Scanning electron microscopy of the mammalian organ of Corti: assessment of preparative procedures.
Different fixation, drying and coating procedures have been applied in preparation of the organ of Corti for scanning electron microscopy (SEM), and structural features of the apical surface of the tissue in unfixed, freeze-fractured preparations used in assessing their effects on morphology. Fixation with glutaraldehyde alone or osmium tetroxide alone causes artefacts that are substantially avoided when tissue is doubly fixed in glutaraldehyde followed by osmium. Significant improvements in preservation are also obtained when tissue is additionally processed through thiocarbohydrazide-osmium (TOTO) processing. In addition to providing a conducting coat, it stabilises the tissue against deformations that might otherwise occur during drying, and reduces the extent of tissue shrinkage. Freeze-drying of TOTO processed tissue produces less tissue distortion than critical point drying (CPD) but is not so easy to apply routinely. The distortions of structure in TOTO-processed CPD tissue are not significant and this may be the preferred procedure for routine use, but air drying from hexamethyldisilazane is a useful alternative, producing results as good as those from CPD samples if TOTO processing is applied beforehand. One particular advantage of freeze-drying, though, is that after freezing, brittle fracture through the tissue can occur making examination of intracellular structure by SEM relatively easy. However, again, TOTO processing prior to freezing is of value as this appears to prevent the formation of large ice-crystal during freezing. Examination of isolated outer hair cells by SEM shows that isolation procedures do not cause significant damage to the stereociliary bundles.
Forge
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Institute of Laryngology and Otology, University College London, United Kingdom.
Nevill
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Zajic
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Wright
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D000818:Animals; D005614:Freeze Fracturing; D006801:Humans; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D009925:Organ of Corti / Q000648:ultrastructure*