Establishment of embedment-free electron microscopy and analysis of the nature of cytoplasmic matrix by this methodology

利用聚乙二醇高水溶性石蜡作为瞬时包埋介质，获得了化学固定组织的非包埋切片，用于电子显微镜检查。经过CO_2的临界点干燥后，这些非包埋切片呈现出与整个贴壁、固定和干燥培养细胞相同的超微结构，并清楚地显示了在传统细胞质基质中未被识别的丝状成分(微小梁)。当用铂和碳旋转复制非包埋切片时，得到的切片图像质量高、分辨率高，与快速冷冻、深度蚀刻复制法获得的图像相当。利用这种新方法，细胞内外表面的膜专门化以及细胞骨架的组织得到了清晰的展示。然而，在通过超速离心法获得细胞器和一些蛋白质在细胞内的位移后，微梁晶格的组织结构没有明显的变化。这与我之前发现的在一定浓度下人工蛋白质溶液呈现与细胞内微梁非常相似的网状结构的发现，反驳了丝状或中间小梁是活细胞的真实结构的观点。无论小梁的真实性或伪影如何，聚乙二醇法也同样适用于一般的扫描电子显微镜，并且扫描和透射电子显微镜与光学显微镜之间的图像的微观关联容易和可靠地进行。此外，聚乙二醇法被证明适用于光镜和电子显微镜免疫细胞化学。因此，这种方法应该被证明是传统显微技术的有价值的补充。

Using polyethylene glycol (PEG), a highly water soluble wax as a transient embedding media, embedment-free sections of chemically fixed tissues are obtained reliably for the electron microscopic examination. After critical-point drying with CO_2, these embedment-free sections present the same aspect of cell ultrastructure as whole mounted, fixed, and dried cultured cells, and filamentous elements (microtrabeculae), which have been unrecognized before in the conventional cytoplasmic matrix, are clearly revealed. Wehen the embedment-free sections are rotaly-replicated with platinum and carbon, resulting images of the sections are of high quality, with excellent resolution and to be quite comparable to those obtained with the rapid freezing, deep etched replica method.With this new methodology, the membrane specializations on the outer and inner cell surfaces as well as the organization of the cytoskeleton is clearly demonstrated. However, after the attainment of the intracellular displacement of cell organelles and some proteins by ultracentrifugation, no marked change in organization of the microtrabecular lattice is discerned.This together with my previous finding that artificial protein solutions at certain concentration exhibit meshworks quite similar to the intracellular microtrabeculae,argue against the idea that gthe filamentous or midrotrabecular strands are the real structure of living cells. Regerdless of the reality or artifact of the microtrabeculae, the PEG method is also applicable to general scanning electron microsoopy, and the intermicroscopic correlation of images between scanning and transmission electron microscopy and light microscopy is easily and reliably performed. Furthermore, the PEG-method is shown to be suitable for light and electron microscopic immunocytochemistry. Therefore this methodology should prove to be valuable adjunct to conventional microscopic techniques.