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首页> 外文期刊>Microscopy research and technique >What we have learned and will learn from cell ultrastructure in embedment-free section electron microscopy
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What we have learned and will learn from cell ultrastructure in embedment-free section electron microscopy

机译：我们在无嵌入截面电子显微镜中从细胞超微结构中学到的知识和将会学到的知识

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摘要

The limitations inherent in conventional electron microscopy (EM) using epoxy ultrathin sections for a clear recognition of biological entities having electron densities similar to or lower than that of epoxy resin have led to the development of embedment-free sectioning for EM. Embedment-free section EM is reliably performed using water-soluble polyethylene glycol (PEG) as a transient embedding medium, with subsequent de-embedment of PEG by immersion into water, followed by critical point-drying (CPD) of the embedment-free section. The present author has stressed that this approach clearly discloses structures whose contours and/or appearance are accordingly vague and/or fuzzy in conventional EM, but does not reveal any new structures. Based on embedment-free electron microscopy (PEG-EM), this article presents five major findings regarding strand- or microtrabecular lattices which have been clearly revealed to occur in the cytoplasmic matrix-an impossibility with conventional EM. These are (1) the appearance of lattices of different compactness in various cells and in intracellular domains of a given cell; (2) the faithful reproduction from an albumin solution in vitro of strand-lattices with correspondingly increasing compactness following increasing concentrations; (3) the appearance of more compact lattices from gelated gelatin than from solated gelatin at a given concentration in vitro; (4) the appearance of either greater or less lattice-compactness by hyper- or hypotonic pretreatments of cells; and (5) the appearance of certain intracellular proteins confined to the centripetal demilune-domain of centrifuged ganglion cells which is occupied with strand-lattices of a substantial compactness. From these findings, questions now arise as to the biological significance of the individual strand itself in the microtrabecular lattices in PEG-EM. In addition, it may be that the appearance of strand-lattices in a given biological domain represents the presence of soluble proteins; the lattice-compactness indicates the concentration of soluble proteins in the domain, and the aqueous cytoplasm is equivalent to the aqueous solution. Further, the appearance of two contiguous lattice domains exhibiting differing degrees of compactness in a given cell indicates that cytoplasmic proteins are solated in a domain with less compact lattices, whereas they are gelated in the other domain. These proposed interpretations need to be confirmed by further studies. If confirmed, the control mechanisms of the localization and movement of intracellular organelles could then be understood on the basis not only of information about the cytoskeletons but also of cell ultrastructure-related information on the concentration and sol-gel states of intracellular proteins. In addition, possible interpretations of the significance of strand-lattices in PEG-EM are also applicable to the nucleoplasm, especially extra-heterochromatin (euchromatin) areas. Finally, several potential uses/advantages of PEG-EM in the cell-ultrastructure have also been demonstrated, especially in three-dimensional reconstructions of nonmembranous structures including stereo-viewing using a pair of EM images with appropriate tilting as well as electron microscopic tomography.
机译：为了清楚地识别电子密度类似于或低于环氧树脂的生物实体，使用环氧超薄切片的常规电子显微镜（EM）固有的局限性导致了无嵌入切片的发展。使用水溶性聚乙二醇（PEG）作为瞬时嵌入介质可靠地执行无嵌入部分EM，随后通过将PEG浸入水中进行去嵌入，然后对无嵌入部分进行临界点干燥（CPD）来可靠地执行无嵌入部分EM 。本作者已经强调，该方法清楚地公开了在常规EM中其轮廓和/或外观相应地模糊和/或模糊的结构，但是没有揭示任何新结构。基于无嵌入电子显微镜（PEG-EM），本文介绍了有关链或微骨小梁晶格的五个主要发现，这些发现已清楚地揭示出存在于细胞质基质中，这是常规EM不可能实现的。这些是（1）在给定细胞的各种细胞和细胞内区域中不同紧实度的晶格的外观; （2）从白蛋白溶液中忠实地从链晶格中体外复制，随着浓度的增加，紧密度相应增加; （3）在给定的浓度下，明胶凝胶比明胶凝胶更紧密的晶格外观; （4）通过高渗或低渗预处理对细胞产生或多或少的晶格致密性; （5）某些细胞内蛋白质的外观局限于离心的神经节细胞的向心半抗原域，其被相当紧密的链状晶格占据。从这些发现出发，现在对PEG-EM的微小梁网格中单个链本身的生物学意义提出了疑问。另外，在给定的生物学域中，链晶格的出现可能表示可溶性蛋白的存在。晶格紧实度指示结构域中可溶性蛋白质的浓度，并且细胞质水溶液等同于水溶液。此外，在给定的细胞中出现两个展现出不同紧实度的连续晶格结构域表明，胞质蛋白在具有较少致密晶格的结构域中被分离，而在另一个结构域中被胶凝。这些建议的解释需要进一步研究证实。如果得到证实，则不仅可以根据有关细胞骨架的信息，还可以根据有关细胞内蛋白质的浓度和溶胶-凝胶状态的细胞超微结构相关信息来理解细胞内细胞器的定位和运动的控制机制。此外，对PEG-EM中链晶格重要性的可能解释也适用于核质，尤其是异染色质外（常染色质）区域。最后，还证明了PEG-EM在细胞超微结构中的几种潜在用途/优点，特别是在非膜结构的三维重建中，包括使用一对具有适当倾斜度的EM图像以及电子显微镜断层扫描进行立体观察。

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