Wheeler E E, Gavin J B, Seelye R N
Stain Technol. 1975 Sep;50(5):331-7. doi: 10.3109/10520297509117083.
The scanning electron microscope appearances and shrinkage of blocks of canine endocardium prepared by freeze-drying directly, by freeze-drying after replacing tissue water with tertiary butanol (2-methyl propan-2-ol) and by critical point drying were compared. All three methods demonstrated endothelial cells which showed nuclear prominences, microvilli and intercellular boundaries. The microvilli varied in size and number from dog to dog but were generally less well defined in specimens freeze-dried from water. Shrinkage due to t-butanol dehydration was significantly less than that which occurred in ethanol in the critical point drying method. Overall the reduction in surface area was significantly less in specimens freeze-dried directly at -65 C (6.8%) than in those dried from t-butanol at -20 C (15.4%) and those prepared bly critical point drying (22.1%). However the amount of shrinkage observed in t-butanol treated tissue was not significantly different from that which was critical point dried. It was not possible to distinguish between comparable samples prepared by these two methods on the basis of their scanning electron microscopic appearances. Thus the relative simplicity and convenience of the t-butanol method, together with its saving of time, its use of standard freeze-drying equipment and the avoidance of ice-crystal artefact justify its consideration as an alternative method of preparing wet biological tissue for scanning electron microscopy.
对通过直接冷冻干燥、用叔丁醇（2-甲基-2-丙醇）置换组织水分后冷冻干燥以及临界点干燥制备的犬心内膜块的扫描电子显微镜外观和收缩情况进行了比较。所有三种方法都显示出内皮细胞，这些细胞表现出核突出、微绒毛和细胞间边界。微绒毛的大小和数量因犬而异，但在从水中冷冻干燥的标本中通常不太清晰。在临界点干燥法中，叔丁醇脱水导致的收缩明显小于乙醇脱水导致的收缩。总体而言，在-65℃直接冷冻干燥的标本（6.8%）的表面积减少明显小于在-20℃从叔丁醇干燥的标本（15.4%）和通过临界点干燥制备的标本（22.1%）。然而，在叔丁醇处理的组织中观察到的收缩量与临界点干燥的组织没有显著差异。基于扫描电子显微镜外观，无法区分通过这两种方法制备的可比样本。因此，叔丁醇法相对简单方便，节省时间，使用标准冷冻干燥设备且避免冰晶假象，这使其有理由被视为一种为扫描电子显微镜制备湿生物组织的替代方法。
