Cryo-liftout system for preparing in situ lamellae in cryo-FIBSEM

用于在冷冻 FIBSEM 中原位制备片层的冷冻提出系统

• 批准号: RTI-2021-00391
• 负责人: McKee, Marc
• 金额: $ 10.4万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=718836
• 关键词: Cryo; liftout; system; preparing; situ

Overview. Several major challenges exist in our biomineralization research for which a cryo-liftout (cryoLO) FIB-SEM nanomanipulator is urgently needed at this time: i) preserving the continuum of 3D information in specimens, ii) precisely navigating size scales from microns to Ångstroms within hard biospecimens to observe hierarchical structure with correlation context of isolated findings, and iii) obtaining high-resolution information on organic and inorganic interfacial relationships in their pristine hydrated state. Bound water is essential in biomineralization processes, where it is a structural component at the interface between biomolecules and mineral surfaces. Water is important in biological systems because it largely defines their biomechanical properties, but is easily lost during conventional sample preparation and in the high-vacuum environment of electron microscopes. Need/Urgency. Given that both the mineral and organics in our samples are hydrated, with interfacial interactions occurring at the nanoscale in the presence of water, a pressing need is to apply cryo-preparation/imaging technology to retain structural water. A cryoLO is urgently needed to fill a significant gap in our correlative cryo-sample preparation. It addresses the challenges listed above, and will keep us internationally competitive in describing structure and function at the nanoscale in our biomineralization research. We have reached a ceiling using conventional electron microscopy methods, and we urgently need the ability to "go fully cryo" for resolving true, hydrated nanostructure. Our students must receive and apply this cryo-training as they develop their careers in a discipline realizing that cryo is the essential and future way to examine biomineralized structure. Suitability. We already have world-leading research programs using advanced ultrastructural approaches to study biomineralized structures using core instrumentation in McGill's Facility for Electron Microscopy Research (FEMR). Impact. A cryoLO nanomanipulator for our existing dual-beam FIB-SEM electron microscope; McKee CFI-LEF #30797) will enable a unique capability (first in Canada) for describing tomographic detail of hydrated biomineralized structure. With this information as acquired by our planned cryo-workflow using the requested cryoLO, we will understand more broadly and comprehensively, and in the pristine hydrated state at the nanoscale, how mineralized tissues/structures and synthesized biomineral assemblies form and are organized hierarchically to provide their exquisitely tuned mechanical functions. This will keep us at the leading edge for nanostructural characterization of biominerals. In addition to facilitating the research of the applicants, it will have added value in providing much-needed infrastructure for the many other structural biologists that access the FEMR whose research would benefit greatly from cryo-examination of their specimens in a pristine state.

概述。在我们的生物矿化研究中存在着几个主要的挑战，此时迫切需要一个冷冻升降式FIB-SEM纳米操纵器：i)保存样品中3D信息的连续体，ii)在坚硬的生物样品中精确地导航尺寸从微米到ngstroms的尺寸，以观察独立发现的分层结构，以及iii)获得关于原始水合状态下有机和无机界面关系的高分辨率信息。结合水在生物矿化过程中是必不可少的，在生物矿化过程中，结合水是生物分子和矿物表面界面上的结构成分。水在生物系统中很重要，因为它在很大程度上决定了生物系统的生物力学性质，但在常规的样品制备过程中和在高真空的电子显微镜环境中，水很容易丢失。 需要/紧急。鉴于我们样品中的矿物和有机物都是水合的，界面相互作用发生在纳米级的水存在的情况下，迫切需要应用冷冻制备/成像技术来保留结构水。迫切需要一种低温液氧来填补我们在相关低温样品制备方面的一个重大空白。它解决了上面列出的挑战，并将使我们在生物矿化研究中在纳米尺度上描述结构和功能方面保持国际竞争力。我们已经用传统的电子显微镜方法达到了天花板，我们迫切需要“完全冷冻”的能力来解析真正的水合纳米结构。我们的学生必须接受并应用这种冷冻培训，因为他们在一个学科中发展他们的职业生涯，意识到低温是检查生物矿化结构的基本和未来的方法。 适宜性。我们已经拥有世界领先的研究项目，使用先进的超微结构方法，使用麦吉尔电子显微镜研究设施(FEMR)的核心仪器来研究生物矿化结构。 冲击力。用于我们现有的双光束FIB-SEM电子显微镜的CryoLO纳米操纵器；McKee CFI-LEF#30797)将使我们能够(在加拿大首个)实现描述水化生物矿化结构的断层细节的独特能力。有了我们计划的冷冻工作流程使用所要求的冷冻LO获得的这些信息，我们将更广泛和更全面地了解，在纳米尺度的原始水化状态下，矿化组织/结构和合成的生物矿物组件是如何形成和分级组织的，以提供其精细调整的机械功能。这将使我们在生物矿物的纳米结构表征方面保持领先地位。除了促进申请者的研究外，它还将为访问FEMR的许多其他结构生物学家提供急需的基础设施，这些生物学家的研究将极大地受益于对其原始状态的标本进行冷冻检查。