, Ken
AU - Lowekamp, Bradley C.
AU - Coffman, Phil
AU - Laquerre, Alexandre
AU - Phaneuf, Michael W.
AU - Hope, Thomas J.
AU - Subramaniam, Sriram
N1 - Funding Information:
We thank Kunio Nagashima for assistance with specimen preparation and Prashant Rao for assistance with image processing. Resin embedded B. subtilis samples, and resin-embedded co-cultures of plasmacytoid dendritic cells and Huh-7 cells were kind gifts from Dr. Kumaran Ramamurthi and Dr. Francis V. Chisari, respectively. This work was supported by funds from the Center for Cancer Research , NCI , NIH .
PY - 2014/3
Y1 - 2014/3
N2 - 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.
AB - 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.
KW - 3D imaging of bacteria
KW - Correlative microscopy
KW - FIB-SEM
KW - HIV-1 core
KW - Ion abrasion scanning electron microscopy
KW - Three-dimensional electron microscopy
KW - Tomography
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U2 - 10.1016/j.jsb.2013.11.008
DO - 10.1016/j.jsb.2013.11.008
M3 - Article
C2 - 24300554
AN - SCOPUS:84894488054
SN - 1047-8477
VL - 185
SP - 278
EP - 284
JO - Journal of Structural Biology
JF - Journal of Structural Biology
IS - 3
ER -
