Direct observation of t-butyl alcohol frozen and sublimated samples using low-vacuum scanning electron microscopy
Direct observation of t-butyl alcohol frozen and sublimated samples using low-vacuum scanning electron microscopy
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DOI：
10.1679/aohc.61.93
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发表时间：
1998-05-01
影响因子：
--
通讯作者：
Ushiki, T
中科院分区：
文献类型：
--
作者：
Hashizume, H;Itoh, S;Ushiki, T
在低真空环境下，在配备有冷却台和高灵敏度YAG型背散射检测器的“湿SEM”或“可变压力SEM（扫描电子显微镜）”中，对叔丁醇中的冷冻生物样品进行了检查。用戊二醛和四氧化锇固定后，大鼠组织块（气管和肾脏）和培养的人癌细胞用叔丁醇梯度系列脱水。样品直接在-10 ℃的冷却台上冷冻，样品室抽真空至20 Pa，在5-6 kV的加速电压下用背散射电子探测器观察。真空度达到20 Pa后20 min图像变得清晰，30 min图像质量良好，这可能是由于叔丁醇在此期间升华的缘故。气管纤毛细胞的纤毛、肾小球足细胞的端足和培养细胞的突起清晰可见，无严重的制备伪影。由于叔丁醇冷冻样品的低真空SERI既简单又提供高的成像质量，因此它有望用于多种生物领域，例如快速病理诊断。
Frozen biological specimens in t-butyl alcohol were examined under a low-vacuum environment in a "wet SEM" or "variable pressure SEM (scanning electron microscope)" equipped with a cooling stage and highly sensitive backscattered detector of the YAG type. After fixation with glutaraldehyde and osmium tetroxide, rat tissue blocks (tracheae and kidneys), and cultured human carcinoma cells were dehydrated with a graded series of t-butyl alcohol. The specimens were directly frozen on the cooling stage at -10 degrees C, evacuated to 20 Pa in the specimen chamber, and observed by detecting backscattered electrons at accelerating voltages of 5-6 kV, The images became clearer 20 min after the vacuum reached 20 Pa and revealed had good quality by 30 min, probably because t-butyl alcohol was sublimated during the time. The cilia of tracheal ciliated cells, end-feet of the podocytes of the renal glomerulus, and processes of cultured cells were clearly observed without any serious preparation artifacts. Since the low-vacuum SERI of t-butyl alcohol frozen samples is both simple and provides high imaging quality, it is expected to be useful in a variety of biological fields such as the rapid pathological diagnosis.