PERSPECTIVE AND LIMITATIONS OF CRYOELECTRON MICROSCOPY - FROM MODEL SYSTEMS TO BIOLOGICAL SPECIMENS
PERSPECTIVE AND LIMITATIONS OF CRYOELECTRON MICROSCOPY - FROM MODEL SYSTEMS TO BIOLOGICAL SPECIMENS
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DOI：
10.1111/j.1365-2818.1991.tb03088.x
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发表时间：
1991-02-01
期刊：
影响因子：
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通讯作者：
VERKLEIJ, AJ
中科院分区：
文献类型：
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作者：
FREDERIK, PM;STUART, MCA;VERKLEIJ, AJ
我们研究了玻璃化温度敏感脂质相以及（小）生物标本的可能性。从双棕榈酰磷脂酰胆碱（DPPC）制备的单层囊泡悬浮液中，在不同温度下形成薄的水膜。在低温电子显微镜下，根据薄膜形成的温度（分别为318、312和296 K），发现囊泡是光滑的、波纹的和多面的或只有多面的。形貌和电子衍射图表明，该膜可以通过玻璃化物理固定在其高温构型中，并在低温下用冷冻电镜进行研究。这一发现表明，通过在生理温度下启动快速冷却，也有可能将生物体中发现的更复杂的膜系统保存在其原始状态。这是通过玻璃化在（人类）血小板粘附在胶原纤维上的标本网格上形成的薄膜来探索的。以120 kV的加速电压进行低温观察，揭示了亚细胞的细节。当使用更高的电子束加速电压（200和300千伏）时，观察到更多的细节。本文的结果表明低温电子显微镜在膜动力学研究中的巨大潜力，无论是在相对简单的模型膜系统还是在更复杂的生物膜系统中。
We investigated the possibility of vitrifying temperature-sensitive lipid phases as well as (small) biological specimens. From a suspension of unilamellar vesicles, prepared from dipalmitoyl-phosphatidylcholine (DPPC), thin aqueous films were formed at various temperatures. With cryo-electron microscopy vesicles were found to be smooth, rippled and faceted or faceted only, depending on the temperature of thin-film formation (318, 312 and 296 K respectively). The morphology and the electron diffraction patterns indicate that membranes can by physically fixed by vitrification in their high-temperature configuration and studied at low temperature by cryo-electron microscopy. This finding suggests that it may also be possible to preserve, in their original state, the more complex membrane systems found in living organisms by initiating rapid-cooling at a physiological temperature. This was explored by vitrification of thin films formed on specimen grids with (human) blood platelets adhering to collagen fibres. Low-temperature observation with an acceleration voltage of 120 kV revealed subcellular details. More details were observed when using higher accelerating voltages (200 and 300 kV) of the electron beam. The results presented in this paper illustrate the great potential of cryo-electron microscopy in the study of membrane dynamics, both in relatively simple model membrane systems and in more complex biological membrane systems.