A LIQUID NITROGEN-COOLED LIGHT MICROSCOPE STAGE TO SCREEN SAMPLES FOR CRYOEM

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 快速冷冻的生物样本显示出近乎自然的结构保存，但这一优点是有代价的；冷冻水合样本的对比度不大，而且样本对电子束非常敏感，因此很难使用电子显微镜来识别这类样本中感兴趣的区域。因此，我们正试图通过光学显微镜对冷冻样品进行预筛选，并将感兴趣的区域绘制在瞬变显微镜-查找器网格上。我们的方法旨在利用细胞生物学中常见的工具，例如GFP标记的感兴趣蛋白质和传统的荧光显微镜。然而，为了在筛选过程中保持样品质量，必须将样品温度保持在低于或等于-130摄氏度。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Rapidly frozen biological samples show near-to-native preservation of structure, but this virtue comes at a price; contrast in frozen-hydrated specimens is modest, and the samples are very sensitive to the electron beam, so it is difficult to use electron microscopy to identify areas of interest in such specimens. We are therefore trying to pre-screen frozen-samples by light microscopy, mapping areas of interest on a TEM-finder grid. Our approach has been designed to make use of tools that are commonly available in cell biology, e.g., GFP-labeled proteins of interest and a conventional fluorescence light microscope. To maintain sample quality during screening, however, one must maintain sample temperature less than or equal to -130 degrees C.