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Elife. 2017 May 13;6. pii: e25916. doi: 10.7554/eLife.25916.

Enhanced FIB-SEM systems for large-volume 3D imaging.

Author information

1
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States.
2
Department of Psychology and Neuroscience, Dalhousie University, Halifax, Canada.
3
Howard Hughes Medical Institute, Molecular and Cell Biology Department, University of California, Berkeley, United States.
4
Howard Hughes Medical Institute, Plant and Microbial Biology Department, University of California, Berkeley, United States.
5
Lawrence Berkeley National Laboratory, Berkeley, United States.
6
Department of Cell Biology and Physiology, University of North Carolina, North Carolina, United States.

Abstract

Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) can automatically generate 3D images with superior z-axis resolution, yielding data that needs minimal image registration and related post-processing. Obstacles blocking wider adoption of FIB-SEM include slow imaging speed and lack of long-term system stability, which caps the maximum possible acquisition volume. Here, we present techniques that accelerate image acquisition while greatly improving FIB-SEM reliability, allowing the system to operate for months and generating continuously imaged volumes > 106 µm3. These volumes are large enough for connectomics, where the excellent z resolution can help in tracing of small neuronal processes and accelerate the tedious and time-consuming human proofreading effort. Even higher resolution can be achieved on smaller volumes. We present example data sets from mammalian neural tissue, Drosophila brain, and Chlamydomonas reinhardtii to illustrate the power of this novel high-resolution technique to address questions in both connectomics and cell biology.

KEYWORDS:

3D cellular structure; FIB; cell biology; connectomics; electron microscopy; imaging; neuroscience

PMID:
28500755
PMCID:
PMC5476429
DOI:
10.7554/eLife.25916
[Indexed for MEDLINE]
Free PMC Article

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