Reger J F, Escaig F
Department of Anatomy and Neurobiology, University of Tennessee, Memphis.
J Submicrosc Cytol Pathol. 1988 Oct;20(4):691-700.
Adult rat (Sprague-Dawley) cerebral cortex was processed by ultra-rapid freezing at liquid helium temperature followed by freeze-substitution, osmium fixation and by other chemical, post-osmication procedures at 0-4 degrees and ambient temperatures as a comparative study for purposes of identifying differences and/or similarities in fine structure following these techniques. Five methods of processing were used: 1) rapid, slam-freezing at liquid helium temperature followed by osmium tetroxide/acetone freeze substitution; 2) perfusion with buffered, 2% glutaraldehyde at ambient temperature followed by post-osmication (2%); 3) en-bloc, buffered 2% glutaraldehyde fixation at 0-5 degrees centigrade and post-osmication (2%); 4) buffered, 2% osmium tetroxide perfusion at ambient temperature; and 5) en-bloc, buffered 2% osmium tetroxide fixation at 0-5 degrees centigrade. In ultra-rapid-frozen cortex good preservation was seen to a depth of 10-15 microns from the surface of the initial, copper-block contact. The tissue processed by ultra-rapid-freeze, freeze substitution demonstrates a general 'smoothness' of plasmalemmal and organelle membranes not observed in tissue prepared by chemical fixation alone. Cellular and organelle morphological differences were minor beyond the general 'smoothness' of membranes and a more intense background, electron density found in tissue prepared by rapid-freeze. Of particular interest was the practically identical images found in the four, chemical techniques not preceded by ultra-rapid freezing. High magnification images also revealed rather minor differences following ultra-rapid-freezing compared to tissue fixed by chemical fixation alone. Although these morphological differences are minimal, there can be no question of the fact that ultra-rapid-freeze followed by freeze-substitution is morphologically superior to chemical fixation alone. Ultra-rapid-freeze, eventually utilizing other substitution agents than osmium tetroxide, will offer several advantages and should be particularly useful for investigators involved in cytochemical and immunochemical methods.
成年大鼠（斯普拉格-道利大鼠）大脑皮层在液氦温度下进行超快速冷冻，随后进行冷冻置换、锇固定，并在0-4摄氏度及室温下进行其他化学后锇化程序，作为一项对比研究，目的是确定采用这些技术后在精细结构上的差异和/或相似性。使用了五种处理方法：1）在液氦温度下快速猛冻，随后进行四氧化锇/丙酮冷冻置换；2）在室温下用2%戊二醛缓冲液灌注，随后进行后锇化（2%）；3）在0-5摄氏度下进行整块2%戊二醛缓冲液固定和后锇化（2%）；4）在室温下用2%四氧化锇缓冲液灌注；5）在0-5摄氏度下进行整块2%四氧化锇固定。在超快速冷冻的皮层中，从最初的铜块接触表面起10-15微米深度范围内可见良好的保存效果。通过超快速冷冻、冷冻置换处理的组织显示出质膜和细胞器膜总体上的“光滑度”，这在仅通过化学固定制备的组织中未观察到。除了膜的总体“光滑度”以及在快速冷冻制备的组织中发现的更强背景电子密度外，细胞和细胞器的形态差异较小。特别有趣的是，在未进行超快速冷冻的四种化学技术中发现了几乎相同的图像。与仅通过化学固定的组织相比，高倍放大图像还显示超快速冷冻后存在相当小的差异。尽管这些形态差异很小，但超快速冷冻后进行冷冻置换在形态学上优于仅进行化学固定这一事实是毋庸置疑的。最终使用除四氧化锇之外的其他置换剂的超快速冷冻将具有多种优势，并且对于从事细胞化学和免疫化学方法研究的人员应该特别有用。
