Inner ear tissue preservation by rapid freezing: improving fixation by high-pressure freezing and hybrid methods.
Inner ear tissue preservation by rapid freezing: improving fixation by high-pressure freezing and hybrid methods.
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DOI：
10.1016/j.heares.2014.06.006
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发表时间：
2014-09
期刊：
影响因子：
2.8
通讯作者：
Forge, A.
中科院分区：
文献类型：
--
作者：
Bullen, A.;Taylor, R. R.;Kachar, B.;Moores, C.;Fleck, R. A.;Forge, A.
在尽可能接近生命状态的组织保存中，快速冷冻固定比常规化学固定具有许多优点。一种可以实现快速冷冻固定的技术，即高压冷冻（HPF），已被证明能够实现无冰晶人工制品的冷冻，并将组织保存到比其他快速冷冻方法更深的深度（超过40 μm）。尽管在电子显微镜中越来越成为常规，但HPF用于内耳组织固定的使用受到限制。保存质量的评估表明，常规HPF技术适用于制备各种物种的内耳组织。整个感觉上皮深度的良好保存是可以实现的。与化学固定组织的比较表明，新鲜冷冻制备物显示出总体上级细胞结构保存。然而，HPF固定引起静纤毛的特征性伪影，表明肌动蛋白束的冷冻质量差。预固定和高压冷冻的混合技术显示出在整个组织中产生细胞保存，类似于在单独的HPF中观察到的。预固定HPF产生一致的高质量的保存静纤毛肌动蛋白束。优化样品的制备，使伪影形成最少，可以分析内耳组织超微结构和功能之间的联系。常规高压冷冻可以保存大深度的内耳组织。快速冷冻保存的立纤毛肌动蛋白显示出特征性的人工制品。混合固定方法改善了静纤毛肌动蛋白的结构保存。改进固定将减少内耳超微结构研究中的伪影。
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. Routine high pressure freezing can preserve large depths of inner ear tissue. Stereocilial actin preserved by rapid freezing exhibits characteristic artefacts. Hybrid methods of fixation improved structural preservation of stereocilial actin. Improved fixation will reduce artefacts in ultrastructural studies of the inner ear.
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影响因子：
5.3
作者：
Dumont, RA;Lins, U;Gillespie, PG
通讯作者：
Gillespie, PG
影响因子：
3
作者：
Ohta, Keisuke;Higashi, Ryuhei;Nakamura, Kei-ichiro
通讯作者：
Nakamura, Kei-ichiro
影响因子：
2
作者：
Leforestier, A;Richter, K;Dubochet, J
通讯作者：
Dubochet, J
DOI：
10.1002/jemt.1060140410
发表时间：
1990-04-01
期刊：
JOURNAL OF ELECTRON MICROSCOPY TECHNIQUE
影响因子：
--
作者：
MEISSNER, DH;SCHWARZ, H
通讯作者：
SCHWARZ, H
影响因子：
2
作者：
Semmler, K;Wunderlich, J;Meyer, HW
通讯作者：
Meyer, HW