C6-O-03Quantifying adult brain ultrastructure using focussed ion beam scanning electron microscopy

Abstract

Electron microscopy is an essential technique for visualising the structure and connectivity of the nervous system. In the past, this was achieved with the slow and error-prone method of serial section transmission electron microscopy. However, more recently, scanning electron microscopy has provided automated methods for imaging significant volumes of tissue. In this talk I will show how focussed ion beam scanning electron microscopy (FIBSEM) can acquire image stacks with near isotropic voxels with which computer algorithms can help speed up the reconstruction and analysis process. With the high precision of this method we can target axons and dendrites that were previously imaged, in vivo, giving insights into the plasticity of adult neural connectivity; as well as revealing morphological characteristics of excitatory, inhibitory, and neuromodulatory axons. Dense reconstructions of all neurites contained within volumes of neuropil allow us to use a 3D approach for making a complete structural analysis. With this data we can quantitatively describe the ultrastructure of a brain region, as well as compare animals at different ages. These experiments show that FIBSEM is a reliable imaging tool, capable of producing a quality of serial images that enable rapid morphological analysis.