A useful method for observing intracellular structures of free and cultured cells by scanning electron microscopy
A useful method for observing intracellular structures of free and cultured cells by scanning electron microscopy
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DOI：
10.1093/jmicro/dfr098
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发表时间：
2012-04-01
期刊：
影响因子：
--
通讯作者：
Ushiki, Tatsuo
中科院分区：
文献类型：
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作者：
Koga, Daisuke;Nakajima, Masato;Ushiki, Tatsuo
使用锇浸渍方法的扫描电子显微镜（SEM）已用于分析组织样品中细胞器的三维结构，但用该技术观察游离和培养的细胞相当困难。本研究的目的是开发一种方法，可以适用于自由和培养细胞的扫描电镜研究细胞内结构后，锇浸渍。该方法也适用于光学显微镜（LM）和透射电子显微镜（TEM）。用0.5%多聚甲醛和0.5%戊二醛的混合物固定HeLa细胞和人白细胞，然后用1%四氧化锇进行额外固定。将这些细胞包埋在低熔点琼脂糖中。在包埋之前，还使用温度响应皿收集培养的细胞。对于LM和TEM，细胞包埋的琼脂糖进一步包埋在环氧树脂中，并常规检查半切片和双切片。对于SEM，琼脂糖在50%二甲基亚砜中冷冻断裂，进行锇浸渍处理，并在高分辨率SEM中观察。低熔点琼脂糖作为包埋介质是有用的，因为它是在长期的渗透扫描电镜保存良好。因此，锇浸渍处理后的细胞器的精细结构，清楚地分析了扫描电镜。这些SEM图像也可以与琼脂糖包埋组织的LM和TEM图像进行比较。
Scanning electron microscopy (SEM) using osmium-maceration methods has been used for analyzing the three-dimensional structure of cell organelles in tissue samples, but it has been quite difficult to observe free and cultured cells with this technique. The present study was performed to develop a method that can be applied to free and cultured cells for SEM studies of intracellular structures after osmium maceration. The method was also applied to light microscopy (LM) and to transmission electron microscopy (TEM). HeLa cells and human leukocytes were fixed with a mixture of 0.5% paraformaldehyde and 0.5% glutaraldehyde followed by an additional fixation with 1% osmium tetroxide. These cells were embedded in low-melting-point agarose. A temperature-responsive dish was also used for collection of cultured cells before embedding. For LM and TEM, the cell-embedded agarose was further embedded in epoxy resin, and semi- and ultrathin sections were examined conventionally. For SEM, the agarose was freeze-fractured in 50% dimethyl sulfoxide, processed for osmium maceration and observed in a high-resolution SEM. Low-melting-point agarose was useful as an embedding medium for SEM, because it was well preserved during prolonged osmication for SEM. Thus, the fine structure of cell organelles was clearly analyzed by SEM after osmium-maceration treatment. These SEM images could also be compared with those of LM and TEM of the agarose-embedded tissues.