Investigation of Focused Ion Beam/Scanning Electron Microscope parameters for Slice and View and Energy Dispersive X-ray Spectroscopy of Embedded Brain Tissue

Abstract

This Thesis investigates the optimization of a Focused Ion Beam/Scanning Electron Microscope (FIB/SEM) Slice and View protocol of brain tissue. Using a Slice and View protocol in a Dualbeam instrument, the ion and electron beams are used in sequence to alternately mill and image the newly exposed surface of a predefined volume. This creates serial section images that may be used for 3D reconstruction. Research questions addressed include finding FIB parameters most beneficial for accurate milling of the tissue, use of different software strategies and image processing for 3D reconstruction of selected subsets enclosed within the volume sectioned, and the use of Energy Dispersive X-ray Spectroscopy for revealing the volume contents \emph{a priori} of the Slice and View. The reults showed that FIB parameters of $30kV$ and $0.9nA$ provided sufficient accuracy and consistency; that the DAB labeling protocol provided characteristics easily identifiable of the morphology of selected interneurons which were reconstructed, but was difficult to use in a generalized semi-automatic reconstruction protocol; and a model created to test EDX tracing did show potential for its use to increase protocol efficiency by narrowing down the regions of interest of the FIB block.