Preparation of inner ear sensory hair bundles for high resolution scanning electron microscopy

Chemical fixation techniques for preservation of sensory hair bundles in the mammalian inner ear for scanning electron microscopy (SEM) are reviewed. Fixatives employed were glutaraldehyde, glutaraldehyde-picrate, glutaraldehyde-tannic acid, glutaraldehyde-formaldehyde, glutaraldehyde followed by postfixation with osmium tetroxide and the osmium thiocarbohydrazide (OTOTO) method. Dehydration was routinely accomplished with ascending grades of acetone followed by critical point drying with liquid CO2 or fluorocarbon sublimation. Specimens other than those prepared by the OTOTO method were metal coated with gold, gold-palladium or platinum. Material was viewed at high resolution (2-3 nm) in a transmission electron microscope (TEM) fitted with a scanning system and an LaB6 filament. A few specimens, which were either coated with platinum, carbon or uncoated, were examined in a field emission SEM. We have concluded that glutaraldehyde fixation followed by critical point drying with CO2 and coating with platinum gives the best general preservation of stereocilia and their cross-links for routine high resolution SEM, but that carbon-coated or uncoated specimens offer potentially better results free from metal coating artifacts when viewed with field emission SEM. These methods have enabled us to make novel observations upon the surface detail and cross-links of stereocilia which have helped considerably in understanding the mechanical properties of hair bundles particularly in relation to sensory transduction. We have found that stereocilial surface detail and cross-links are sensitive to fixation regimens. In particular they are degraded by exposure to osmium tetroxide; they are also highly labile since deleterious changes in their appearance can be detected as early as 15 minutes following death.