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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.