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的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 快速冷冻的生物样品显示出接近天然的结构保存，但这种优点是有代价的;冷冻水合样品的对比度适中，并且样品对电子束非常敏感，因此很难使用电子显微镜来识别这些样品中的感兴趣区域。 因此，我们正试图通过光学显微镜对冷冻样品进行预筛选，在TEM取景器网格上绘制感兴趣的区域。我们的方法旨在利用细胞生物学中常用的工具，例如，GFP标记的感兴趣的蛋白质和常规的荧光显微镜。然而，为了在筛选期间保持样品质量，必须将样品温度保持在小于或等于-130 ℃。

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.