Cryo-SEM and subsequent TEM examinations of identical neural tissue specimen
Cryo-SEM and subsequent TEM examinations of identical neural tissue specimen
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DOI：
10.1016/j.brainresprot.2004.12.004
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发表时间：
2005-02-01
期刊：
影响因子：
--
通讯作者：
Hashikawa, T
中科院分区：
文献类型：
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作者：
Nakatomi, R;Hayashida, T;Hashikawa, T
对冷冻断口标本在低温保护、非脱水、无腐蚀湿“条件下的低温扫描电子显微镜，即直接低温扫描电子显微镜下，对相同的神经组织标本进行了透射电子显微镜观察。与复制的电子显微镜相比，直接低温扫描电子显微镜可以获得对比度渐变平滑的图像。直接低温扫描电子显微镜与透射电子显微镜相结合的主要优点是节省了扫描电子显微镜的制备时间。它在具有较少伪影的多维样本结构的大范围调查中具有很高的潜力。由于样品是在“湿”条件下尽可能快地制备的，因此可以在较低至较高的放大倍数下检查目标结构。在本研究中，神经元和神经胶质成分，如质膜和包括突触小泡的细胞器，定位于骨折表面。在随后的透射电子显微镜检查中，证实了潜在的内部结构可以从细胞学和分子生物学方面进一步表征。此外，直接冷冻扫描显示膜内颗粒细小(直径约10 nm)。然而，由于电子的透明性，它们不能在重新处理的透射电子显微镜样品中得到证实。应用本方案，神经组织的体视学和内部结构检查已在超精细水平上同时进行。(C)2005 Elsevier B.V.保留所有权利。
Low temperature scanning electron microscopy of frozen-fractured specimens under cryo-protecting, non-dehydrating, and non-etching wet" conditions, that is, direct cryo-SEM, was followed by transmission electron microscopy (TEM) with the same neural tissue specimens. In comparison to replica TEM, direct cryo-SEM can obtain images with a smooth gradation of contrast. The major advantage of direct cryo-SEM combined with TEM was that time was saved in SEM preparation. It had a high potentiality at a wide-range survey of multidimensional specimen structures with less-artifacts. Because the specimens were prepared as quickly as possible under "wet" conditions, the target structures could be examined under lower through higher magnifications. In the present study, neuronal and glial elements, such as plasma membranes and cell organelles that include the synaptic vesicles, were localized on the fractured surface. In subsequent TEM examination, it was confirmed that the underlying internal structures could be further characterized from cytological as well as molecular biological aspects. In addition, direct cryo-SEM distinctively demonstrated small intra-membrane particles (ca. 10 nm in diameter). However, due to electron lucency, they could not be confirmed in the re-processed TEM specimens. Applying the present protocol, stereological and internal architectural examinations of the neural tissues have been simultaneously conducted at ultra-fine levels. (c) 2005 Elsevier B.V. All rights reserved.