Large-volume reconstruction of brain tissue from high-resolution serial section images acquired by SEM-based scanning transmission electron microscopy.

With recent improvements in instrumentation and computational tools, serial section electron microscopy has become increasingly straightforward. A new method for imaging ultrathin serial sections is developed based on a field emission scanning electron microscope fitted with a transmitted electron detector. This method is capable of automatically acquiring high-resolution serial images with a large field size and very little optical and physical distortions. In this chapter, we describe the procedures leading to the generation and analyses of a large-volume stack of high-resolution images (64 μm × 64 μm × 10 μm, or larger, at 2 nm pixel size), including how to obtain large-area serial sections of uniform thickness from well-preserved brain tissue that is rapidly perfusion-fixed with mixed aldehydes, processed with a microwave-enhanced method, and embedded into epoxy resin.