A new procedure for high resolution light microscopy on peripheral nervous tissue

Abstract

▼Basic research into clinical and therapeutical problems of nervous pathways requires increasingly differentiated information about detailed structures. Light microscopical devices commonly in use do not allow good simultanous discrimination between myelin sheaths, intracellular space of Schwann cells, or interstitial structures and especially between different fibre types (Ref. 1). However, automatic detection by image analysis is often necessary for statistical validation of results (Ref. 2). Discrimination procedures applied to images obtained by conventional light microscopical techniques often fail. In particular, small nerve fibres cannot be detected reliably (Ref. 3). These features can be detected by evaluation of extensive areas by transmission electron microscopy but this is very expensive. Reflection contrast microscopy (RCM) achieves better resolution than conventional light microscopy (Ref. 4, 5, 6). Furthermore, images obtained by RCM have an intense contrast and are thus very suitable for automatic image analysis (Ref. 7). Therefore, we sought to adapt this procedure to histology of neural tissues. This tip describes a method to investigate fixed peripheral nervous tissue by means of RCM. We found that RCM applied on highly osmificated (by the OTOTO method) (Ref. 8, 9, 10) and subsequently counter-stained specimens: