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

概况. 在我们的生物矿化研究中存在几个主要挑战，目前迫切需要一种低温抬出（cryoLO）FIB-SEM纳米操纵器：ii）在硬生物样本内精确地导航从微米到微米的尺寸尺度，以观察具有孤立发现的相关背景的层次结构，和iii）获得关于在其原始水合状态下的有机和无机界面关系的高分辨率信息。结合水是生物矿化过程中必不可少的，它是生物分子和矿物表面之间界面的结构组分。水在生物系统中很重要，因为它在很大程度上决定了它们的生物力学特性，但在传统的样品制备过程中和电子显微镜的高真空环境中很容易丢失。 需求/紧迫性。 考虑到我们样品中的矿物和有机物都是水合的，在水的存在下，界面相互作用在纳米级发生，迫切需要应用低温制备/成像技术来保留结构水。急需一种cryoLO来填补我们相关的冷冻样品制备中的一个重大空白。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级描述结构和功能方面保持国际竞争力。我们已经达到了使用传统电子显微镜方法的上限，我们迫切需要能够“完全冷冻”来解析真正的水合纳米结构。我们的学生必须接受和应用这种低温培训，因为他们在一个学科中发展自己的职业生涯，认识到低温是检查生物矿化结构的必要和未来的方式。 适宜性。我们已经拥有世界领先的研究计划，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）的核心仪器研究生物矿化结构。 冲击我们现有的双束FIB-SEM电子显微镜的cryoLO纳米操纵器; McKee CFI-LEF #30797）将实现描述水合生物矿化结构的断层扫描细节的独特能力（加拿大首次）。通过我们计划的冷冻工作流程使用所需的cryoLO获得的这些信息，我们将更广泛和全面地了解，在纳米级的原始水合状态下，矿化组织/结构和合成的生物矿物组件如何形成并分层组织，以提供其精致的机械功能。 这将使我们在生物矿物的纳米结构表征方面处于领先地位。除了促进申请人的研究外，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大大受益于对原始状态下标本的冷冻检查。

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.