Bridgman P C, Lewis A K, Victor J C
Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110.
Microsc Res Tech. 1993 Apr 1;24(5):385-94. doi: 10.1002/jemt.1070240504.
In order to test the ability of freeze substitution to accurately preserve the ultrastructure of the actin component of the cytoskeleton, the structure of rotary shadowed actin filaments was compared following preparation by glutaraldehyde fixation and freeze etch or freeze substitution. Freeze substituted actin filaments were further processed by either etching away frozen organic solvent or critical-point-drying before rotary shadowing. Comparison of filament diameters showed no significant difference between actin filaments that were directly etched and those that were freeze substituted and then etched. However, freeze substituted and then critical-point-dried filaments were significantly larger in diameter than filaments that were directly etched in water. The long pitch (right-handed) two start helix was not affected by the different methods of preparation. However, the left-handed "genetic" helical repeat that was prominent in actin filaments prepared by freeze etch was more difficult to detect in freeze substituted specimen, especially following critical-point-drying. Although the organization and distribution of actin filaments in extracted cells was similar in both freeze substituted and freeze etched specimens, there were some detectable differences. In cells that were freeze substituted and then critical-point-dried, filaments appeared to intersect at greater angles and seemed more "taut." These results suggest that freeze substitution can preserve the overall morphology of actin filaments, but some chemical or physical modification of macromolecular surface structure may occur during the substitution process and these changes may be further exaggerated by subsequent processing steps.
为了测试冷冻置换准确保存细胞骨架肌动蛋白成分超微结构的能力，在通过戊二醛固定、冷冻蚀刻或冷冻置换制备后，比较了旋转阴影肌动蛋白丝的结构。冷冻置换的肌动蛋白丝在旋转阴影前，通过蚀刻掉冷冻有机溶剂或临界点干燥进行进一步处理。细丝直径的比较表明，直接蚀刻的肌动蛋白丝与冷冻置换后再蚀刻的肌动蛋白丝之间没有显著差异。然而，冷冻置换后再临界点干燥的细丝直径明显大于直接在水中蚀刻的细丝。长螺距（右手）双起始螺旋不受不同制备方法的影响。然而，在冷冻蚀刻制备的肌动蛋白丝中突出的左手“遗传”螺旋重复在冷冻置换标本中更难检测到，尤其是在临界点干燥之后。尽管在冷冻置换和冷冻蚀刻标本中，提取细胞中肌动蛋白丝的组织和分布相似，但仍有一些可检测到的差异。在冷冻置换后再临界点干燥的细胞中，细丝似乎以更大的角度相交，并且看起来更“紧绷”。这些结果表明，冷冻置换可以保留肌动蛋白丝的整体形态，但在置换过程中可能会发生大分子表面结构的一些化学或物理修饰，并且这些变化可能会被后续处理步骤进一步放大。
