Abstract
In the preservation of tissues in as ‘close to life’ state as possible, rapid freeze fixation has many benefits over conventional chemical fixation. One technique by which rapid freeze-fixation can be achieved, high pressure freezing (HPF), has been shown to enable ice crystal artefact-free freezing and tissue preservation to greater depths (more than 40 μm) than other quick-freezing methods. Despite increasingly becoming routine in electron microscopy, the use of HPF for the fixation of inner ear tissue has been limited. Assessment of the quality of preservation showed routine HPF techniques were suitable for preparation of inner ear tissues in a variety of species. Good preservation throughout the depth of sensory epithelia was achievable. Comparison to chemically fixed tissue indicated that fresh frozen preparations exhibited overall superior structural preservation of cells. However, HPF fixation caused characteristic artefacts in stereocilia that suggested poor quality freezing of the actin bundles. The hybrid technique of pre-fixation and high pressure freezing was shown to produce cellular preservation throughout the tissue, similar to that seen in HPF alone. Pre-fixation HPF produced consistent high quality preservation of stereociliary actin bundles. Optimising the preparation of samples with minimal artefact formation allows analysis of the links between ultrastructure and function in inner ear tissues.
Highlights
Preservation of biological structure in a state as ‘close to life’ as possible is best achieved by fixation of all components of the sample at the same time
The preservation of inner ear tissue by high pressure freezing (HPF) was assessed in a number of ways, using the tissues of different species under different freezing conditions
Utricular maculae can be isolated from mice as well as guinea pigs and gerbils, allowing testing across several mammalian species to ensure there were no speciesspecific differences in preservation
Summary
Preservation of biological structure in a state as ‘close to life’ as possible is best achieved by fixation of all components of the sample at the same time. From this perspective, the use of chemical fixatives is not desirable due to the time taken to diffuse fixatives through the sample and the specificity of the chemical crosslinks formed (Gilkey and Staehelin, 1986). Samples are cooled at a rate sufficient for water to be frozen in a vitreous state, without the formation of ice crystals (“vitrification”). Structures are preserved hydrated, and in as close to their native state as possible.
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