What we have learned and will learn from cell ultrastructure in embedment-free section electron microscopy
What we have learned and will learn from cell ultrastructure in embedment-free section electron microscopy
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DOI：
10.1002/jemt.20572
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发表时间：
2008-06-01
影响因子：
2.5
通讯作者：
Kondo，Hisatake
中科院分区：
文献类型：
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作者：
Kondo，Hisatake
传统电子显微镜（EM）使用环氧树脂切片来清晰识别电子密度类似于或低于环氧树脂的生物实体的固有局限性导致了EM无包埋切片的发展。无包埋切片EM使用水溶性聚乙二醇（PEG）作为暂时包埋介质，随后通过浸入水中使PEG脱包埋，然后对无包埋切片进行临界点干燥（CPD）。本文作者强调，这种方法清楚地揭示了其轮廓和/或外观在传统EM中相应地模糊和/或模糊的结构，但没有揭示任何新的结构。基于无包埋电子显微镜（PEG-EM），这篇文章提出了关于链或微小梁晶格的五个主要发现，这些发现已经清楚地显示出发生在细胞质基质中-这是常规EM不可能的。这些是：（1）在不同细胞和给定细胞的细胞内区域中出现不同致密性的晶格;（2）在体外白蛋白溶液中忠实地再现了随着浓度增加而相应增加致密性的链晶格;（3）在体外给定浓度下，来自凝胶化明胶的晶格比来自分离明胶的晶格更致密;（4）通过细胞的高渗或低渗预处理，出现更大或更小的晶格致密性;和（5）出现某些细胞内蛋白质，局限于离心神经节细胞的向心半月形结构域，该结构域被占据相当致密的链晶格。从这些发现中，现在出现了关于PEG-EM中微小梁晶格中单个链本身的生物学意义的问题。此外，在给定的生物结构域中出现链格可能代表可溶性蛋白质的存在;晶格紧密度表明结构域中可溶性蛋白质的浓度，而含水细胞质相当于水溶液。此外，在给定的细胞中表现出不同程度的紧凑性的两个连续的晶格结构域的外观表明，细胞质蛋白孤立在具有不太紧凑的晶格的结构域中，而它们在其他结构域中凝胶化。这些建议的解释需要进一步的研究来证实。如果得到证实，那么细胞内细胞器的定位和运动的控制机制不仅可以基于细胞骨架的信息，还可以基于细胞超微结构相关的细胞内蛋白质浓度和溶胶-凝胶状态的信息来理解。此外，对PEG-EM中链格的意义的可能解释也适用于核质，特别是异染色质外（常染色质）区域。最后，PEG-EM在细胞超微结构中的几个潜在用途/优势也得到了证明，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的EM图像以及电子显微镜断层扫描的立体观察。Microsc. Res. Tech. 2008.© 2008 Wiley‐利斯公司
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. Microsc. Res. Tech. 2008. © 2008 Wiley‐Liss, Inc.