Three Dimensional Ultrastructural Analysis of CNS Axons Using Serial Ion- Abrasion Scanning Electron Microscopy (SIA-SEM) G.J. Kidd*, A. Avishai**, N. Ohno*, X. Yin*, N. Avishai**, A.H. Heuer**, and B.D. Trapp* *Department of Neurosciences, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44120 **Department of Materials Science and Engineering, Case Western Reserve University, 10900 Euclid Ave, Cleveland, OH, 44106 Axonal dysfunction and degeneration underlie many central nervous system (CNS) diseases, including those affecting the myelin sheath in individuals with multiple sclerosis [1]. Studies of the pathogenesis of CNS axonal damage in human tissues and experimental animal models are often impeded by the small size of most CNS axons (0.5-2 m). Myelinated axons in CNS white matter tracts are densely packed with little extracellular space between them. Key axonal organelles, including mitochondria and microtubules, are at or beyond light resolution limits, which makes it difficult to differentiate them from one another using light and confocal microscopy techniques. TEM provides high resolution in single slices but obtaining three dimensional (3D) data, such as organelle lengths, shape information and distribution along each axon is not straightforward. Recent advances in serial ion–abrasion SEM (SIA-SEM) imaging have provided new options for generating serial images with 10 nm resolution or better. In dual beam focused ion beam systems, the surface area of the region of interest is milled with a focused Ga ion beam and then imaged with an SEM using backscattered electrons [2-4]. This method offers several advantages for imaging CNS axons: data collection is automated, data sets allow reconstruction of axons and their organelles in three dimensions, and the tissue can be prescanned to locate regions of interest, such as areas in which axons are in longitudinal orientation. Another advantage is availability of dual beam microscopes, which are common in nanofabrication and material science labs. In previous work, we have investigated some technical issues in applying a FIB approach to examination of CNS white matter axons [5]. In this paper we focus on the practical issues of obtaining and using this approach as a routine analysis method in the lab. Samples of cerebellar white matter were obtained from three wild-type rats aged 30d old, an age at which myelination of CNS axons is largely complete, and optic nerves from two 6 month wild type optic nerves. Animals were perfusion fixed with 2.5% glutaraldehyde and 4% paraformaldehyde, then treated with buffered 0.4% osmium tetroxide, washed and stained in ethanolic uranyl acetate. Samples were dehydrated and embedded in Durcupan resin as previously described [2, 5]. To enhance cytoskeletal and mitochondrial contrast at low magnification, samples were also tannic acid treated prior to osmification [5]. Blocks were trimmed, sputter coated with 30nm palladium, and examined in a Nova-200 Nanolab Dual Beam FIB, (FEI) system at the Swagelok Center for Surface Analysis of Materials at Case Western Reserve University. Milling of the tissue block face was performed with a 1nA ion beam current and imaged in high- resolution mode (immersion lens) using the through lens detector in backscattered electron mode. The images were acquired in a high resolution format of 2048-1768 pixels and scanning time of 188 doi:10.1017/S143192761006143X Microsc. Microanal. 16 (Suppl 2), 2010 © Microscopy Society of America 2010