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.
Key words3D reconstruction Hippocampus Microwave-enhanced processing Neuropil Perfusion Scanning electron microscopy Scanning transmission electron microscopy Serial sections Structural plasticity Ultramicrotomy
We thank Drs. Cliff Abraham and Jared Bowden for tissue samples from which images in the Figs. 2, 3, and 4 were taken. We also thank Laurence Lindsey for help with computational tools and Patrick Parker for help in preparing the manuscript. This work was supported by the US National Institutes of Health grants NS021184, NS033574, and EB002170 to K.M.H. and the Texas Emerging Technology Fund.
- 17.Cantoni M, Genoud C, Hébert C, Knott G (2010) Large volume, isotropic, 3D imaging of cell structure on the nanometer scale. Microsc Anal 24:13–16Google Scholar
- 19.Knott G, Rosset SP, Cantoni M (2011) Focussed ion beam milling and scanning electron microscopy of brain tissue. J Vis Exp 53:e2588. DOI: 10.3791/2588Google Scholar
- 23.Mendenhall JM, Yorston J, Lagarec KG, Bowden J, Harris KM (2009) Large volume high resolution imaging of brain neuropil using SEM-based scanning electron microscopy. Program No. 484.17. 2009 Neuroscience Meeting Planner. Society for Neuroscience, Chicago, IL (Online)Google Scholar