Non-coating fixation techniques or redundancy of conductive coating, low kV FE-SEM operation and combined SEM/TEM of biological tissues.

Non-coating fixation methods, in particular the tannic acid/arginine/osmium tetroxide procedure, are employed for a number of reasons on the guinea-pig organ of Corti hair cell stereocilia glycocalyx and the imprints of the stereocilia at the bottom side of the tectorial membrane, and on the rat and cat intestinal epithelial microvilli glycocalyx and mucus-producing goblet cells. These methods are used firstly to confirm that non-coating prepared specimens can be embedded for TEM observation at 60-100 kV without loss of detail information, and these images can be compared with cryo-FE-SEM images of the same structure/tissue. Secondly, they show that specimens treated according non-coating techniques become optimally preserved and electrically conductive, so that no external conductive coating is required. In this way a comparison of images of subsequent fresh fracture faces is possible without a decrease in information on detail, which otherwise could happen after subsequent coating layers required after standard fixation. Thirdly, they show that non-coating methods can be used quite well with low accelerating voltages because the osmium-tannic acid complex in the specimen surface produces a large number of backscattered and secondary electrons in the surface layer, showing in particular surface phenomena. Fourthly, they show that with an optimal non-coating preservation, in combination with a well-balanced pre-fixation mixture, preparation artefacts due to extraction and even dehydration and drying are minimized. This is compared with images of the organ of Corti hair cells treated with a so-called three-aldehyde pre-fixation mixture, which causes disrupted stereocilia to cling onto the bottom side of the tectorial membrane.