TOOLS AND RESOURCES

Enhanced FIB-SEM systems for largevolume 3D imaging C Shan Xu1*, Kenneth J Hayworth1, Zhiyuan Lu1,2, Patricia Grob3, Ahmed M Hassan3†, Jose´ G Garcı´a-Cerda´n4, Krishna K Niyogi4,5, Eva Nogales3,5, Richard J Weinberg6, Harald F Hess1 1

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

*For correspondence: xuc@ janelia.hhmi.org Present address: †Department of Biomedical Engineering, University of Texas at Austin, Austin, United States

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 mm3. 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.

Competing interests: The authors declare that no competing interests exist.

DOI: 10.7554/eLife.25916.001

Funding: See page 34

Introduction

Received: 10 February 2017 Accepted: 09 May 2017 Published: 13 May 2017

Many modalities of electron microscopy (EM) can probe cellular structure at the nanometer scale. However, despite considerable progress over the past decade in developing high-resolution 3D imaging, there remain important limitations, reflecting an inherent trade-off between resolution and the size of the 3D volume. Different currently available EM methods, and their advantages and disadvantages have been reviewed recently (Briggman and Bock, 2012; Titze and Genoud, 2016). For demanding applications such as tracing neuronal processes in three dimensions, high resolution in the z axis, in addition to the xy plane, is critical (Lichtman and Denk, 2011; Meinertzhagen, 2016). FIB-SEM offers exactly this capability, with x, y, and z resolution all

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

Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) can automatically generate 3D images with superior z-axis resolution, yielding data that needs...
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