The use of low vacuum scanning electron microscopy (LVSEM) to analyze peripheral nerve samples

Abstract

The use of electron microscopy allows analysis of the microarchitecture of peripheral nerves both during development and in the processes of nerve regeneration and repair. We describe a novel method for the rapid analysis and quantification of myelin in peripheral nerve using a low vacuum scanning electron microscopy protocol. For this methodology, excised nerves are prepared for traditional transmission electron microscopy (TEM) imaging, but at the stage where semi-thin sections would be taken, the resin block is instead imaged at low vacuum in the scanning electron microscope (SEM) using the backscattered electron signal. Any features in the tissue which have incorporated high concentrations of osmium from the fixation process (e.g., myelin) appear as bright regions in the image. Myelin therefore is easily identifiable in the images, and since there is a high contrast difference between it and the surrounding tissue, automated measurements for myelin thickness (e.g., G ratio) using standard and freely available image analysis software (e.g., ImageJ) are easily achievable, consistent, and repeatable. This method therefore greatly speeds up the analysis of nerve samples and, in many cases, will obviate the need for ultrathin sections and TEM for analyzing nerve morphology. Low vacuum (LV) SEM imaging has benefits compared with light microscopy; magnification is continuous, resolution is higher, and contrast and brightness are controllable. Since the resin block does not need a metal coating for imaging in LV mode, once the images have been collected, the block is still ready to section for both light microscopy (LM) and TEM.