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We demonstrate gas cluster ion beam scanning electron microscopy (SEM), in which wide-area ion milling is performed on a series of thick tissue sections. This three-dimensional electron microscopy technique acquires datasets with

We demonstrate gas cluster ion beam scanning electron microscopy (SEM), in which wide-area ion milling is performed on a series of thick tissue sections. This three-dimensional electron microscopy technique acquires datasets with 10 nm isotropic resolution of each section, and these can then be stitched together to span the sectioned volume. Incorporating gas cluster ion beam SEM into existing single-beam and multibeam SEM workflows should be straightforward, increasing reliability while improving z resolution by a factor of three or more.

Focused-ion-beam scanning electron microscopy (FIB-SEM) has become an essential tool for studying neural tissue at resolutions below 10 nm x 10 nm x 10 nm, producing data sets optimized for automatic connectome tracing. We present a technical advance, ultrathick sectioning, which reliably subdivides embedded tissue samples into chunks (20 mu m thick) optimally sized and mounted for efficient, parallel FIB-SEM imaging. These chunks are imaged separately and then 'volume stitched' back together, producing a final three-dimensional data set suitable for connectome tracing.