HIGH PRESSURE CRYOCOOLING FOR BIOLOGICAL X-RAY DIFFRACTION MICROSCOPY

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。 我们开发了低温X射线衍射显微镜(Cryo-XDM)，用于对水合的、未染色的生物样品进行成像，最近报道了首次使用8keV x射线在30至50 nm的分辨率下冷冻水合的耐辐射金黄色葡萄球菌的图像。在展示了低温XDM在生物成像中的可行性后，我们希望将分辨率提高到10 nm，以可视化精细的细胞结构，可能是在分子水平上。到目前为止，实现这一目标的主要挑战之一是冰层条件。在骤降-冻结阶段，样品可能会被结晶冰污染，这可能会导致噪声水平增加，使分相算法无法找到解决方案。为了解决这个问题，我们计划与康奈尔大学的S.Gruner博士和C.U.Kim博士合作，利用高压冷冻样本将它们保存在无定形冰态下。我们要求1天的光束时间，以测试高压冷冻应用于生物XDM的可行性。

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. We have developed cryogenic x-ray diffraction microscopy (cryo-XDM) for imaging hydrated, unstained biological samples and recently reported the first frozen-hydrated images of D. radiodurans with 8 keV x-rays at a resolution of 30 to 50 nm. Having shown the feasability of cryo-XDM in biological imaging, we hope to improve the resolution toward 10 nm to visualize fine cellular structures, possibly at the molecular level. So far, one of the main challenges in reaching this goal has been the ice condition. During the plunge-freezing stage, samples can become contaminated with crystalline ice which can result in increased noise levels preventing the phasing algorithm from finding a solution. To solve this problem we plan to utilize high-pressure freezing samples to preserve them in the amorphous ice state in collaboration with Dr. S. Gruner and Dr. C.U. Kim at Cornell. We request 1 day beamtime to test the feasibility of applying high-pressure freezing for biological XDM.