Large Volumes in Ultrastructural Neuropathology Imaged by Array Tomography of Routine Diagnostic Samples
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Routine samples in pathology and neuropathology are usually prepared according to certified standard sample preparation protocols that do not necessarily introduce the large amounts of heavy metals required to generate optimized contrast and to render the final resin block conductive. Imaging of such samples by volume electron microscopy (EM) methods such as serial block face scanning electron microscopy (SBFSEM) or focused ion beam scanning electron microscopy (FIB-SEM) can thus be challenging due to both contrast and charging issues. Array tomography on the other hand, where hundreds of ultrathin serial sections are deposited on conductive substrates and imaged in a modern field emission scanning electron microscope (FESEM) does not encounter such problems: Section arrays may be poststained with heavy metals leading to superior imaging contrast even from weakly stained blocks. Using a sample from a patient with a congenital myopathy (nebulin-related myopathy) characterized by the so-called electron-dense nemaline rods in muscle fibers we describe preparation of section arrays and how they are imaged in a FESEM in an automated way using a typical, commercially available software platform. We further demonstrate how we can target individual cells by hierarchical imaging cascades. Alignment/registration of image stacks using freeware packages such as Fiji and its TrakEM2 plugin and semiautomated single plane-based segmentation of the nemaline rods using IMOD are also explained.
Key wordsVolume electron microscopy Array tomography (AT) Serial sections FESEM 3D pathology Muscle pathology Myopathy Nemaline
Hierarchical imaging using Atlas 5 AT. Starting from an overview of the entire array zooming in stepwise first to one section, then to one cell, finally to the branched nemaline rod at high magnification and moving from there to a region with distorted filaments (MP4 14,098 kb)