PT  - JOURNAL ARTICLE
AU  - K.J. Hayworth
AU  - D. Peale
AU  - M. Januszewski
AU  - G.W. Knott
AU  - Z. Lu
AU  - C.S. Xu
AU  - H.F. Hess
TI  - GCIB-SEM: A path to 10 nm isotropic imaging of cubic millimeter volumes
AID  - 10.1101/563239
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 563239
4099  - http://biorxiv.org/content/early/2019/02/28/563239.short
4100  - http://biorxiv.org/content/early/2019/02/28/563239.full
AB  - Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) generates 3D datasets optimally suited for segmentation of cell ultrastructure and automated connectome tracing but is limited to small fields of view and is therefore incompatible with the new generation of ultrafast multibeam SEMs. In contrast, section-based techniques are multibeam-compatible but are limited in z-resolution making automatic segmentation of cellular ultrastructure difficult. Here we demonstrate a novel 3D electron microscopy technique, Gas Cluster Ion Beam SEM (GCIB-SEM), in which top-down, wide-area ion milling is performed on a series of thick sections, acquiring < 10 nm isotropic datasets of each which are then stitched together to span the full sectioned volume. Based on our results, incorporating GCIB-SEM into existing single beam and multibeam SEM workflows should be straightforward and should dramatically increase reliability while simultaneously improving z-resolution by a factor of 3 or more.