title: Inner ear tissue preservation by rapid freezing: improving fixation by high-pressure freezing and hybrid methods.
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.
authors.affiliation: Centre for Auditory Research, UCL Ear Institute, London WC1X 8EE, UK. Electronic address: a.bullen@ucl.ac.uk.
authors.forename: A
authors.initials: A
authors.lastname: Bullen
authors.affiliation: Centre for Auditory Research, UCL Ear Institute, London WC1X 8EE, UK.
authors.forename: R R
authors.initials: RR
authors.lastname: Taylor
authors.affiliation: Laboratory of Cell Structure and Dynamics, NIDCD, National Institutes for Health, Bethesda, MD 20892-8027, USA.
authors.forename: B
authors.initials: B
authors.lastname: Kachar
authors.affiliation: Institute of Structural and Molecular Biology, Birkbeck College, London WC1E 7HX, UK.
authors.forename: C
authors.initials: C
authors.lastname: Moores
authors.affiliation: National Institute for Biological Standards and Control, Potters Bar EN6 3QG, UK.
authors.forename: R A
authors.initials: RA
authors.lastname: Fleck
authors.affiliation: Centre for Auditory Research, UCL Ear Institute, London WC1X 8EE, UK.
authors.forename: A
authors.initials: A
authors.lastname: Forge
mesh_terms: D000818:Animals; D016477:Artifacts; D015925:Cryopreservation / Q000379:methods*; D007758:Ear, Inner / Q000473:pathology* / Q000648:ultrastructure; D005849:Gerbillinae; D006168:Guinea Pigs; D051379:Mice; D008810:Mice, Inbred C57BL; D008854:Microscopy, Electron; D023421:Models, Animal; D009673:Notophthalmus viridescens; D009926:Organ Preservation / Q000379:methods*; D059547:Stereocilia / Q000473:pathology / Q000648:ultrastructure; D013997:Time Factors; D016707:Tissue Fixation / Q000379:methods*