Lindroth M, Bell P B, Fredriksson B A, Liu X D
Department of Pathology II, Faculty of Health Sciences, Linköping University, Sweden.
Microsc Res Tech. 1992 Jul 1;22(2):130-50. doi: 10.1002/jemt.1070220203.
Today's electron microscopes have a resolution sufficient to resolve supramolecular structures. However, the methods used to prepare biological samples for electron microscopy often limit our ability to achieve the resolution that is theoretically possible. We use whole mounts of detergent-extracted cells grown on Formvar-coated gold grids as a model system to evaluate various steps in the preparation of biological samples for high resolution scanning electron microscopy (SEM). Factors that are important in determining the structure and composition of detergent-extracted cells include the nature of the detergent and the composition of the extraction vehicle. Chelation of calcium is extremely important to stabilize and preserve the cytoskeletal filaments. We have also demonstrated both morphologically and by gel electrophoresis that treatment of cells with bifunctional protein crosslinkers before or during extraction with detergent can significantly enhance the preservation of both proteins and supramolecular structures. The methods used to dry samples are a major determinant of the quality of structural preservation. For cytoskeletons freeze-drying (FD) is superior to critical point-drying (CPD), one reason being that CPD samples have to be dehydrated, thereby causing more shrinkage as compared to FD samples. The high pressures to which samples are exposed during CPD may also cause increased shrinkage, and water contamination during CPD causes severe structural damage. We have obtained the best structural preservation of detergent-extracted and fixed cells by manually plunging them into liquid propane and drying over night in a freeze-dryer. The factor that most limits achievement of high resolution in SEM is the metal coat, which has to be very thin, uniform, and free of grain in order not to hide structures or to create artifactual ones. We have found that sputter-coating with 1-3 nm of tungsten (W) or niobium (Nb) gives extremely fine-grained films as well as satisfactory emission of secondary electrons. These samples can also be examined at high resolution by transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). The best preservation and visualization of supramolecular structures have been obtained using cryosputtering, in which the samples are freeze-dried and then sputter-coated within the freeze-dryer while still frozen.
如今的电子显微镜具有足以分辨超分子结构的分辨率。然而，用于制备生物样本以进行电子显微镜观察的方法常常限制了我们达到理论上可能的分辨率的能力。我们使用在福尔马林中涂覆金网的洗涤剂提取细胞的整装片作为模型系统，来评估用于高分辨率扫描电子显微镜（SEM）的生物样本制备中的各个步骤。在确定洗涤剂提取细胞的结构和组成方面重要的因素包括洗涤剂的性质和提取介质的组成。钙的螯合对于稳定和保存细胞骨架丝极其重要。我们还通过形态学和凝胶电泳证明，在用洗涤剂提取之前或期间用双功能蛋白质交联剂处理细胞，可以显著增强蛋白质和超分子结构的保存。用于干燥样本的方法是结构保存质量的主要决定因素。对于细胞骨架，冷冻干燥（FD）优于临界点干燥（CPD），一个原因是CPD样本必须脱水，因此与FD样本相比会导致更多收缩。CPD过程中样本所承受的高压也可能导致收缩增加，并且CPD期间的水污染会造成严重的结构损伤。我们通过将洗涤剂提取和固定的细胞手动投入液态丙烷并在冷冻干燥机中过夜干燥，获得了最佳的结构保存效果。在SEM中最限制实现高分辨率的因素是金属涂层，它必须非常薄、均匀且无颗粒，以免隐藏结构或产生人为结构。我们发现用1 - 3纳米的钨（W）或铌（Nb）进行溅射镀膜可以得到极细颗粒的薄膜以及令人满意的二次电子发射。这些样本也可以通过透射电子显微镜（TEM）和扫描透射电子显微镜（STEM）进行高分辨率检查。使用低温溅射镀膜获得了超分子结构的最佳保存和可视化效果，即在冷冻干燥机中对样本进行冷冻干燥，然后在仍处于冷冻状态时进行溅射镀膜。
